J. Mol. Biol. (1992) 224, 659-670

Probing Protein Structure by Solvent Perturbation Magnetic Resonance Spectra

of Nuclear

Nuclear Magnetic Resonance Spectral Editing and Topological in Proteins by Paramagnetic Relaxation Filtering Gennaro

Mapping

Esposito

Istituto di Biologia, Universita

Facolta di Medieina Viale Gervasutta, 33100 Udine,

di Udine,

Arthur

Italy

M. Lesk

of Haematology,

Department

MRC

University of Cambridge Clinical Centre, Cambridge, CB2 2QH, U.K.

Henriette

School

Molinari

Dipartimento di Chimica Organica e Industriale Via Golgi 19, 20133 Milano, Italy

Andrea Istituto

Motta

di Molecole di Interesse Biologic0 6, 80072 Arco Felice, Napoli, Italy

per la Chimica

CNR,

Via Toiano

Neri Niccolai Dipartimento di Biologia Molecolare Centro Didattico dell ‘Universita “Le Scotte “, 53100 Siena, Italy

and Annalisa

Pastore

European Molecular Biology Laboratory Meyerhofstrasse 1, Postfach 1022.09, 6900 Heidelberg,

Germany

(Received 13 November 1990; accepted 3 December 1991) Soluble spin labels, which “bleach” the surface proton resonances of a protein to n.m.r. measurements, can provide useful information about protein conformation and dynamics. The use of the soluble nitroxide, TEMPOL, has been explored to show the correlation of the paramagnetic perturbations of protein two-dimensional n.m.r. data with proton exposure to the free radical in hen egg-white lysozyme. The results demonstrate that the nitroxide approaches the protein randomly, and that the extent of the observed paramagnetic effects reflects the native folding pattern of the protein. A correlation of spectral simplification with the known tertiary structure establishes the feasibility of new strategies for topological mapping of surface and buried protons of the protein. Application to the elucidation of protein structure and to the study of dynamical processes is discussed. Keywords:

0022-2836/92/070659-12

$03.ctqO

TEMPOL;

n.m.r.;

e.s.r.; protein

surface

659 @? 1992 Academic Press Limited

660

G. Esposito

1. Introduction Information on structure, dynamics and biological functions of relatively small proteins may be obtained from 2D n.m.r.t techniques (Ernst et al., 1987; Wiithrich, 1986). In this approach interactions through space, detected by NOESY (Jeener et al., 1979) and ROESY (Bothner-By et al., 1984; Bax & Davis, 1985) experiments, form the basis for the structure determination (Wagner et al., 1986), often in conjunction with distance geometry (Williamson et aE., ‘1985) or restrained molecular dynamics calculations (Kaptein et aZ., 1985). This approach has been successfully applied to proteins mass up to with relative molecular 10,000. However, overlap of resonances complicates the assignment procedure, requiring technical and/or chemical methods for spectral simplification. Techniques (3D and 4D) have been recently introduced for removing ambiguities in resonance identification (Griesinger et al., 1989; Kay et al., 1990), but the 2D approach is still the most widely used. In 2D spectroscopy, spectral simplification has been achieved by multiple quantum spectroscopy (Mareci & Freeman, 1983), and multiple quantum filtering (Piantini et aZ., 1982; Shaka & Freeman, 1983). A chemical approach to spectrum simplification is based upon the perturbation of a specific region of the macromolecule; for instance, using nitroxidestable spin labels covalently bound to proteins or nucleic acids (Wien et al., 1972; Dwek et al., 1975; Schmidt & Kuntz, 1984; Moonen et al., 1984; Kosen et al., 1986; Anglister et al., 1984a,b; De Jong et al., 1988), or employing extrinsic probes, such as paramagnetic metal ions (Campbell et al., 1975; Arean et al., 1988; Williams, 1989). In these experiments the simplification arises from dipolar interactions between the unpaired electron spin of the label and proximal protein hydrogen atoms. These interactions, which increase the relaxation rate of the protons, broaden and therefore induce a local bleaching of the resonances in the binding site (Solomon & Bloembergen, 1956; Berliner, 1976). Difference spectra computed from observations in the presence and in the absence of the bound spin label identify the resonances of the residues forming the binding site. Studies on the effects of soluble spin labels on aromatic and paraffinic hydrocarbons (Qui et al., 1982), as well as on protic solvents (Nientiedt et al., 1981), have also been reported. As a development of the chemical perturbation 7 Abbreviations used: lD, 2D, 3D and 4D, 1, 2, 3 and 4-dimensional; n.m.r., nuclear magnetic resonance; NOESY, 2-dimensional nuclear Overhauser enhancement spectroscopy; ROESY, rotating-frame Overhauser effect 2D spectroscopy; TOCSY, 2-dimensional homo-nuclear Hartman-Hahn; D&F-COSY; double-quantum-filtered correlated spectroscopy; e.s.r., electron spin resonance; p.p.m., parts per million; CPMG, Carr-Purcell-Meiboom-Gill; HEW, hen egg-white; DSS, sodium 2,2-dimethyl2-silapentane-5-sulfonate.

