Carbon-13 nuclear magnetic resonance studies of proteins.

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Ann. Rev. Biophys. Bioeng. 1977. 6:383-417 Copyright © 1977 by Annual Reviews Inc. All rights reserved

Annu. Rev. Biophys. Bioeng. 1977.6:383-417. Downloaded from www.annualreviews.org Access provided by University of Michigan - Ann Arbor on 02/20/15. For personal use only.

CARBON-13 NUCLEAR

MAGNETIC RESONANCE

+9098

STUDIES OF PROTEINS William Egan, Heisaburo Shindo, and Jack S. Cohen

Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014

INTRODUCTION Nuclear magnetic resonance (NMR) methods, in principle, are capable of resolving resonances of single atoms in protein molecules. In practice, most of the resonances from hydrogen nuclei (lH NMR) in proteins overlap to produce a broad unresolved envelope (1). Carbon-13 NMR (13C NMR) has an intrinsically higher resolving power than proton NMR by a factor of about 20 because of the greater range of electronic configurations experienced by carbon atoms (2). Consequently, initial results on amino acids were interpreted to indicate that I3C NMR would be a potentially valuable tool in studies of proteins in solution (3). Initially, sensitivity problems made this potential unrealizable. The sensitivity of carbon-13 relative to proton NMR at the same magnetic field strength is 1:62.9. If the fact that I3C has a natural abundance of 1.1 % is taken into account (12C has no resonance condition), this leads to a relative sensitivity of 1:5716. This sensitivity problem in l3C NMR, however, has to a large extent been overcome by the introduc tion of the pulse Fourier transform method (4). Further losses of signal intensity in BC NMR spectra arise as a result of coupling between 13C and IH nuclear spins. These couplings can be removed by the applica tion of a second radiofrequency field at the resonance value for protons. If this frequency has sufficient power and is spread over the entire range of proton absorp tions, a process often accomplished by noise modulation, then complete decoupling of the proton nuclear spins from the 13C nuclear spins results. Each carbon atom in the molecule then gives rise to a single resonance, and the resultant proton decoupled I3C spectra are relatively simplified. In addition, applying a IH decou piing frequency leads to changes in the relative populations of the energy levels for the DC nuclear spins; this phenomenon, known as the nuclear Overhauser effect 383

384

EGAN, SHINDO & COHEN

(NOE), results in an enhancement of the integrated areas of the 13C resonances, the maximum value of which is 1.98, and this yields a maximum value of 1:1918 for the relative sensitivity for I3C and IH NMR.


Rationale for the Application of J3c NMR in Protein Chemistry

The two most important problems in protein chemistry are how proteins fold and how they function. Forty years of physicochemical work has not sufficed to answer these questions in satisfactory detail. A great deal is known of the molecular architecture of some 40 proteins from their X-ray crystallographic structures (5). However, such structures do not provide evidence for the mechanism of folding of the polypeptide chain to form a globular protein in solution (6). The standard spectroscopic techniques used to study gross conformational changes in solution (UV, CD-ORD, fluorescence) do not generally provide sufficient structural resolution to allow a detailed understanding of the mechanism of protein folding. There has been a tendency to move from experimen tal to theoretical approaches in considering the problem of protein folding. How ever, any solution suggested by theoretical analysis must be tested experimentally before it can be considered acceptable. In many cases the X-ray crystallographic structures of protein have suggested possible mechanisms of enzymatic action involving specific amino acid residues. Often these groups have been implicated by prior chemical modification studies in solution, but this method provides ambiguous results of the uncertainty principle type since they involve disturbing the native structure one wishes to study. The precise roles of the groups suggested by the chemical modification and X-ray crystal lographic studies involved in the mechanism of action need to be confirmed by solution methods. Ideally, a technique is required that in some way would provide a synthesis of the physical resolution of X-ray crystallography with the dynamic solution characteris tics of the spectroscopic methods. BC NMR is such a synthesis; it not only can give kinetic data if spectra are taken as a function of time but it also can give information on dynamic equilibria in solution. It should not be construed, however, that I3C NMR is considered an alternative to these other methods but rather that it repre sents a complementary technique filling a gap between their respective optimum capabilities. Nevertheless, at the present, 13e NMR must be considered the method of choice for studying many problems in protein chemistry. IH NMR has been most valuable in studies of proteins where resonances that can be assigned to individual atoms in the molecule are involved (7). Such resonances then provide information on the electronic configuration of the atom when the protein is subjected to a perturbation, such as change in temperature, solution pH, or inhibitor concentration. In effect such resonances are sensitive, nondisturbing probes of their own microenvironment within the protein molecule. Since the resolution of carbon atoms of distinct chemical types is far greater than that of protons, it is to be expected that a wider range of carbon atom resonances are likely to be resolvable in protein 13e NMR spectra. This would presume that

NMR PROTEIN STUDIES

385

line widths are not greater for 13C resonances than for IH resonances (they are usually sharper). Results published in the past 3 years have provided clear evidence for this proposition. Recently, the resolution of single carbon atom resonances have been reported for tryptophan, tyrosine, arginine, phenylalanine, histidine, and lysine residues (8, 9). In addition, the resolution of glutamic and aspartic acid carboxyl carbon resonances have recently been reported (10).


Use of Carbon-13 Labeling One unique advantage of I3C NMR, and indeed NMR methods of any stable isotope existing at low levels of natural abundance, is the ability to utilize enrichment of the isotope. This is not possible for IH NMR, although the reverse negative labeling with deuterium to remove proton resonances is a standard technique. The capability of labeling selected carbon atoms in a molecule with high I3C enrichment has two distinct advantages-direct assignment and improved sensitivity. If a carbon atom is enriched to n% its individual resonance will be n times the intensity (area) in the resulting spectrum and consequently it will be readily assignable. Indeed, with high enrichment a high order of resolution is not required, provided the enriched resonance is distinguishable above the background of multiple natural abundance resonances. For this purpose, a minumum of about 15% is needed for i3C NMR protein studies (11). One further advantage of higher enrichment of I3C is the possibility of measuring I3C-I3C coupling constants (12). Of course, at natural abundance there is only a 0.01 % probability that two I3C atoms will be adjacent to each other, thus making detection virtually impossible. At 10% enrichment this figure has risen to I %, at 50% enrichment to 25%, and of course at 100% to 100% probability. Since these couplings are expected to contain information on the relative electronic and angular configurations of adjacent bonded carbon atoms (13), it is clear this could be a distinct advantage of higher i3C enrichment. However, this matter must be qualified by several considerations. At high abundance levels three-bond and higher i3C couplings begin to be observed. Although these may contain much more information than the direct two-bond couplings, they also give rise to much more complex spectra. In addition, the further couplings of the carbon resonances reduces the simplicity of the spectrum by increasing the number of lines. This can lead to problems of resolution and assignment, the very advantages for which I3C labeling can be so valuable. Consequently, multiple carbon atom enrichment . must be ap proached with caution. Clearly there are competing effects that must be evaluated for each type of investigation. Apart from the above potential pitfalls there is the basic question of how does one label a specific carbon atom in a molecule as complex as a protein. Logically there are three approaches: biosynthesis, chemical synthesis, and chemical modification. Utilizing the biosynthetic approach will lead to all groups of a given type in the molecule being labeled (14), so it is not as specific as one might generally desire. In addition, multiple metabolic pathways in the organism can lead to a loss of the very expensive 13e metabolite.


386


Chemical syntheses in the case of proteins implies peptide synthesis, and there are limitations in this approach due to the less than perfect yields obtained at each step in the peptide bond synthesis. With the solid phase method (15) it is possible to synthesize peptides containing up to 20 amino acids in reasonable yields. In such cases, where yields may be low, high 13C enrichment is most important. With the chemical modification approach it is possible to attach a standard group, such as a carboxymethyl group, to a protein, but with one or more of the carbon atoms in the group enriched ( 1 6). However, this approach is open to the same criticisms that can be leveled against chemical modification without stable isotope labeling. A more subtle extension of this approach, which might be termed isotopic substitution, is the substitution by chemical methods of one or more of the I3C atoms in the intact protein (17).

