Conformational Analysis of a lgCl Hinge Peptide Derivative in Solution Determined by NMR Spectroscopy and Refined by Restrained Molecular Dynamics Simulations * H O R S T KESSLER,' SlGGl M R O N G A , ' G E R H A R D MULLER,' LUIS M O R O D E R , ' and ROBERT HUBER'

'Organisch-Chemisches Institut, TU-Munchen, D-8046 Garching, and 'Max -Planck lnstitut fur Biochemie, D-8033 Martinsried, Germany

SYNOPSIS

The hinge region links the antigen binding Fsbpart to the constant F, domain in immunoglobulins. For the hinge peptide derivative [ AcThr ( OtBu) -Cys-Pro-Pro-Cys-Pro-AlaProNHPI2the assignment of the 'H and 13C resonances was achieved by two-dimensional nmr techniques: total correlation spectroscopy (TOCSY), nuclear Ovethauser enhancement spectroscopy (NOESY), rotating frame nuclear Overhauser enhancement spectroscopy (ROESY), heteronuclear multiple quantum coherence ( HMQC ) transfer, and a HSQC (modified Overbodenhausen experiment) with high resolution in F, , which was several times folded in F1 but still phase correctable. Conformational relevant parameters ( 78 nuclear Overhauser effect distance restraints, 35HH for prochiral assignments, temperature gradients) were determined by nmr and served as input data for molecular dynamics ( M D ) structure refinement. A simulated model compound corresponding to the [ Cys-Pro-ProCysI2 core elongated by the peptide chains in the Fahand F, direction served as a starting structure for the final MD run. The conformation calculated in in uucuo does not agree with the C, symmetry required from nmr data, but the structure obtained by a water simulation fulfills the requirement. Here the core of the hinge peptide derivative adopts a polyproline I1 double helix as in the x-ray structure of IgG1. Hence, segments responsible for the internal flexibility are located outside the core as confirmed by the flexibility of the solvent exposed C termini.

I NTRO DUCTI 0N In immunoglobulins the hinge region links the antigen binding Fabpart to the constant Fc domain' (Figure 1) . T h e hinge peptide part is represented by a cyclic double chain bis-cystinyl octapeptide in parallel alignment containing the two adjacent interchain disulfide bridges, which are the only covalent interconnections of the two heavy chains. The hinge part is selected as a core peptide to mimic the spatial structure of the protein in the region that is responsible for Fah-F, relative movements.2-5 Here we report the conformational analysis of a Hic,pol\mers, Vol. :31, 1189-1204 (1991) K- 1991 .John Wiley & Sons, Inc.

ccc ooo6-:3:,25/9 I / 1 0 1 1x9-I fi$n4.00

* Dedicated to Prof. Dr. E. Wunsch on the occasion of his retirement as director a t the Max-Planck Institut fur Biochemie.

hinge peptide derivative in solution (Figure 2 ) , which differs from the native hinge peptide in one protecting group ( OtBu) and additional acetyl and NH2 groups a t the N and C termini, respectively. T h e free peptide has been studied p r e v i ~ u s l yThe .~ charges at the C- and N-terminal ends may have a strong influence on the conformation of the hinge region. So we decided t o study the peptide with varied capping groups. T h e designed peptide should represent the structure of the hinge peptide part in the immunoglobulin better than the above-mentioned ~ e p t i d e .The ~ introduced acetyl and NH2 group should additionally reduce the conformational space, which is already restricted by the abundance of proline residues and cystine bridges. A general procedure of conformational analysis was carried o ~ t .Homo~ , ~ and heteronuclear (inverse) two-dimensional ( 2 D ) nmr techniques were used for complete 'H and I3C assignment, including stereospecific 1189

1190

KESSLER ET AL.