et al. approach, soluble radicals have been recently used for mapping protein surfaces (Esposito et al., 1989; Petros et al., 1990). This approach, an extension of a 1D relaxation method previously proposed (Niccolai et al., 1982, 1984), holds the promise that nitroxideprotein interactions might be developed into a general approach for the study of protein folding and dynamics. In this paper, the first of a series, we establish the basis of the method and report necessary controls. Based on the increased relaxation of protons interacting with spin labels, we present a method for 2D spectral simplification that allows a correlation between nitroxide-exposed and solventexposed protons. Hen egg-white lysozyme was used as a model because of the availability of detailed n.m.r. data, including the complete assignment of the spectrum (Wien et al., 1972; Redfield & Dobson, 1988), and X-ray crystal structure (Imoto et al., 1972; Artymiuk & Blake, 1981). The spin label TEMPOL (4 - hydroxy - 2,2,6,6 - tetramethylpiperidine - 1 - oxyl) was employed because of its solubility in water, the solvent of choice of most protein n.m.r. investigations. 2. Materials

and Methods

Hen egg-white lysozyme was purchased from Sigma (Chemical Co.) and dialyzed at pH 3.0 for 48 h before use. A 5.0 x 10m3 M mother solution in *H,O was prepared at pH 3.9 (uncorrected pH-meter reading). A few ~1 of a 20 M-4-hydroxy-2,2,6,6-tetramethylpiperidine-I-oxyl (TEMPOL, Sigma) solution were added directly to the n.m.r. tube containing 760 ~1 of mother solution, in order to reach the desired TEMPOL/lysozyme ratio (R). The remaining portion of “undoped” mother solution was used without further additions as a blank. A similar procedure was employed for the preparation of the 90 x 10m3 M solution in 99% H,O/lO% ‘H,O. Exactly the same protocol was followed for the preparation in *H,O of the samples both with and without TEMPOL, so that the effects of amide ‘H-‘H exchange, taking place before and during data acquisition, could be comparable. e.s.r. spectra were recorded with a Bruker ER 2661) spectrometer operating at X band (978 GHz) and equipped with a Bruker variable-temperature unit and a digital thermometer. The modulation frequency was 166 kHz and the modulation amplitude was 65 G. ‘H-n.m.r. spectra were acquired at 566 MHz on a Varian VXR spectrometer, connected to a SUN 3/l 10 computer, and at 566 MHz on a Bruker WM-596 spectrometer interfaced to an Aspect 2096. An internal standard for chemical shift measurements was not used, to avoid having to correct for differential effects of the free radical upon the solute and the reference compound (Qiu et al., 1982). Chemical shifts were then always referred to the computer memory location corresponding to the resonance of Leul7 upfield ring-current shifted d-methyl resonance (-0-66 p.p.m.) in the label-free lysozyme solution (Redfield & Dobson, 1988). All 2D experiments were run at 308 K with R = 6. Such a nitroxide/protein ratio was chosen as a good compromise between broadening due to transverse relaxation and signal-to-noise ratio in 2D experiments. We monitored the extent of resonance attenuation after 46 to 56 ms transverse relaxation upon changing the nitroxide concentration (up to a ratio of 20).