CONSIDERATIONS OF SENSITIVITY AND RESOLUTION Due to the large number of resonances occurring in restricted spectral regions and the limiting amounts of material usually available or desirable for study, consider ations of resolution and sensitivity assume a special importance for 13e NMR investigations of biomacromolecules. In this section, several factors that influence these parameters are discussed. We define resolution as the ability to separate single carbon atom resonances and sensitivity as the ability to detect a given resonance above the background noise level. The total effective magnetic field, Heff' acting on a nucleus in an atom or molecule is proportional to the applied field, Ho: Heff = Ho(l- cr) where cr is the screening factor, a dimensionless constant whose value is independent of Ho but dependent on electronic (chemical) environment. This screening factor derives from the mag netic fields set up in the molecule by the circulation of electrons induced by Ho. As a result of screening, the resonance condition is modified relative to a free nucleus, becoming u = 'YHcoff/27T, where u is the resonance frequency and 'Y is the mag netogyric ratio, a nuclear constant (for a discussion of magnetic resonance parame ters see 1 8, 19). The frequency at which a particular nuclear spin resonates is therefore a function of its electronic microenvironment. Assuming that NMR line widths are independent of Ho (a point to which we will shortly return), then the frequency separation between absorptions and conse quently the ability to separate single carbon atom resonances is directly proportional to Ho. Therefore, from the viewpoint of spectral resolution, operation at the highest field strength seems advantageous (Figure I). High resolution 13e spectrometers operating at field strengths between about I.S and 8.6 T ( 1 T 104 gauss) are now commercially available; those operating above about 2.5 T are superconducting magnet systems. The question of sensitivity in comparison to resolution is more complex, as it is subject to a number of variables deriving from both the intrinsic properties of the spin system and external technological constraints. From Faraday'S law, the voltage induced in the receiver coil [whose axis is positioned perpendicular to Ho ( =H.)] is proportional to the strength of the initial nuclear magnetization and the rate of =

NMR PROTEIN STUDIES

387

16% HEW I.YSOZYME


at pH 3.9,38°C

1 80

Figure I

(J 6%

175

Chemical Shift in ppm

165

170

I3C NMR spectra of carboxyl and carbonyl resonances of hen egg white lysozyme

solution in 0. 1 M NaCl) at differing field strengths.

(Top trace)

15-MHz observing

frequence, 4-kHz sweep width, 8-k data points, 32-k scans with 1 .5-sec delay time.

trace)

25-MHz,

6.25-kHz

0.2-sec delay time.

(Middle

sweep width, 8-k data points with 8-k zero filling, 36-k scans with

(Bottom trace)

scans with l.S-sec delay time.

68-MHz,

15.2-kHz

sweep width,

32-k

data points, 36-k


388

EGAN. SHINDO & COHEN

change of the magnetization (19). Since the initial magnetization in the high temper ature limit (kT}> yHo h) and the Larmor precessional frequency are each linearly proportional to Ho. the induced signal voltage is expected to be proportional to H;. In practice. the signal strength must be considered in conjunction with the noise level. In a tuned circuit, the mean square noise is a linear function of the frequency (19); then:fore, we expect the signal to root mean square (rms) noise [the signal- to-noise ratio (S/N)] to be proportional to H!·s. Recently Hoult & Richards (20) re-examined the factors governing this 3/2 dependence and concluded that a 7/4 field dependence is more general. For a variety of reasons, however, a less than 3/2 or 7/4 dependence is usually observed. As indicated in Table I an approximately linear dependence is observed, with the dependence somewhat greater than linear in the lower frequency electromagnet range and less than linear at the higher frequencies on going from an electromagnet to a superconducting magnet. However, it should be noted that significant improvements in sensitivity for the commercial systems are possible, as the modifications made here at the National Institutes of Health with the TT-14 and WH-270 systems indicate, and the figures quoted in Table 1

Integral S INa for ethylene g lycol-d2 at differing magnetic field strengths Observing frequency

Spectrometer

(MHz)

FX�Of

15

TT-14

15

TT-14

FX-IOO

(ml)

Normalized relative SIN ratiod

IJlnc

Obs.

H0e

3/2e Ho

1.0

1.0

1.6

1.0

10.0 (20)

4.0

0.77

15

1.5 (10)

3.0

I.S

25

0.95(10)

3.0

1.9

1.1

2.2

68

1.2 (10)

6.5

3.6

4.5

9.6

68

1.9 (12)

15.0

6.6

(home-

built probe)

S am ple

v olume b

0.95(10)g

NIH-270 (Bruker magnet and probe;

home-built spectromete r)

NIH-270 (Bruker magnetic and homebuilt probe and spectrometer)

aThe sample used for measuring the quisitions were collected with a

4-kHz

SIN ratio

was neat ethylene glycol-d2. Eight ac

sweep and audio filter. The signal intensity, Is. is

given by integration of the Fourier Transformed signal; the

rms noise, In. is given by

integral value of the power spectrum of the noise component over bEstimated effective sam ple volume.

1024

the

data points.

CEither crystal filtering or quadrature phase detection was used in al\ cases. d The standard SIN ratio was based on an effective volume of 1 ml at a IS-MHz ob

serving frequency for the

FX�O spectrometer. eTheoretical values assuming the SIN ratio to

of the applied magnetic field strength. f Data courtesy of K. Goto (JEOL, USA).

gNMR tube outer diameter (in millimeters).

vary either linearly or with the

3/2

power


NMR PROTEIN STUDIES

389

Table 1 should not be taken as definitive. We can additionally expect that commer cial probe designs for superconducting systems will improve in the near future as they did for electromagnet systems in the past. In the following discussion, a first power dependence of the SIN ratio on the field strength is assumed. Thus far it appears that the greater the magnetic field strength, the better both spectral resolution and SIN ratio. However, for I3C studies at low concentration it is often necessary to accumulate a large number of free induction decoys (FIOs); accordingly, a consideration of the time required for the spin system to return to equilibrium (the relaxation times) before the next pulse can be applied is required [a discussion of relaxation mechanisms in BC NMR spectroscopy may be found (21)] (cf 19). We initially consider only the case of 13C_IH dipole-dipole (�O) relaxation for an isotropically reorienting C-H vector. For biomacromolecules, at the usual \3C resonance frequencies (about 15-90 MHz) the extreme narrowing condition (6lc'Tc < 1) is not met and Tl becomes a function of Ho. In these circumstances a longer time is required for the magnetiza tion to return to its equilibrium value at the higher field strength. Accordingly, fewer FIOs can be collected per unit time; alternatively, for given delay periods between pulses, a smaller portion of the magnetization will have returned to equilibrium at the higher field strength, resulting in a comparatively weaker free induction decay signal after a subsequent pulse. The dependence of T1 on correlation time as a function of Ha is given in Figure 2a. As mentioned, a contribution to 13C NMR signal strength derives from the NOE. This enhancement, like the dipole-dipole relaxation to which it is so closely asso ciated, is both field and correlation time dependent. The 13C(IH) NOE decreases from its extreme narrowing value of 1.98 to a non-extreme narrowing limit of 0.15 (Figure 2c). Finally, the field and correlation time dependence of T2 should be considered. Although T2 does not influence the integrated absorption intensity of a resonance, it can lead to a field-dependent broadening of resonances, thus affecting sensitivity by making a signal difficult to detect above the noise level. This broadening can, of course, adversely affect resolution (Figure 2b). The salient features of the dependence of Tl> T2, and the NOE on correlation time and field strength have previously been discussed (22, 23). Assuming that the SIN ratio is directly proportional to Ho> the effects of Th T2, and the NOE can be combined and a simple expression for the intensity, l of a resonance is derived (equivalent to the SIN ratio at a given level of the noise): I K(NOE + I) HaT2 ( l/T\)lIZ, where K includes such factors as sample volume, concentration, and the quality of the probe (10). Taking all these potential variables to be constant, this equation results in the theoretical plot of signal intensity as a function of 'Tc that is presented in Figure 2d. It is clear from Figure 2d that for the detection of resonances in many proteins higher field strengths are desirable. The framework in which we have so far detailed the effects of relaxation behavior on NMR signal sensitivity, namely isotropic reorientation, is not always realistic. For the interpretation of backbone carbonyl and aCH resonances of globular pro teins, an isotropic reorientation model is probably satisfactory. For side-chain car bon atoms and resonances in nonglobular proteins, a more complex model including =

390


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10 8 6 4 2 0 -

10

-

9 .5

-9

-8.5

-8

-'7.5

-1

-7.5

-1

CORRELATIOM TInE EXPII01

'\,\'\

1.8

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1.6 1.4 III

i

1.2

.8

(el

15 ""Z

68 "HZ

.6

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.4

�--

.2 0 -10

-9

-8.5

-8

CORRELRTION TInE EXPIIOI

Theoretical plots of relative spin·lattice (liT,) and spin·spin (l!Tz) relaxation NOE, and relative sensitivity as a function of correlation time, TC' for four commonly

Figure 2

times,

-9.5

NMR PROTEIN STUDIES

391

144 128 112

(8)


96

"-

N

.-

80 64 48 32 16 0 -10

4.6

"\

3.5 3 2.6 2 1.6

-8

1 S

-

.