cyclohexyl-carbodiimide a t molar ratios of 1 : 6 : 4 according t o the procedure of Ronai e t a1." Precipitation with ether followed by reprecipitation from MeOH / AcOEt led to the desired hinge peptide derivative in 65% yield over the two steps as wellcharacterized product. Amino acid analysis of the acid hydrolysate ( 6 M HCI, 24 h, 110OC): T h r 1.65 ( l ) , Pro 8.01 ( 8 ) , Ala 2.00 ( 2 ) , Cys 3.79 ( 4 ) . T h e low value of T h r is hydrolysis dependent as determined p r e v i o ~ s l y ,homogeneous ~ on high performance liquid chromatography ( p-Bondapak (218; eluent: CH3CN/0.1M sodium phosphate, p H 3.5; linear gradient from 10 to 40% CH3CN in 50 min; flow rate: 2 mL/min; detection: uv at 214 nm) and high performance thin layer chromatography (CHC13/MeOH/H20/pyridine, 60 : 40 : 10 : 5; CHC13/MeOH/HzO/HzO/AcOH, 60 : 25 : 4 : 2 ) . The molpeak of the fast atom bombardment mass spectrum was consistent with the hinge peptide derivative. Prior to its use for nmr spectroscopy, a 20mg sample of the compound was dried over Pz05 for 30 h in vacuo and then dissolved in degassed DMSO-d,. The nmr spectra were acquired on Bruker AMX 500 and 600 spectrometers (500 and 600 MHz for 'H, respectively), and processed on a X32 computer. We recorded total correlation spectroscopy (TOCSY)"J' spectra (20- and 80-ms spin-lock period), a "three-spin'' exclusive copy ( E . COSY) l3 spectrum, nuclear Overhauser enhancement spectroscopy ( NOESY ) 6,14 ( Figure 3 ), and rotating frame nuclear Overhauser enhancement spectroscopy ( ROESY) I 5 , l 6 spectra ( 120 ms mixing time). T h e spectra were recorded with 512 tl increments, 4K data points, and a spectral width of 4600 Hz.

Figure 1. The encircled hinge region represents the core peptide of human G1 immunoglobulins connecting the Fsb (antigen binding) and F, (constant domain) part of IgG1.

assignments. The results of molecular dynamics calculations (vacuum and water) were compared to the x-ray structure.8

MATERIALS A N D METHODS The hinge peptide derivative [ Ac-Thr ( OtBu) -CysPro-Pro-Cys-Pro-Ala-Pro-NHzI2 was prepared by acetylation and amidation of the double-chain biscystinyl peptide [ H - T h r ( OtBu) -Cys-Pro-Pro-CysPro-Ala-Pro-OH l2 in parallel alignment synthesized according to procedures described p r e v i o ~ s l yIt . ~was acetylated with a n excess of N-hydroxysuccinimido acetate and the resulting bis-acetyl derivative was isolated as homogeneous material by simple precipitation from dimethylformamide ( D M F ) with ether. The acetylated compound was then reacted in DMF with 1-hydroxybenzotriazole/ NH3 complex and di-

AC

Thr'(tBu)

CySe

CH, / \ Cp, /CHz\/CH2 CH, CH, \

CH,CO

FH,\ cH\z /CH,

cys5

I

CH,

~

1

~

17

cpz

\

/CH,

- NH - FH - CO - NH - FH - CO - N - CH - CO - N-CH - CO - NH - CH - CO-N-CH-CO-NH-CH - CO - N-CH - CO - NH, I I CH-0. tBu

YH2

y

S

S

I

I

S

sI

I

CH3

I

7%

CH, I

CHSCO - NH - CH - CO- NH - CH - CO - N - CH - CO - N-CH I

CH-0. tBu

/

\

CH\, ,CH,

I

CH3

CHZ

/

CH, \

\

/w, CH, \

I

/

CH,

cp,

CH,

\

/CH2

- CO - NH - CH - CO-N-CH-CO-NH-CH - CO - N-CH

CH,

/

CH3

2

I

CH3

CH,

Figure 2. Constitution of the hinge peptide derivative, which differs from the native hinge peptide in the additional acetyl and NH2 group at the N and C terminus, respectively.