Mapping

Protein

Surfaces by Paramugnetic

661

Probes

Table 1 A survey of the effects of TEMPOL on the amide protons compared with the exposed surface areald2, the presence of a hydrogen bond in the crystallographic structure and the protonjdeuterium exchange rates IO

5 KVFG

20

15

30

25

LAAAMKRHGLDNYRGY

40

35

S LGNWVCAAKFE

45

S NFNTQATNR

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

H bond Structural

RCE

element

‘H/*H rxrhange Exposed surfwe area TEMPOT, exposure

Ezm’lf-ll00~0000~~~~~~0~0~000~~~~~~~~~~~~~~~~~~~~~~~~~

mrazzzl

004000044404404040440040004444444444044444~~0

-mmm--mmmmmmmm0mmm~~~~m---mm~mmmmmmm~~mmmm~m~ 50

55

70

65

60

80

75

85

90

NTDGSTDYGILQINSRWWCNDGRTPGSRNLCNIPCSALLSSDITA H bond Structural

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA element

‘H/*H exrhange Exposed surface area TEMPOI, exposure

I

!a

ifmom

P

SV

Strwtural

element

‘H/‘H exchange Exposed surface area TEMPOL exposure

m

q mmm-mmm0mm0qmmn~---n--mmm~m--nmmmm~m--o loo

95

H bond

n

I

0000o0ooooooo0oo0ooooo-o-oo-oeooo-~oooooooooo 40040444444440444444~~40~044444440~0444@00040

NCAKKIV

105

115

110

125

120

SDGNGMNAWVAWRNRCKGTDVQAWI

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA -1IIIIzI I 0oooooooo000-oo-0o00ooooooooooooooooooo

RGCRL

I

I

I

I

044444444440004444044444440400044440400

Filled symbols in the respective lines indicate inaccessibility either to TEMPOL OP water OP the presence of hydrogen bonds: open symbols indirate wcessihility or abnerwe of hydrogen bonds. Half-filled symbols indicate partial accessibility or a dintorted hydrogen bond. The elements of Rerondary strurtwe we also indirated along the amino acid sequence. A H bond A Partial

H bond

A No H bond t Fast ‘H/*H exchange unaffected by TEMPO12

m

a-Helix

.

m

/?-Sheet

@Medium

Slow exchange

-

B-Turn

+ Not exposed

exchange OPartially

0 Fast exchange t Slow ‘H/‘H

exposed

0 Exposed

0 Exposed

exchange TEMPOL

A conventional CPMG spin-echo sequence was employed for this purpose. For the TOCSY experiments (Braunschweiler & Ernst, 1983) in 90% H,O/lO% ‘H,O a sweep width of 6 kHz was used in both dimensions, the acquisition time was @171 s in t,, 0085 s in t, and 128 transients were collected for each t, increment. The solvent was suppressed with a DANCE preseturation pulse train (Morris & Freeman, 1978) applied during the relaxation delay (typically 15 s). A 15 ms MLEV-17 spin-lock mixing train (Bax BEDavis, 1985), preceded and followed by a 2 ms trim pulse, was applied. The experimental data were weighted in both t, and t, with 67.5” shifted sine-squared bell-functions and zero filled to 2 K in t, before Fourier transformation. The NOESY experiments (Macura & Ernst, 1980) in ‘H,O were run in experimental conditions identical to those of TOCSY, except for the irradiation of the residual ‘H,O signal, which was achieved by coherent irradiation (Zuiderweg et al., 1986) during the relaxation and the mixing times (100 ms). Sine-squared-bell window functions (10” shift) were applied before transformation in both dimensions. Decreasing of peak intensities caused by TEMPOL, shown in Table 1, was evaluated from a TOCSY 2D experiment in H,O processed with the same parameters. The classification wm done using the same vertical scale.

n Not exposed q Partially exposed

exposed

The refined co-ordinates of hen egg-white lysozyme were given to us by Dr P. Artymiuk. Hydrogen atoms were added using the INSIGHT graphics program (Dayringer et aE., 1986). Exposed surface areas were calculated by the method of Shrake t Rupley (1973) using a probe radius of 1.4 a (1 A = @l nm) and hydrogen bonding was analysed using programs written by A.M.L. Amide exchange data were taken from Redfield & Dobson (1988). The secondary structure assignment is given according to DSSP (Kabsch t Sander, 1984). Peak volumes were calculated by standard VARIAN software.

3. Results The effects of TEMPOL on lysozyme mined by monitoring the radical and of the protein spectra, respectively.

were deterenvironments of the by e.s.r. and n.m.r.