-

1

CORRELATION TlhE EXPIIOI

"-

4

,.. ... ... II> z .... ... z ... .... ,.. ... ... 1% ..J .... '"

-8.5

-9

-9.S

(0)

68 11HZ

"'"

,,�'0 \ -"'\ -------- _____

2611HZ

'

-----

1611HZ

.6 o -10

-9.6

-9

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7 S

-

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7

CORRELATION TInE EXPIIOI

used observing frequencies (as indicated). Plots are based on isotropicaliy reorienting C-H vector ( 1.09 A) relaxing exclusively by the dipole·dipole mechanism.


392


the effects of internal rotation and anisotropic reorientation is needed. These effects have previously been discussed (21-23). We should additionally point out that dipolar relaxation is not limited to l3C_IH couples. l3C_14N interactions and l3C_l3C interactions (for studies with highly enriched materials) can lead to relaxation and must be considered when appropriate (24, 25). So far we have centered our discussion on dipolar relaxation. In proteins, this is certainly the most significant relaxation mechanism for protonated carbon atoms. However, for non-protonated unsaturated carbon atoms the chemical shift aniso tropy (CSA) mechanism can become important, particularly at high magnetic field strengths (26). Recently it has been shown that the CSA mechanism dominates the relaxation of certain aromatic and carbonyl resonances at 68 MHz in hen egg white lysozyme (R. S. Norton, A. O. Clouse, R. Addleman, and A. Allerhand, unpub lished results). The CSA mechanism, when applicable, will shorten Tl preferentially at high field, thereby partially removing one of the disadvantages of high field NMR; however, CSA will also lead to a broadening of resonances (the CSA contribution to T2). Theoretical plots of line widths (W) and the Tl relaxation time deriving from the CSA mechanism are shown in Figure 3. A value of aa 200 ppm was used for the calculations. It appears that aromatic carbon atoms have aa values of about 200 ppm (27, 28); awvalues for carbonyl carbons appear to be closer to 145 ppm (29). Additionally, to the extent that the CSA mechanism is operative, the NOE will be reduced. The importance of other Tl mechanisms should be minimal (21). Spin rotation is usually of importance only for very small molecules and scalar coupling requires a rapidly relaxing nucleus with y close to carbon (e.g. Br). These circumstances are not likely to be met in proteins. Obviously the interaction of factors (field variation of Tb T2, NOE, induced receiver coil voltage, and relaxation mechanism) involved in determining the optimal field strength for use in l 3C NMR studies of proteins is complex; consequently, one cannot recommend one field strength as ideal for all protein studies. For example, it may be most advantageous to study carbonyl resonances at 68 MHz but non-protonated aromatic resonances at 45 MHz. The experimenter must determine which conditions are best suited to the particular task at hand. Having illustrated the variables that must be considered for resolution and sensitivity optimization, we now turn to several predominantly instrumental consid erations. Due to the low sensitivy of I3C nuclei, the majority of I3C work has been per formed with highly concentrated solutions in large sample tubes (10 mm or more in diameter) with closely coupled receiver coils. This propensity to use large sample =

Figure 3

�

Log-log plots of Tt (seconds) and line widths (hertz) for a t3C nuclear spin relaxing by the chemical shift anisotropy mechanism (�(T= 200 ppm) (solid lines), and by the dipole dipole mechanism from 3 hydrogen atoms 2.16 A away (dashed lines). Isotropic rotational reorientation was assumed for both relaxation mechanisms. The plots are determined for four magnetic field strengths (in kilogauss), as indicated. (Norton, Clouse, Addleman, and Aller hand, reproduced with permission.)

---r---'---,---,

1000 ��-

/. 63.�


100

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(A)

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1000 �-----.---.r---�--'--��

100

10 w 1,0

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0.1

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0.0 II

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6

394



sizes still persists (30, 31). If, however, only a small quantity of sample is available, then a considerable sensitivity advantage can be gained by using a properly designed microcell (32) or, more effectively, through use of a small, closely coupled receiver coil (microprobe) (H. Shindo and T. C. Farrar, unpublished results). The use of a microprobe presumes that sample solubility is not a problem. Earlier workers have shown (33, 34) that the SIN ratio can be expressed as follows: SIN

=

(Tlj) [O.51)(V/�V)QT(XoH�Vslkn]O.5,

l.

where f noise other than thermal, vsl Vc = filling factor (TJ), VS sample volume, Vc = coil volume, Q quality factor of the tuned receiver circuit, lJ = spectrometer operating frequency, �lJ = filter bandwidth, X· magnetic susceptibility of the sample, and Ho magnetic field strength (gauss). Taking f, T, and �lJ to be constant, the following convenient form of equation I is derived: =

=

=

=

=

SIN

=

KQO.5 1/0.5 v�·5 H�'S c:

2.

where K is a constant and C is the concentration. Several points derive from equation 2: first the sample should contain as many nuclei as possible within the effective volume of the receiver coil; second, the SIN ratio increases by the 3/2 power of Ho ; third, if the receiver coil volume is held constant then the sensitivity is proportional to the filling factor (however, if the sample volume is held constant, the sensitivity is proportional to the square root of the filling factor); fourth, if the amount of sample is limited then the sensitivity is proportional to the reciprocal of the square root of the coil volume, independent of the sample volume. This last observation means that optimum sensitivity will be obtained with a microprobe. These observations, however, are not strictly followed in practice; the variables found in equation 1 and 2 are not totally independent and therefore one must consider these as equations of rather limited use. In this regard, the recent reformu lation of Hoult & Richards (20) may be more rewarding. As we have previously mentioned, SIN ratios using superconducting magnet systems are not as large as might have been anticipated. The reason for this behavior stems, as has been pointed out (20), from the use of saddle-shaped (Helmholz) receiver coils in the superconducting system in place of solenoidal coils. Hoult & Richards (20) have indicated that the performance of the solenoidal coil should be approximately three times better than the Helmholz coil. Therefore, it is possible that, if technical problems can be overcome, the wedding of solenoidal coils to superconducting magnet systems will produce a . significant breakthrough in sensitivity. Finally, we would like to mention a few other developments in the area of signal sensitivity improvement. The first involves the use of either a crystal filter (35) or quadruture phase detection (36), both techniques producing J2 improvements in the SIN ratio. A recently reported improvement on the Bruker WH270 system involves the insertion of a high frequency transistor circuit between the probehead and the preamplifier in place of the 1.14 cable (37).

NMR PROTEIN STUDIES

395

BACKGROUND STUDIES ON AMINO ACIDS AND PEPTIDES


Amino Acids

The first extensive listing of BC chemical shift values of amino acids was reported in 1970 (3). Further listings, including values for the less soluble aromatic amino acids tyrosine (38) and tryptophan (39) and the sulfur-containing amino acids (40), were subsequently reported. The hitherto unreported BC NMR spectrum of "/ carboxy glutamic acid (41), including resonance assignments, is given in Figure 4; the presence of three carboxyl groups can be seen at a glance. The ready exchange of C(4}-H for C(4}-D allowed the assignment of the C(4) and C(2) resonances. The pH dependencies of the l3C resonances of most amino acids have been reported; these indicated a potential value for studies of peptides and proteins. Thus, each carbon atom in histidine was found to exhibit an inflection for each of the three titrating groups present (carboxyl, imidazole, and amino), even when the carbon atoms were distant from the titrating group (42). The opposite directions of chemi cal shift for the C2 and C4 carbons for the same titration process indicate the existence of at least two opposing effects. These are considered to be through space (electric field) and through bond (inductive) effects, producing downfield and upfield shifts, respectively. The direction of these shifts, although not their precise values, were predicted by molecular orbital calculations (43). These and other results indicate that the i3C chemical shift is roughly proportional to the electron density at a carbon atom (44). l3C NMR titration curves have also been reported for tyrosine (45), proline (46), and the sulfur-containing amino acids (40). The differential chemical shift changes of side-chain carbon atoms on titration of a particular group have been used to indicate conformation in solution (12, 47). Coupling constants between adjacent BC atoms in 85% BC-enriched amino acids are likewise pH dependent. For example, JCo-(;a changes by approximately 6 Hz from low to high pH ( 12). The chemical shift of the carbon atoms also vary with pH, and a linear correlation of the coupling constant and chemical shift is observed (47, 12). The probabilities of line intensities as they depend on percentage of BC enrichment have been presented graphically (12). The ring 13C_IH coupling con stant in histidine is found to reflect the imidazole titration but is relatively insensitive to the titration of other groups (48). A certain amount of confusion has surrounded the question of the relaxation behavior of the carboxyl carbon atom in glycine. The discrepancies in results and conclusions have largely resulted from the effects of low levels of paramagnetic metal ion impurities (49). As a result of this, Roberts and co-workers re-investigated (50) their original conclusion that a minimum in the TI value represented a major spin rotation (SR) contribution to relaxation (51). Egan & Cohen (unpublished results) observed two minima in the pH TI profile (Figure 5). These were attributed to the effects of low levels of paramagnetic metal ion and the titrations of the carboxyl and amino groups. The increase in TI values on increasing the pH at intermediate values of pH may be attributed to slow exchange of paramagnetic ions between binding sites (R.E. Wasylishen and J. S. Cohen, submitted for publication).


' (5 )

(51

CO2

",(4)/ C

W \0 0--

CO2

H (0)

!;l

1(3)

CH

1(2)

�

(I)

H2 N-C - CO 2

I

J,.L.

..

,, ... �",.,,,,,,,,",.�.,

> .Z

2

I

H

J, � �

..

...

OJ

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OJ ... 0 ... .,; cD ... ...

trJ

...

...

C (4)

'"

...

C(l)

'"

C(3)

C(I}

of the carbonyl carboxyl region

7.4). Spectra Figure 4 llC NMR spectra (68 MHz) or 'i-carboxy glutamic acid (0.1 M, pH C-4 is deuterated). (Bottom trace) Spectrum at atom hydrogen (the 020 in recorded Spectrum trace) (Top . are expanded recorded in H20. (Egan, Stenfio, and Fernlund.)

� lIP

8::£: �


NMR PROTEIN STUDIES

397

Bubbling H2S through the solution (P. Hazan, R. Deslaurier, and I. C. P. Smith, personal communication) caused the T, value at intermediate pH to increase to 85 sec (yI. Egan and J. S. Cohen, unpublished results), the same value reported by Roberts and co-workers for a wide range of pH values (50). This independence of the T, value as a function of pH can be taken as a monitor of the absence of contaminating paramagnetic metal ions. Additionally, the fact that the NOE value at 1 5 MHz for the carboxyl carbon atom of glycine in the absence of paramagnetic metal contaminants is only 60% of the maximum value indicates a significant SR contribution to the relaxation mechanism; little CSA contribution is expected at 15 MHz. The above considerations generally apply to carbon atoms or groups that bind metal ions, that do not contain bound hydrogen atoms, and therefore that have relatively long relaxation times. Protonated carbon atoms in amino acids undoubtedly relax predominantly by the DD mechanism. Non-protonated, unsaturated carbon atoms probably relax by some mixture of DD, SR, and CSA depending on field strength and correlation time.

70 60 50

C) Q)

,!!J. I-

40 30 20

..D--Q. "-

10 0

3

7

5

9

11

pH Figure 5 Spin lattice relaxation times of the carboxyl carbon atom of 90% I3C enriched glycine (1.0 M in D20) as a function of pH, alone (0) and in the presence of (A) EDTA (0. 1 M). The open circles represent the T\ values obtained on reversing the pH.

398



Peptides \3C NMR studies of peptides have recently been reviewed (52), and therefore only the most important results are presented here. The end effects on the Co, Ca, and Cp carbon atoms on forming a peptide bond between two amino acids have been reported by Christl & Roberts (53). Gurd and co-workers have reported the chemical shift and relaxation times as a function of pH for the side-chain carbon atoms of most amino acids in pentapeptides of the form · Gly-Gly-X-Gly-Gly (54). There have been many studies of the l3C NMR characteristics of proline incorpo rated in different peptides (53, 55, 56, 57). Clearly the ability to determine the proportion of syn and anti forms by direct observation of the 13C spectrum provides a valuable conformational probe for proline-containing peptides. A large number of\3C NMR studies of linear peptides, including many of biologi cal interest, have been reported (52). At natural abundance of 13C these consist mainly of a comparative analysis of chemical shifts and relaxation times to define conformational mobility. In this respect, I3C NMR may be less useful than IH NMR, where coupling constants such as Ca-NH more readily provide conforma tional information. In addition, it may be the case that linear peptides are flexible in solution but form a more rigid, specific structure only on binding to their receptor site. One of the structural restraints that has been used to reduce the number of possible conformers is cyclization of the peptide chain (58). In this respect the work of Bovey and Smith and co-workers has most clearly demonstrated the changes in chemical shift values that can ensue on fonning a cyclic peptide, oxytocin (59, 60). A detailed analysis of diketo-piperazines has also been reported (61). The use of 13C_13C and 13C_1H coupling constants to provide conformational information is only now beginning. Feeney et al (62) have used the CO-Cil coupling to obtain information on the side chains. Enriched amino acids have been incorpo rated into small peptides (63), ribonuclease S-peptide (64), and oxytocin (65, 66). Initial \3C NMR studies of polypeptides concentrated on the gross changes ob served for the helix-random coil transition (67, 68, 69). Allerhand et al (69) have measured the flexibility of backbone and side-chain carbon atoms in poly-Y-benzyl glutamate in both conformational forms from the Tl relaxation times. Not surpris ingly, the backbone carbon atoms exhibited longer correlation times in the helical form, whereas the side-chain carbon atoms showed very little change in either case. The changes in chemical shift values attributable to the helix-coil transition are relatively small (up to a few parts per million). A study of gelatin has shown that the \3C NMR spectrum is essentially what one would expect on the basis of a random coil structUfl: (70). The pH dependence of carboxyl groups in peptides has been studied. Effects on the terminal carboxyl group titration have been interpreted in terms of the structure of the carboxyl terminal end of the peptide (71). The carbonyl-carboxyl resonances in the spectrum of the tripeptide, glutathione, containing two carboxyl groups have been assigned (72). The backbone carbonyl group resonances showed a significant pH dependence.

NMR PROTEIN STUDIES

399

CARBON-13 NMR STUDIES ON GLOBULAR PROTEINS

Studies at Natural Abundance of 13C

The first

13e NMR spectra of proteins were published in 1970 (38,73); three further (74, 75, 76). In 1972-73, serious analyses of

reports appeared the following year

these spectra and of their consequences began to be seen. As with proton NMR studies, the

\3e NMR investigations of proteins can be divided into three periods:


presentation of initial protein spectra; division of spectra into regions of different chemical types, and comparison of the spectra of native and denatured forms; and detailed analysis of single atom resonances. The first comparison of the spectra of native and denatured protein, ribonuclease A, was reported in

1971 (76). This preliminary report was followed a year later by (77). The transition from native folded form at pH 6.55

a more extended analysis

to denatured unfolded form at pH

1.45

was clearly observed as a sharpening of

resonances. This sharpening derives from two effects:

(a)

the "equivalence" of

resonances of the same chemical type, which are non-equivalent in the folded globular form of the protein due to differences in local microenvironment; and

(b) narrower line widths due to increased mobility of the polypeptide in the random coil (unfolded) form. In these studies and

(77) partially relaxed spectra of ribonuclease were also presented,

Tl values for many types of carbon atoms in various states of ribonuclease were

determined. These were average values for peaks containing multiple carbon atom resonances. A greater mobility was exhibited by the denatured (oxidized) form, with an increase in segmental motion. However, values for the side chains of various residues, such as arginine, showed them to be almost as mobile in the native as in the fully oxidized form. Although the inability to study individual carbon atom resonances prevented this study from arriving at a more definitive conclusion, the point was made that local correlation times could be determined for a protein in different conformational states.