(a) e.s.r. spectra exclude speci$c TEMPOL-protein site binding The e.s.r. spectrum of TEMPOL in aqueous solution (10m4 M, at 308 K) is shown in Figure l(a). Such

662

G. Esposito

et al. 9mM HEW Lysozyme + 54m~ TEMPOL

(b)

II.5

II

I’:

IO.5 IO 9.5 p.p.m. 0

I

,r.*,m

-0.5 -I i-5

-2 -2.5

(a) I

9 mm HEW Lysozyme

6m

Figure 1. e.s.r. spectra of (a) 10e4 M-TEMPOL in ‘H,O at 308 K. e.s.r. spectra of TEMPOL-lysozyme (b) R = 1 and (c) R = l/10.

system at

a spectrum is characteristic of a small nitroxide spin label freely tumbling with an effective rotational correlation time z, of approximately lo- l2 s (Dwek, 1973). When the spin label is in the presence of both an equimolar amount of lysozyme (R = 1, Fig. 1: spectrum (b)), or of a large excess of the protein (R = l/10, Fig. 1: spectrum (c)) its motion is not restricted, thus giving spectra identical to that of free TEMPOL (Fig. l(a)). A solution with an excess of TEMPOL (R = 6) was also studied, giving an e.s.r. spectrum identical to those of Figure 1. The identity of all the recorded spectra indicates freely tumbling nitroxide species both in the absence and in the presence of the protein, excluding strong interactions between the free label and the protein, and preferential binding sites. (b) TEMPOL

does not alter the conformation protein

of the

Possible perturbing effects of TEMPOL on the conformation were assessed by of lysozyme searching for chemical shift variations in the n.m.r. spectrum of lysozyme upon TEMPOL titration. Figure 2 compares high and low-field regions of lysozyme in the presence (R = 6; Fig. 2(a)) and in the absence (Fig. 2(b)) of the radical. These two regions, containing isolated peaks, were chosen for the following reasons. The high-field spectrum contains ring-current shifted peaks, the chemical

I-T”’

Il.5

‘/

I

II

I”,

“I”“”

IO.5 IO 9.5

p.p.m. 0 -05

-I

-1.5 -2 ->5

(b)

Figure 2. 1D spectrum of lysozyme, 9 mM in ‘H,O at 500 MHz at 308 K, in (a) the presence and (b) the absence of spin-label (R = 6). ‘H, 400 MHz; H,O/‘H,O, 90/10; T = 308 K pH = 3.84.

shift variation of which is diagnostic of conformational changes caused by aromatic side-chain reorientations (Jardetzky & Roberts, 1981). The low-field region contains the N”1 indole resonances of six Trp residues, which have been reported to interact differentially with spin labels (Cassels et al., 1978). As is apparent in Figure 2, the small amount of radical used mainly brings about two effects: a generalized small down-field shift for most of the resonances (the maximum shift observed was 006 p.p.m.) and a broadening up to disappearance for some indole resonances (Fig. 2(a)). The effect on the chemical shift was analysed by TEMPOL titration. Figure 3 reports the ‘H-n.m.r. chemical shift variation of selected lysozyme resonances as a function of free radical concentration. For concentrations of the free radical up to 1.0 M, the experimental points can be fitted by a linear function of the concentration. Since all chemical shifts to Leul7 b-methyl resonance were referred (-966 p.p.m. from DSS), any variation is relative to this resonance. The linear dependence of Figure 3, however, would be conserved also on an absolute scale. The y proton of Ile98, for instance, resonates at -2.04 p.p.m. in the absence of spin label (Redfield

Mapping

I.0

0.0

I I 1 I I I I I

-

2 5

2 E 8 e $ t

the Met105 Hfl protons are not affected by the presence of (low concentration) TEMPOL (Fig. 2), as judged by the resonances at -0.97 p.p.m. These results imply that TEMPOL does not perturb the native structure of the protein under the conditions of these measurements (see Discussion).

I

t

0.6-

I I I I I I I I

0.4-

663

Protein Surfaces by Paramagnetic Probes

(c) TEMPOL

1D experiments can provide information only in regions containing isolated peaks. We therefore explored the effect of TEMPOL in 2D maps. Figures 4 and 5 compare significant regions of NOESY and TOCSY experiments in the absence (Figs 4 and 5(a)) and in the presence (Figs 4 and 5(b)) of TEMPOL. Even though the cross-peak patterns are identical, except for the small shift already observed in the 1D spectra, TEMPOL notably broadens and reduces the number of correlations in the 2D contour map. This is particularly evident in the TOCSY fingerprint region (Fig. 5). Paramagnetic relaxation filtering is stronger in TOCSY than in NOESY experiments. The observation of differential effects on the resonances affords the possibility of using backbone amide protons for investigating protein secondary structure.

i

P.P m

Figure 3. Chemical shifts of some representative high and low-field resonances of lysozyme as a function of freeTEMPOL concentration. The abscissa indicates the chemical shift values of the resonance lines with respect to the &methyl resonance of Leu17 (-066 p.p.m.). The molar concentration of the free radical is indicated on the y-axis.