A comparison of i3e NMR spectra of native and

denatured hen egg white lysozyme, a-lactalbumin, and several other proteins have been made at 23 (39), 25 (42), and 68 (78) MHz. However, observation of single carbon atoms were not reported in these studies, even at high field strengths. Comparison of the \3C NMR spectra of globular proteins of different sizes (molecular weight) or overall correlation times showed that the line widths in general increased with molecular size (24). This behavior is expected on the basis of the relationship, W11

oc

1 IT2

oc

1" C"

This relationship was used to investigate

the conformation and self-aggregation properties of histones by proton and 13e NMR (79). Changes in line widths of regions of the spectra as a function of salt and protein concentration were estimated by a comparison of experimental with simu lated spectra. No quantitative criteria of best values for line widths, however, were given. Further, the relationship of line width to

T2

can only be regarded as a first

approximation. The line-broadening simulation approach also assumes simple equivalence of resonances of a given chemical type, which is known not to be the case for native globular proteins. The conclusions based on such evaluations can at best be regarded as qualitative.


400


Much of the advantage associated with the 13C NMR method relates to the ability to observe single carbon atom resonances. Therefore, the initial step in any detailed analysis of the I3C NMR spectrum of a protein is the attempt to assign specific carbon atoms in the molecule, with particular emphasis on the designation of resolved single carbon atom resonances. In the earlier studies on proteins it was pointed out that the most promising region of the spectrum for the anticipated study of single carbon atom resonances was the region containing the aromatic and arginine side-chain resonances (42). The first unequivocal report of single carbon atom resonances was made by Allerhand, Childers & Oldfield in 1973 (8). They observed 22 sharp peaks, some of which derived from more than one carbon atom, in the aromatic region of the I3C NMR spectrum of hen egg white lysozyme at 15 MHz with a large size probe. They attributed these peaks to 28 non-protonated atoms of 3 Tyr, 1 His, 3 Phe, and 6 Trp residues present in lysozyme. Even at a protein concentration of about 20 mM with the large size probe (requiring about 10 ml of solution) they still required about 100,000 pulses, taking several days to obtain a satisfactory SIN ratio. Subsequent improvements enabled them to reduce the time taken to accumulate a spectrum by several factors. Another approach that enabled them to obtain clearer spectra was the introduc tion of the so-called convolution-difference spectroscopy (80). This consists of the manipulation of a single data set (FID), so that one version is multiplied by a large exponential factor. This results in the Fourier transformed lines being extensively broadened. The resulting Fourier transformed spectrum is subtracted from the original; the difference then consists of only the sharp resonances, with all the broad components subtracted out (Figure 6). This is considered a useful way to directly distinguish resonances of carbon atoms with and without bound hydrogen atoms, i.e. those with long relaxation times (and hence sharp lines) and with short relaxa tion times (and hence broad lines). Of course there can be dangers in basing whole analyses on such manipulated data; however, the results appear to be excellent with the parameters chosen (9). Allerhand et al (9) showed that the carbon resonances they were observing in hen egg white lysozyme were non-protonated by applying off-resonance, noise modulated proton decoupling. Since there were no changes in the 22 sharp reso nances, it was clear that these resonances derived from carbon atoms without bound hydrogens. The resonances were initially separated into groups according to their chemical types on the basis of known chemical shift values of amino acid carbons. Studies of the relaxation behavior of the resolved resonances indicated that the dipole-dipole mechanism dominates at 15 MHz. At the higher field strength of 6.3 T (68 MHz), the CSA mechanism is found to be dominant for unsaturated, non protonated carbon atoms in proteins. At high field strengths, the CSA mechanism will have adverse effects on the resolution of these type of carbon atom resonances (see Figure 3). The assignment of resonances to specific carbon atoms in several proteins was arrived at by Allerhand and his co-workers by several different strategies (9). The methods adopted are summarized here. (0) Perhaps the most direct approach is the

NMR PROTEIN STUDIES

401


HEN EGG-WHITE LYSOZYME

Figure 6 Regions of aromatic carbon atoms and C C of arginine residues in the convolution difference natural abundance llC Fourier transform NMR spectra of hen egg white lysozyme. Each spectrum was recorded at 15 MHz under conditions of noise modulated, off-resonance proton decoupling using 8192 time domain addresses, a spectral width of 3787.9 Hz, 49,152 accumulations, a recycle time of 2.205 sec (30 hr total time). The convolution-difference procedure was used with Tl 0.72 sec, T2 0.036 sec, and K 1. (A) 14.6 mM protein in HzO, pH 3.05, 0.1 M NaCI, 44°C. (B) 13.8 roM in protein in DzO, pH 3.08, 0.1 M NaCI, 42°C. (Reproduced with permission, J. Bioi. Chern.) =

=

=

comparison of spectra of homologous proteins that differ by one or more mutations, so that only one or a few amino acids in the sequence are different. In this way, the C'Y resonances of Trp residues in hemoglobin were assigned (81). Human fetal hemoglobin has a substitution of a tyrosine residue at position 130 in the /3 chain. A comparison of the tryptophan region showed a single resonance corresponding to the C'Y carbon atom of Trp 130 (9). (b) Comparison of the spectra of the diamagnetic and paramagnetic forms of the same heme protein in different oxidation states provides a means of distinguishing between those residues of a given kind that are close to, and therefore broadened or shifted, in the paramagnetic form or distant from, and therefore unaffected, in the region of the heme group, e. g. ferro-, ferri-, and cyanoferricytochrome c (9). (c) The addition of paramagnetic metal ion, which is known from other evidence to bind to one strong site in the protein, provides an analogous means of using the distance dependence of the paramagnetic effect. The selective assignment of resonance 22 to Trp 108 C'Y in lysozyme on the addition of Gd3+ is an excellent example of this approach. (d) Chemical modification of a single


402


group provides a means of distinguishing between resonances of groups of the same type. However, if several changes are observed this can provide ambiguous results. On the other hand, the oxidation of Trp 62 in lysozyme to the oxindole form (9) provided a specific assignment of several carbon resonances to that residue (9). (e) The pH dependence of certain resonances provides a prima facie means of identifying them. For example, the characteristic downfield NMR titration shift of histidine resonances was used to assign the C,,! resonances to the single histidine resonance present in lysozyme: His 26 and 33 in cytochrome c (9) were distin guished on the basis of their known titration characteristics (82). (j) One of the NMR methods used is the selective decoupling of protons on carbon atoms of different chemical types. This approach was used to distinguish the resonances of tyrosine residues from those of arginines, which absorp in the same region of the \3C spectrum. (9). (g) Another method is to use known differences in relaxation times between resonances of different chemical types. These can be rationalized on the basis of the differing number of protons providing the dominant dipole-dipole relaxation to unsaturated carbon atoms in aromatic side-chain groups (24). Adja cent \4N atoms can also result in a reduction of T values of carbon atoms (24). It \ is not always necessary to determine an accurate T value, which is very time \ consuming, to utilize this approach since the partially relaxed spectra (PRFf) allow a distinction of T\ values to be made. In this way, the CE atoms of Trp residues were distinguished from the C,,! atoms of Phe and His residues in lysozyme (9). (h) Changing from HzO to D20 solvent can affect both the chemical shift value and the relaxation time of those carbon atoms that bind a deuteron. In this way, the C E resonances of Trp were distinguished from those of Phe and His in lysozyme (9). Although many of the above methods may distinguish between resonances of different chemical origin and others require detailed knowledge of the protein sequence and three-dimensional structure, it is suggested that a combination of the above approaches may well yield unique results in the assignment of many resolved resonances in the \3C NMR spectra of proteins. The assignment of carbon resonances to three tyrosine residues in myoglobins has been accomplished by comparing the spectra of sperm whale, horse, and red kan garoo myoglobin, which have specific substitutions (83). The \3C NMR titration curves of the tyrosine residues have been determined. It is noteworthy that tyrosine 146 does not titrate, even up to pH 12; this is consistent with other evidence of a nontitrating tyrosine in these proteins (84). Differences were reported in the \3C NMR spectra of lysozyme at 20° and 30°C, particularly in the Cy Trp resonances (85). These differences were interpreted to indicate that a conformational change occurred for lysozyme between these two temperatures. However, no controls for the intrinsic temperature dependence of the NMR signals in question were reported (86). In addition, it is well known that a tempera ture and pH-dependent self-association process occurs for lysozyme in solution (87). Consequently, the small differences observed in the \3C NMR spectra of lysozyme could be attributed to changes in self-association rather than a conformation change (i.e. to quaternary rather than tertiary structure). Evidence for this possibility comes