& Dobson, 1988), i.e. at a field approximately 3 p.p.m. higher than its random-coil position (Bundi & Wiithrich, 1979). Its spectral occurrence, caused by ring-current shifts brought about by the proximity of Trp63 and Trp108 (Imoto et aZ., 1972), is not drastically changed by the presence of spin label either at low or high concentrations (Figs 2 and 3). This implies that, up to 1.0 M, TEMPOL does not change the orientation of the Trp indole rings, leaving the Ile98 environment unaffected. Similarly, r

in 20 spectra jiltering

(d) Analysis of intensity changes A classification of the observed TEMPOL-induced intensity changes of the fingerprint region crosspeaks is reported in Table 1. A ternary model (exposed, intermediate, buried) is adopted here to allow detailed comparison with the exposed surface areas of the amide groups, the hydrogen bond distribution and the amide proton hydrogen/deuterium

-2.4

-2.4 198yCH

-2.2 B

B &

4

D

-2.0

-2.2

- -2.0 - -1.8

- I.8 -1.6

- -1.6

- I.4

- -1.4

-1.2

-- -1-z

-1.0 -0.8

-0.6

2

- -1.0 - -0.8

E;

- -0.6

-0.4

E. 2

- -0.4 ‘I

/

- -0.2 - o-0 - 0.2

0.8

16 A

0.4 0.6

0.2

- 0.4 0.6

- 0.6

0.8

-- 0.8 1’1 I I I I III I I I I I I I I I I I I I J I / I I I / I I I I.0 0.8 0.4 0.0 -0.4 -0.8 -1.2 -1.6 -2-O -2.4 0.6 0.2 -0.2 -0.6 -I*0 -1.4 -1.8 -2.2

UAJ , I, I , I, I I I Irl I I I I, I.0 -0.4 -0.8 -1.2 -1.6 o-0 -2.0 -2.4 -0.2 -0-6 -1-O -1.4 -1.8 -2.2

Pm (a)

P.P m. (b)

Figure 4. Up-field region of the 500 MHz ‘H NOESY spectra of lysozyme in (a) the presence and (b) the absence of TEMPOL with R = 6. The connectivity Ile98 y-methyl proton (missing in (b)) shows up at increased contour levels (see also Fig. 2).

664

G. Esposito et al.

K33 I96 0

Q @

4.5

4-o

3.5

3-o

2.5

FI (p.p.m.1 (a 1

Fig. 5. exchange rates. In future developments a binary model might be sufficient. Amide protons were considered exposed if they were not observable in the presence of TEMPOL (open symbols), not exposed if more than two levels were observed in the 2D spectrum contour plot (filled symbols in Table 1) and partially exposed if they presented one or two levels. Although the experiments with and without TEMPOL were performed in exactly the same experimental conditions, water suppression might also account for the partial reduction of some of the intensities. The same applies for half-slow amide exchange rates taken from the literature and reproduced on our samples. Amide protons were considered hydrogen bonded when a distance of up to 3.0 A with the acceptor and an N-O-C angle larger than 135” was measured. Allowing for distorted hydrogen bonds, we considered as an intermediate class those cases in which

the distance is 3-O to 3.5 A and the angle between 135” and 115”. The three classes considered in the exposed surface areas correspond to the following: completely buried if the proton has no exposed area (filled symbols); partially buried if the area is between 91 to 69 A2 (half-filled symbols); exposed otherwise (open symbols) (Table 1). Volume ratios are illustrated more quantitatively in the histogram of Figure 6, where the attenuation was evaluated from the volume ratios of the corresponding cross-peaks in the presence and in the absence of TEMPOL. The ternary classification adopted in Table 1 corresponds here to cross-peak attenuation from 90 to 100% (exposed), 0 to 50% (not exposed) and 50 to 90% (partially exposed). Fingerprint region cross-peak volumes could be safely measured only for 76 connectivities out of the 110 identified in our spectra, yet enough to establish a firm quantitative test of the analogic classification criterion used with the contour map inspections.

Mapping

Protein

Surfaces by Paramagnetic

665

Probes

e

\A10 A14 0

K33 0 c30

0 1176

QV99 0

1119

0121 "QSLX

L56.

I

I

of3

b

I

I

I

495

I

4-o FI (p.p.m.