NMR PROTEIN STUDIES

403

from the essentially similar changes observed in specific residues, such as the Trp Cy resonances upon changing pH (H. Shindo, J. S. Cohen, and J. A. Rupley, unpublished results). Such changes are not observed for the internal ester-35, 108 of lysozyme, or in the presence of di-N-acetyl glucosamine inhibitor (H. Shindo, J. S. Cohen, and J. A. Rupley, unpublished results), when lysozyme is known not to self-associate. Resolved resonances of single carboxyl groups in lysozyme have been described by Shindo & Cohen (10) (Figure 7). In view ()f the number of such resonances in proteins ( 1 1 in lysozyme) this work was carried out at high field strength to provide high resolution and sensitivity. Several carboxyl resonances were distinguished against the background of carbonyl side-chain and backbone resonances by their change in chemical shift on changing pH. Reasonable continuities in several NMR titration curves could be delineated. The use of C02+ ion, which is known to bind in a single site in the active site of lysozyme, enabled the continuity of one of the curves to be delineated from the effects on resonances at low and high pH values. One resonance was found to be absent in material that had been dialyzed extensively at low pH (C I). This was attributed to the carboxyl group of bound acetic acid, the presence of which was also revealed by proton NMR (88). Since resonance Cl was also affected by CoH, the acetic acid must be bound in the active site region. It was expected that the resonances of the side-chain carboxyl groups of aspartic and glutamic acids would be resolved from each other and from the terminal carboxyl group on the basis of their characteristic properties in the free amino acids (3). Subsequent 13C NMR studies of guanidination and cleavage of the terminal leucyl residue of lysozyme (H. Shindo and J. S. Cohen, unpublished results) show that peak C2 and C3 were originally misassigned and instead should be attributed to the carboxyl carbon resonances of 01u-7 and Leu- 129, respectively. Additionally, the NMR titration behavior of lysozyme and its guanidination derivative indicates that, as in the crystal (87), G1u-7 forms a strong salt bridge with Lys- l ; however, the terminal leucyl carboxyl group may not be involved in the salt bridge. The active site Glu-35 carboxyl carbon resonance was not observed in the region S 1 80-184 ppm; this is probably due to an upfield shift and/or broadening resulting from the lysozyme self-association (89). The second resonance, C4/5, affected significantly by COH and therefore close to the active site region, has been tentatively assigned to Asp 52, which is known to be involved in the mechanism of action of lysozyme. Resonances corresponding to glutamic acid also have been observed in spectra of ribonuclease A, where five to seven titrating carboxyl resonances have been resolved (Figure 8). The observation of individual carboxyl resonances for two, albeit small, stable and well-characterized proteins nonetheless provides another valuable probe technique for protein structure and function, and particularly for a group, the carboxyl side chain, for which no other amenable spectroscopic technique exists.

13C-Enriched Proteins There are four possible approaches that can be taken in the preparation of BC-enriched proteins.

404



pH

3.45

3.89

""",-""-JVI..NV

4.74

."""-'�" 00J'...

5.55

"""--""""",,,

5.90 6.58

1 80

1 76

1 72

168

ppm

Figure 7 Carboxyl and carbonyl carbon resonances of natural abundance IlC NMR spectra of hen egg white lysozyme (10 mM) in H20 containing (DzO (4: 1), and 0. 1 M NaCI. The peaks labeled CI-C1O were observed to titrate on change of pH (10; reproduced with permission,

Proc. NaIL Acad. Sci. USA).

NMR PROTEIN STUDIES

PH

R I BONUCLEASE A


405

4 . 44

4 . 04

2 . 78

190

180

1 60

170 C h em i cal S h i f t

150

( ppm)

Figure 8 Natural abundance t3C NMR spectra (68 MHz) of carboxyl, carbonyl, Arg-C and Tyr-C carbon resonances of bovine pancreatic ribonuclease A ( 10 mM, 0. 1 M NaCl) as a function of pH. Spectra were recorded at about 28°C using 32,768 acquisitions, 32-k data points, 15-kHz spectral width. Several peaks, most likely due to the 5 Glu 'Y-carboxyi carbon atom resonances and the C-terminal carboxyl carbon atom resonance, are observed as single carbon atom resonances in the down-field portion of the spectra and are seen to alter chemical shift on change in pH.

406


INTRODUCTION OF A "C-LABELED CHEMICALLY MODIFYING GROUP The preparation and observation of DC NMR spectra of [I3C]carboxymethyl histidine myoglobin and ribonuclease were reported by Gurd and co-workers (16) and this approach is expected to be extended to other selective reagents (90). The amino terminal groups of hemoglobin have been carbamylated with [13C]cyanate, and the derivatives have been used to probe the local environments (91). Although the chemical modification approach in principle is not a good one for the study of native protein conformation, it does appear to be a very valuable one for clarifying the number and type of chemically modified products produced in a given reaction. An excellent example of this is the analysis of the spectrum of the mixture �btained on treating cytochrome c with DC-enriched bromoacetate (92), which showed that the protein was much more extensively modified than had previously been assumed (Figure 9). Thus, the use of isotopically labeled reagent and the observation of the spectrum of the resulting mixture, or after purification, provides an excellent means to characterize the number and nature of the protein derivatives. This approach has been utilized by Norton & Allerhand (93) in their study of the formation of acetoxy tryptophan-62 on the oxidation of lysozyme by N-bromosuccinimide in acetate


.

tA)

( 8)

200

180

160

Figure 9

140

120 ppm

The 5000-Hz 13C NMR spectra of [2-13C]carboxymethyl cytochrome c. (A) [2-13C]carboxymethylfenicytochrome c, 10,000 pulses. Carboxymethylation time was 6 days at pH 7 in 0.1 M cyanide (B) Fenicytochrome c, 49,69 1 pulses (92; reproduced with permis sion, Biochemistry). ..

NMR PROTEIN STUDIES

407


buffer. By using enriched acetate buffer they were able to follow the time course of the decomposition of the acetoxytryptophan intermediate formed during the reac tion. Such applications are unique to stable isotope NMR spectroscopy. CHEMICAL SYNTHESES Since the total synthesis of a protein is an enormous task, the application of chemical synthesis to incorporate l3C-enriched amino acids into proteins consists mainly of tacking an extra amino acid onto the end of a protein chain (94), removing an amino acid, and replacing it with a l3C-enriched analogue, or the synthesis of a peptide that can later be used to form a globular protein by noncovalent complexation (95). The covalent joining of a chemically synthesized peptide, containing a selectively enriched amino acid, with peptide(s) derived from a protein, to form a globular active protein, has been discussed (96) but not accom plished. Apart from this difficult possibility these approaches are limited in that the terminal amino acids of proteins are generally not of interest, and that not many peptide complexes are known that give rise to highly active globular proteins that resemble a native protein. In this respect, the ribonuclease S system is perhaps the best example, of which three S peptide derivatives have been reported, containing 1 5% l3C (uniformly labeled) Phe-8 ( 1 1), 90% l3C C2 His (97), and about 5% l3C2 Gly replaced for Ala at position 6 (98). Unfortunately, the results of these experiments were somewhat disappointing. Although 15% l3C enrichment was sufficient to allow clear observation of the aromatic resonances, it was insufficient to provide unequivocal observation of the enriched aliphatic resonances above the background of the many aliphatic natural abundance carbon atom resonances present in the spectrum of ribonuclease (95). The preparation of the l3C C2 His derivative proved to be difficult due to the low yields of the blocked histidine derivative required for protein synthesis, so that only incomplete results could be obtained in this case (97). Finally, the l3C NMR spectrum of the l3C Gly ribonu clease S were rather poor, and the changes in chemical shift observed (98) in a situation in which a random coil (S peptide) to helix (ribonuclease S) transition is known to occur (99) were rather small (42). Nevertheless, there is little doubt that these preliminary studies showed the feasibility of the approach, and with improved instrumentation the possibility of significant conformational insights can be ex pected. BIOSYNTHESIS The first example of a selectively l3C-enriched protein prepared by biosynthesis, Escherichia coli tryptophan synthetase (14) containing 13C2 His (syn thesized chemically), which was fed to the bacterium, did not give clear-cut results; this definitely resulted from a poor choice of amino'acid for an initial study. Thus, the C2 resonance of histidine is broadened by both a 14N dipolar coupling and, probably, by the effects of paramagnetic metal ion contamination. In addition, the protein chosen for study is of medium size, which will also result in line broadening, and has many histidine residues. Since histidine residues can be quite effectively studied by IH NMR, it seems less important to study these same groups by l3C NMR but rather to choose those groups that cannot be effectively studied by other methods. A further example of the selective use of l3C2 His in the biosynthesis of