I

I

I

II

II

11

3-5

11

3-O

“1

1

2.5

1

(b)

Figure 5. Fingerprint with-R = 6.

-

region of the 500 MHz ‘H TOCSY spectra in (a) the absence and (b) the presence of TEMPOL

-

4. Discussion (a) Nature

of the interactions

For confident application of our results to structural or dynamical problems, we must understand the mechanism of the interaction between TEMPOL and the protein. There is consensus that even merely a collision or a very weak transient complex between a protein and a radical is sufficient for dipolar interactions between the unpaired electron of the radical and hydrogen spins of solute, which are modulated by relative translational motion of the random molecules. Indeed, the utility of such a method depends on the absence of specific interactions between the spin label and the macromolecule that perturb the conformation. Furthermore, the possibility of penetration of the label to locations in the protein not accessible to the solvent, and vice versa, must be evaluated when

correlating interactions with protein conformational features. The comparison of our results with quantities derived from the crystal structure of hen eggwhite lysozyme resolves this question. Evidence for weak, random interactions between TEMPOL and lysozyme is contained in the linear dependence of chemical shifts on TEMPOL concentration shown in Figure 3. This result can be interpreted as follows (Qiu et al., 1982; Draney & Kingsbury, 1981). All models for explaining paramagnetic shifts require the physical proximity of the free radical, L, and the interacting protein, P. The following equilibrium expression, equivalent to a Langmuir absorption isotherm, will thus exist for each molecule: P+LSPL; K =

[PLI

[Pl[Ll’

666

G. Esposito

et al. (Draney

& Kingsbury,

6 = L

0.6 04 0.2 0.0

.

.

.

t

a.z-l......................................l SYNCI~~,YSDONOMNAW”*~~~~~~=~~“~~~,~~~~~ AA911129

Figure 6. Normalized cross-peak volumes of lysozyme spectrum HN-a connectivities in the presence of TEMPOL (R = 6) are reported versus the primary sequence number. Normalized volumes VJV,,, are the ratios of the corresponding cross-peak volumes measured in 2D TOCSY spectra of paramagnetic and diamagnetic solutions. When the experimental volume determination was not available, the maximum value was given to the corresponding normalized cross-peak volume (filled bars), according to the ternary classification adopted in Table 1, i.e. V&V, = 1, for not exposed; VJV, = 05, for partially exposed and VP/V, = 01, for exposed. Average normalized values are reported for partially overlapping crosspeaks, entailing in some cases a classification other than that obtained by direct inspection of the contour maps (Table 1). This applies to the cross-peak pairs N44-D87, N27-G102, Gl02-AlO, A95G117, S86-G4, G16-W123 and AllO-N77. Negative bars were used for unidentified 0, quantitative; q , not or absent cross-peaks. quantitative. AA, amino acid residue.

If intermolecular interactions involve only weak van der Waals forces, then to a good approximation, ideal solution behaviour may be assumed and the various equilibrium constants will be small

NL’

l+K[L]

1981). Therefore,

6 + pL

l

1 +K[L]

6

p

where 6, and 6,, are the limit free and complexed protein chemical shifts, respectively, and 6, the observed average chemical shift. This equation implies that the chemical shift is independent of the protein concentration [P] and linearly proportional to the free radical concentration [L] (Draney & Kingsbury, 1981), as observed in Figure 3. This is consistent with our observations. Absolute referencing is not necessary to infer weak interactions from the observed linearity. These findings also indicate the nature of the intermolecular radical-protein interactions. A n-stacking model, that is. direct spin transfer between n-electrons of aromatic rings and the spin label (Morishima et al., 1977), has been rejected by Qiu et al. (1982), who reported a linear dependence of 13C chemical shifts on free radical concentration even for highly conjugated aromatics. This conclusion is consistent with both our e.s.r. spectra (Fig. 1) and the linear dependence of the resonances in Figure 3. An alternative to n-stacking is suggested by studies of the interactions between the unpaired electron of the nitroxide radical and the OH or CH, protons of protic solvents (Nientiedt, et al., 1981). According to these authors, proton-electron interactions occur by two mechanisms: by dipolar and scalar coupling via hydrogen bonds, with a time dependence given by anisotropic rotation of t,he complex and by exchange: and by dipolar couplings during occasional collisions of the radical and solvent molecules. The former interaction dominates for hydroxyl protons, whereas the latter interaction (“translational motions”) relaxes the methyl proton spin. The effects involving, for instance, the Trp indole protons and the y-methyl group of Ile98. previously discussed (see Results), may be explained by this model. From our results, we conclude that the relaxation (l/T,) is determined by dipolar couplings modulated by the translational motions with elastic collision (Nientiedt et al., 1981). (b) Spectral

simpli$cation

From what we have demonstrated, spin labels do not perturb the structure of lysozyme. Therefore, no variation of the J coupling is expected. This implies that the intensity reductions in the 2D experiments shown in Figures 4 and 5 are not caused by variation of the J,,, in the density operator D expression (Ernst et al., 1987), but by spin label induced relaxation. Since antiphase cancellation is absent in TOCSY and NOESY experiments, no additional intensity reduction effects in the data sets with and without TEMPOL are expected. Paramagnetic relaxation filtering effects are

Mapping

Protein

Surfaces by Parawuzgnetic

19

667

Probes

.19

Figure 7. A stereo drawing of the structure of lysozyme with the TEMPOL-accessible amide protons indicated. The filled and half-filled circles at the amide proton positions correspond to the filled and half-filled squares in Table 1. stronger in TOCSY than in NOESY experiments. This arises from the different effect of the mixing process in the NOESY and in the TOCSY experiments. The MLEVl7 pulse train used throughout the TOCSY mixing time entails a magnetization leakage equally governed by longitudinal and transverse relaxation processes, with rates l/T, and l/T,, (approximately equal to l/T, in liquids), respectively. On the contrary, in the NOESY experiment. the mixing time acts as a pure T, filter, because it is T, that governs the filtering during the mixing period. For macromolecules the 7; filtering is less effective than the T, filtering, because the ratio T,,JT,, is less than 1, T,, and T2,,, being the longitudinal and the transverse relaxation times in the presence of paramagnetic species (Solomon & Bloembergen. 1956). The different effect found in different 2D experiments might become important in the evaluation of 2D maps used in further developments and applications of this technique.

(c) Mapping

the protein

surface

To show the actual feasibility of this method for mapping the exposed and buried residues, we consider one of the classes of protons, the amides, and correlate the spectral perturbation of TEMPOL with elements of secondary and tertiary structure. A good correlation may be observed between amide exchange rates and exposure to TEMPOL (see Table 1). Of the 14 exceptions in Table 1, 11 are in fast exchange although inaccessible to TEMPOL. This may easily be explained considering that sites exposed to water may be completely sheltered to the bulkier spin-label. It is commonly assumed that protons with slow exchange rates are hydrogen bonded. However, it has already been pointed out that this inference is not always valid (Redfield & Dobson, 1988). Some amide protons of non-hydrogen bonded residues have slow exchange rates and, conversely, fast exchange can sometimes be observed in hydrogen bonded protons. The factors governing proton exchange rates are more complicated than is

generally acknowledged. Similar anomalies may therefore be expected also for the exposure to TEMPOL. Lysozyme is a typical globular protein (Fig. 7). The secondary structure shows four helices at positions 5 to 15 (Hl), 25 to 35 (H2), 80 to 84 (H3) and 89 to 101 (H4). The first two helices pack together, with the second mostly buried in the core of the protein. Helices 3 and 4 are partially buried. A triple-stranded antiparallel b-sheet and a very short two-stranded b-sheet are present in the regions 42 to 46, 50 to 54 and 57 to 60, and 1 to 3 and 38 to 40, respectively. The general exposure of the secondary structure element does not necessarily imply, of course, that all the amide groups of the region are exposed to the solvent; indeed many helices in proteins have a hydrophilic face exposed to solvent and a hydrophobic face buried in the protein interior. This feature shows up quite clearly in Figure 6, where the amide protons located in helical regions exhibit an alternating pattern of TEMPOL exposure, in good agreement with the alternating pattern of the corresponding computed surface areas or exchange rates (see Table 1). In addition, the overall decreasing trend of H2 amide exposure parallels nicely the progressive burying of this region in the structure. The TEMPOL exposure of the b-sheet regions reflects both the hydrogen bond network and the location of these elements with respect to the overall protein surface. Within the triple-stranded b-sheet region 42 to 60, for instance, the H-bonded amide protons (44, 46, 51, 52, 53, 57 and 60) invariably exhibit lower TEMPOL exposures and the overall exposure level appears higher for the segment 42 to 46, consistent with its outer location. Most of the tight turns appear quite accessible to the spin label, according to the current views about their role in surface recognition. This could bear some relevance for the identification of antigenic determinants by means of possible aspecific paramagnetic probes. The exceptions can be seen, however, in Figure 6, i.e. the 74 to 77 and 115 to 118 turns (type I and type II, respectively). The first can be ascribed to steric hindrance due to the covering loop 61 to 64 from one side, and Leu75 and Cys76 side-chains from the other.