408


a protein is the preparation of a-lytic protease from Myxobacter ( 100, 101). This was chosen for a study of the serine proteases because it contains only a single histidine residue; consequently there could be no ambiguity in the assignment of the His resonance. The \3e NMR titration curve for the selectively enriched protease was considered to reflect the titration of the aspartic acid carboxyl group in the presumed charge relay system (102). However, this curve is extremely similar in chemical shift and pK (6.75) value to the titration curve of histidine in peptides. On this basis alone one would have to conclude that a-lytic protease had a normal histidine residue. However, the 13C_1H coupling constant in a-lytic protease was found to be pH independent in the pH range 5.2-8.2, and this was interpreted to indicate that the pK value observed for the I3C chemical shift must arise from a group other than histidine ( 100, 101). However, the accuracy of such an evaluation, particularly where the data analyzed was the difference between proton-coupled and proton-decoupled spectra, cannot be considered to be much greater than 5%. The maximal difference quoted in the coupling constants at low and high pH values was only 8% ( 100, 101), and it has been shown that the 13C_1H coupling constant is not very susceptible to the titration of other groups (48). Therefore, it would appear that the basis for the conclusion that the titration curve observed in a-lytic protease represents anything other than a normal histidine residue is not sufficiently estab lished. Other biosynthetic preparations utilizing BC enrichment for high resolution studies include ferredoxin grown in media containing 50% 13C Gly (103) and cytochrome c from Neurospora grown in 90% [methyf-13C]methionine (104). ISOTOPE SUBSTITUTION Isotope substitution involves preparation of a protein normal in every respect except that a group (or groups) is enriched in an isotope other than that normally found. In the work under discussion this would be substitu tion of I3C for 12C. An example of this approach is the preparation of [methyl-BC] methionine by substitution in the intact protein myoglobin by the use of [methyl13C]iodide (17). A similar preparation has been made of [ methyJ-13C]methionine cytochrome c (A. Schjeter, A. Lanir, I. Vig, and J. S. Cohen, unpublished results). Both myoglobin and cytochrome c contain two methionine residues. However, in the case of cytochrome c one of these is directly bound to the heme iron atom. Thus, only one methionine was observed in ferricytochrome c, since that of the heme bound methionine was shifted downfield by the paramagnetic effect of the iron atom. However, when cytochrome c was reduced or the pH was raised to give the hy droxyl form two resonances were observed. This approach provides nondisturbing probe of the nature of the immediate heme environment in methionine-bound heme proteins, as well as in other cases. A further approach not precisely covered by the categorization given above is the use of I3C enrichment in studies of enzyme mechanism. For example, glutamate aspartate transaminase has been labeled at Cys 390 with I3C-enriched cyanide ( 105). The mechanistic pathway of the serine protease, chymotrypsin, is thought to involve an acyl-enzyme intermediate (106). Acetyl chymotrypsin has been known to exist for some time now from enzyme activity measurements and kinetics (106). Recently, direct fluorescence observation of the presence of such an acyl intermediate was


NMR PROTEIN STUDIES

409

obtained (107). The use of p-nitrophenyl l3C2 acetate enabled the intermediate to be prepared, as indicated by enzyme activity measurements, and to be directly observed by l3C NMR spectroscopy (C. H. Niu, H. Shindo, J. S. Cohen, and M. Gross, submitted for publication) (Figure 10). There is little doubt that such meth ods will be extended to a wide range of detailed studies, including pH, temperature, and chemical effects, to probe the active site of enzymes in solution. The preparation of highly l 3C-Iabeled proteins, together with the current im provements in NMR technology, will also lead to studies of proteins in matrices other than free solution, such as membrane-bound states, which are hardly amena ble to other physical techniques.

1 3C NMR STUDIES OF STRUCTURAL PROTEINS Collagen and elastin are the major fibrous constituents of connective tissue. In living systems, these proteins exist as structured entities embedded in a ground-substance matrix. As a consequence of their structure and function, BC NMR studies of these connective tissue proteins are presently directed towards the determination of their rotational reorientation rate constants, R (= liT c) .

Collagen

The biological function of collagen is primarily structural. In the form of filaments it possesses a high tensile strength and a low coefficient of elastic extensibility. The characteristic mechanical properties of collagen are a consequence of its chemical composition and molecular organization. The structural unit common to all colla gens at the molecular level is the triple-stranded helix, tropocollagen. Each of the chains that comprise the triple helix consists of approximately 1000 amino acids, predominantly arranged in the triplet sequence Gly-X-Y, where X and Y are usually pyrrolidine (proline and hydroxyproline) residues. These triple-stranded units band together to form collagen fibrils, whose exact dimensions are a function of source. Since the amino acid side chains of tropocollagen residues are at the surface of the collagen helix, their mutual interactions determine the structure of collagen fibers (108). These interactions may be investigated by using BC magnetic reso nance. Collagen line widths are too broad to detect with high resolution techniques if the usual decoupling field (y H2/21T 2 kHz) is used (109). However, application of a large decoupling field (yHz/21T 75 kHz) at the proton frequency eliminates the static I3C-IH dipolar interactions ( 110) [which makes the dominant contribution to the I3C line widths, at least at low magnetic field strengths (1.4 T)] and spectra can be obtained by using a high resolution pulse Fourier transform spectrometer. The dipolar decoupled spectra of collagen fibrils (calf achilles tendon and rat tail tendon) are shown in Figure 1 1 (110). The integrated signal intensity from the tendon spectra showed that the majority of l3C nuclei contributed to their un saturated signal intensity to the spectra. This result, coupled with the fact that spectra were obtained with a 90-T-90 (T 3.0 sec) pulse sequence, demonstrate that the majority of collagen fibril carbon atoms possess rotational reorientation correla tion times several orders of magnitude smaller than those found in rigid solids (1 10). =

=

=

410


(Al


CH31300-E (j CH'3gOH 3

(8 )

1 "16

1 74

1 72

1 70

Figure 10 Proton-decoupled 68-MHz 13C NMR spectra. (A) a-Chymotrypsin (2.0 mM) plus p-nitophenyl- J-I3C-acetate (3.0 mM) at room temperature in 0.2 M phosphate buffer (pH 5.1). After 2 hr, the pH of the solution was adjusted to 3.2. (B) 3.7 mM p-nitrophenyl-J -I3C- acetate under the same conditions after 3 days.


NMR PROTEIN STUDIES

41 1

The spectra shown in Figure 1 1 were taken at natural abundance and are there fore a composite of many unresolved side-chain and backbone carbon resonances. The extraction of the side-chain and backbone reorientational rates from the natural abundance spectra is therefore a difficult problem. To circumvent this resolution problem, Torchia & VanderHart (1 10) prepared reconstituted chick calvaria colla gen containing I3Ca- and I3Co-labeled glycine. At 20°C, the Ca glycine carbon has a Tl and NOE values of 0. 1 sec and 1 .35, respectively; the corresponding Co values are 1 . 5 sec and 1.6. These data (small TI and nonminimal NOE values) indicate that the backbone reorientation over certain directions must be rapid (R > 106 sec -I), whereas the broad line widths (due primarily to unaveraged chemical shift anisotropy) indicated that reorientation over certain other directions must be slow (R < 10-3 sec-I). A more detailed analysis of the data reveals that the reorientation rate about the long axis of the collagen fiber is in the range 10"6-10-8 sec-I with angular excursions greater than about 300 (1 10). This value for the reorientation rate is comparable to that expected for tropocollagen (dimensions 3000 X 15 A), indicat ing that the intermolecular forces that stabilize the structure of the reconstituted collagen fibrils are not strongly angle dependent and hence do not arise from a unique set of interactions between the side chains ( 1 10).