668

G. Esposito

For all but two of the amide protons in lysozyme (those of residues 63 and 86) the exposure to nitroxide parallels exposure to the solvent and/or the presence of hydrogen bonds. TEMPOL exposure of the Trp63 amide proton, despite the hydrogen bond and the small exposed surface area, is likely to arise from the penetration of the spin label in the catalytic cleft of lysozyme. In contrast, the Ser86 HN is inaccessible to TEMPOL despite its large solvent exposure and lack of a hydrogen bond. Reduced spin label response is indeed noticeable for the whole segment 85 to 87 connecting the two helices 80 to 84 and 89 to 100 and does not adopt, any regular secondary structure. Inspection of the molecular structure shows that a steric barrier created by the P-sheet 42 to 60 prevents the penetration to this region of the nitroxide probe, but not of water.

(d) Extension

to other proton classes

Although the method may be extended to all of the observable protons to give a more complete description of the molecular surface, our present choice was dictated by the possibility of correlating our results with detailed information available specifically about amide protons. Furthermore, because of the backbone rigidity, the electronamide proton dipolar interactions are modulated by the same correlation time and a direct comparison between differential paramagnetic effects and nitroxide accessibilities may be attempted (Table 1). Protein surface mapping has been recently addressed also by Petros et al. (1990), using paramagnetic-probe-induced perturbation of 2D DQFCOSY spectra of ubiquitin and lysozyme. Their experimental observations were focussed only on side-chain connectivities (mostly methyl groups). They propose that association occurs to some extent between lysozyme and TEMPOL, based on the degree of cross-peak attenuation observed using a lower spin probe concentration (10 mM) than that employed for ubiquitin (20 mM). While valuable and complementary to our data on the back-bone, sidechain nitroxide exposure is more difficult to analyse in structural terms. Moreover, care should be exerted before inferring association from nitroxide induced attenuation of the side-chain connectivities. The increased sensitivity to the paramagnetic agent of the side-chain protons observed by Petros et al. (1990) for lysozyme as compared with ubiquitin may arise, not only from a significantly different overall protein tumbling rate (because of the higher molecular weight of lysozyme and the different temperatures used in the two measurements, 35°C and 50°C for lysozyme and ubiquitin, respectively), but also from the differential specific effects of local motions superimposed on the molecular tumbling. that demonstrate conclusively Our data TEMPOL-lysozyme interactions are very weak, even at high R values.

et al.

5. Conclusions Our results have established t,he structural and dynamic interpretation of the spectral changes produced. These are for the moment restricted to amide protons. A comparison of our results with proton/deuterium exchange rates shows that the detection of a paramagnetic perturbation in this class of protons allows the distinction between amides completely exposed, involved in superficial hydrogen bonds located in a relatively rigid portion of the macromolecule, and buried, whether hydrogen bonded or not. From these observations one may adduce valuable information on the location of specific amide protons within the molecule. A detailed correlation of the n.m.r. results with detailed structural information was possible here only because the structure of lysozyme is known and the proton n.m.r. spectrum has been fully assigned. Dealing with a protein of unknown structure, we could obtain the amino acid map of the TEMPOL-exposed residues. We are currently investigating how to derive information about the structures of unknown proteins from these methods. The possibility of mapping protein surfaces might also be applied to studies of the mechanism of folding and unfolding. The results might shed new light on the controversy between local unfolding and water penetration models (Woodward et al., 1982; Englander & Kallenbach, 1984). In conclusion, the spin labelling method described here, in combination with 2D spectroscopy, seems to be suited for (1) editing subspectra of the exposed regions and of the protein core with the aid of proper weighting factors; (2) investigating the mechanism of folding; (3) studying dynamical processes requiring detection of solvent exposure of portions of the protein, or monitoring changes in them, and (4) elucidation of surface exposure regions in proteins. We thank the Eniricerche Research Center, Monterotondo (Roma), Italy, where this work was initiated, Mr Gianni Mezza (Eniricerche) for technical assistance and Dr Marco Tato (VARIAPU’ spa) for help in reprocessing part of the data. A.M.L. thanks the Kay Kendall Foundation for generous support.

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by M. F. Moody