Elastin Elastin is the other major protein of connective tissue. Tissues containing elastin fibers have the rubber-like mechanical properties of high elasticity and small elastic modulus. Several lines of evidence have suggested that elastin consists of cross linked polypeptide chains that alter configuration (extend) when stretched. In an ideal rubber, elasticity can be explained in terms of changes in configurational entropy. The question may be posed whether the mechanical properties of elastin can be explained in a similar fashion. Thermochemical data have revealed large changes in the internal energy of elastin on stretching; however, since the number and extent of water-protein interactions vary with stretching, the thermochemical data are extremely difficult to interpret. The high resolution \3C NMR spectrum of native elastin, unlike collagen, may 2 kHz) (109). Since the be observed with a normal decoupling field (yH2/21T majority of the BC resonances are observed (determined from a comparison of native and denatured elastin) one may immediately infer that substantial segmental motion is present. An initial value of the reorientational correlation time of approxi mately 4 X 10-8 sec for native elastin was derived from the carbonyl carbon TI value ( 1 . 1 sec); KCNS-denatured elastin had a slightly shorter carbonyl TI value (0.7 sec) and consequently a shorter correlation time, approximately 2 X 10-8 sec. The initial estimate of the correlation time of elastin, derived from the assumption that the carbonyl carbons are relaxed primarily by the nearest neighbor protons, Ha and NH, and that the rotational reorientation of these C-H vectors is isotropic. This initial study (109) indicated that configurational entropy has an important role in determining the configurational properties of elastin. A more thorough analysis, including Th T2, and NOE values, of ligamentum nuchae has recently appeared (l 1 1). In this analysis, the relaxation data and NOE =


o Figure 11

1 00 p.p.m .

200

Comparison of dipolar decoupled I3C NMR spectra of various collagens obtained

at 20°C using 90-t-90 pulse sequences.

(a) Calf tail tendon, t = 3.0 sec, 19,500 scans; (b)

reconstituted chick calvaria collagen, I3C enriched at C(O),

t =

reconstituted chick calvaria collagen, I3C enriched at the glycine Ca,

(d)

7.0 sec, 4,900 scans; (c)

t=

1 .0 sec, 1 8,350 scans;

sample used in (c), dehydrated, -95"C, 3.0-sec repetition time, 1 5,700 scans, 0.7-msec

polarization time. In each case a field of 17 gauss was applied in the rotating frame of the protons to remove the dipolar coupling and a spectral window of 20 kHz with a sampling delay of 50 Il-sec was employed. Chemical shifts in parts per million from external CSz ( 1 10; reproduced with permission, J.

Mol. Bioi. ).


NMR PROTEIN STUDIES

413

enhancement were analyzed in terms of a single correlation time model and a log-X2 distribution of correlation times. Employing a log-X2 distribution of correla tion times, a self-consistent analysis ofthe Th T2, and NOB data was obtained, with the average backbone-carbonyl value of T approximately 8 X 10-8 sec. From analysis ofthe chemically shifted carbonyl resonances, it was further concluded that the Gly, Pro, and Val residues are significantly more mobile than the Ala residues (I l l); these later residues are located in the cross-linking area. As is apparent from the preceeding two examples, rotational reorientation corre lation times (and the anisotropy of the motion) are directly derivable from \3e NMR studies. This information may be used to propose and assess models.

CONCLUDING REMARKS Every review of a relatively new technique applied to biological problems can be counted on to laud the potential value of that technique in its concluding remarks. In this respect l3e NMR is certainly no exception. However, it is well to mention the severe limitations of this technique in studies of proteins at this point, most notably the poor sensitivity. It still takes many hours to obtain the 13C NMR spectrum ofa protein at a relatively high concentration (10 mM) with adequate SIN ratio. To be more generally applicable to many proteins, not only to those that are stable, soluble, and readily available, \3e NMR needs to be improved in sensitivity by a factor of one to two orders of magnitude. It is gratifying to know that some of the best qualified persons in the field are addressing themselves to the serious technical questions raised by this requirement. From the results presented here it seems likely that considerations of resolution, sensitivity, and sample size will require superconducting high field magnet NMR spectrometers for high resolution studies on proteins. This will naturally require careful analysis by granting agencies, since these instruments are too expensive currently to allow every laboratory to possess its own. However, it is hoped that there will be a reduction in price of superconducting magnets when it is realized what the potential advantages are over electromagnets in terms of applications, apart from the lower running costs since power and water requirements are unneces sary with superconducting magnets (more than compensating for the cost of liquid helium and nitrogen). Rather than discourse in general about the future value of Be NMR in studies of proteins, it may be preferable to show one possible example. Glycoproteins are often difficult to crystallize. Also, there is no adequate physical means to study them directly in detail because of the absence of a carbohydrate chromo phore. However, a He NMR spectrum of a glycoprotein, horseradish peroxidase, shows that the anomeric sugar carbon atom resonances are in a region of the spectrum totally devoid of protein resonances (ca 100 ppm) (Figure 1 2). In view of the successful results reported in the analysis of the structure and micrody namic behavior of polysaccharides, it is not difficult to predict that 13C NMR has a very important role to play in solution studies of glycoproteins and indeed in general.


HORSERADI SH PEROXIDASE

J

200

1 50

100

50

0.0

ppm

Figure 12 Proton decoupled natural abundance \3C NMR spectrum (68 MHz) of horseradish peroxidase, a glycoprotein with 48 sugar residues per molecule (molecular weight, ca 40,000). The arrowed resonances are attributed to the anomeric carbon atoms of the sugar moieties; several sugar resonances additionally appear in the spectral region of the protein alpha carbon resonances. (Horseradish peroxidase courtesy of Dr. I. Morishima.)

415

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Proton nuclear magnetic resonance studies of Bence-Jones proteins.

Nuclear magnetic resonance studies of amphiphile hydration.

Nuclear-magnetic-resonance studies of Pseudomonas aeruginosa cytochrome c-551.

31P nuclear magnetic resonance studies of Ehrlich ascites tumor cells.

Phosphorus nuclear-magnetic-resonance studies of compartmentation in muscle.

Fluorine-19 nuclear magnetic resonance studies of Escherichia coli membranes.

High-resolution nuclear magnetic resonance studies of double helical polynucleotides.

Conformational studies of alanine oligopeptides by nuclear magnetic resonance.

31P nuclear magnetic resonance studies of HeLa cells.

13C nuclear magnetic resonance studies of egg phosphatidylcholine.

Proton and phosphorus nuclear magnetic resonance studies of ribonuclease T1.

Nuclear magnetic resonance studies of phenylthiohydantoin amino acids.

Positive theta-angles in proteins by nuclear magnetic resonance spectroscopy.

lac repressor: 3-fluorotyrosine substitution for nuclear magnetic resonance studies.

Preliminary nuclear magnetic resonance studies on human saliva.

Evanescent Waves Nuclear Magnetic Resonance.

Nuclear magnetic resonance studies of the copper binding sites of blue copper proteins: oxidized, reduced, and apoplastocyanin.

From Nanodiscs to Isotropic Bicelles: A Procedure for Solution Nuclear Magnetic Resonance Studies of Detergent-Sensitive Integral Membrane Proteins.

Probing protein structure by solvent perturbation of nuclear magnetic resonance spectra. Nuclear magnetic resonance spectral editing and topological mapping in proteins by paramagnetic relaxation filtering.

[Diagnose of traqueomalacia with nuclear magnetic resonance].

Proton nuclear magnetic resonance study of cobrotoxin.

15N nuclear magnetic resonance of living cells.

15N nuclear magnetic resonance of flavins.

Magnetic resonance studies in stroke.