- CO - NH,

ANALYSIS OF A IgGl HINGE PEPTIDE DERIVATIVE

Thr' COCH3

1191

2.0

3.0

CONE2 4.0

Pro8 Thr' , Pro4,Pro6 I,

cys2

JPm

NH

Pro3

5.0 PPm 7.5

6.5

5.5

Ha

Figure 3. Part of the 500-MHz NOESY spectrum of the hinge peptide derivative. Sequence NOE connectivities are connected by lines starting with the Thr' NH/COCH3 cross peak and ending with the Pro8 C"H/NH2 cross peaks. Interruption naturally occurs at the proline residues, but all Pro CaH/C6H connectivities are visible in the 80-ms TOCSY spectrum. Peaks marked by asterisks correspond to Ala7 and Thr' resonances of a very low represented ( < 5 % ) second conformation. The peak at 6.33/3.35 is an artifact (antidiagonal peak).

Prior to Fourier transformation the data were multiplied by a ~ / 4 - s h i f t e dsquared sine-bell window function and zero filled t o 1K in the F1dimension. Random variation of the T,,, ( i n 10% of T,,,) for the NOESY spectrum served to suppress zero quantum coherences. A 600-MHz heteronuclear multiple quantum coherence spectrum (HMQC) 17-*' (Figure 4 ) and a 500-MHz HMQC with TOCSY transfer2" (pulse sequences, acquisition, and processing parameters as described before) " were recorded with additional GARP 22 decoupling during acquisition. A modified2' 600-MHz HSQC ( Overbodenhausen) experiment",25 (see Figures 5 and 6 ) with 1500-Hz (10-ppni) and 4200-Hz (70-ppm) sweep widths in

F1and F2,respectively, was recorded in 45 min. Data points in tzwere l K , 453 tl increments, 8 scans, and 4 dummy scans were acquired. The transmitter frequency in Flwas set a few hertz high field from the C 6 resonances of the prolines t o allow a nonoverlapping folding of all resonances. T h e resulting digital resolution in F1was 6.6 Hz/pt. For all spectra quadrature detection was used in both dimensions with time proportional phase incrementation (TPP)26 for t l . A 500-MHz HMBCS spectrum27~28 (selective 'inverse COLOC') with a 270' Gaussian pulse for the excitation of the carbonyl resonance region was recorded and processed with similar parameters as described before.'l

1192

KESSLER ET AL.

20

6

(13C)

40 1

B I

Q Ala7Ca @

tp

0 0

3

ProC6

I

I

cysca

50

1 d

s Pro Ca ChrlCa

0

60

I

@ ThrlCO

'I L

70

I

I

1

----r------

Figure 4.

1

-

1

-

7

-

,

p

PPm

___c 1

The 600-MHz HMQC spectrum of the hinge peptide derivative.

T o translate 'H- 'H distances into a three-dimensional structure, we used the method of restrained molecular dynamics simulation^.^^^"' All energy minimizations ( E M ) and molecular dynamics ( M D ) simulations were performed on Silicon Graphics 4D/ 25GT, 4D/70GTB, and 4D/240SX computers using the software package INSIGHT (BIOSYM ) for

graphical display and interactive modeling. For all calculations we used the programs from the Groningen molecular simulation system ( GROMOS ) software package."'"* The energy function is composed of bonding terms representing internal constraints as bond lengths, bond angles, torsion angles, improper dihedrals, and nonbonded terms consisting

180"

90"

180" 90"

180"

I

I

I I

I

CARP

Figure 5. Pulse sequence for the folded Overbodenhausen experiment. It differs from the original ~ e q u e n c e * ~ in. 'the ~ implemented r'''C pulse directly after tl and the succeeding 6 delay, which serves for refocusing the chemical shift evolution taking place in the dead time before tl and in the 180" proton pulse duration period. This modification'" allows a correct phasing of all signals including the folded resonances in the phase-sensitive ( TPPIZfi) spectrum.

ANALYSIS OF A IgCl HINGE PEPTIDE DERIVATIVE

L

40

1

H 41

Pro Ca

L

1193

c y s CP 42

6

I

6

Thrl Ca

h

43

(13C)

1

44 I

00

45

4

I

46

47

48

49

ppm

4.5

4.0

3.5

3.0

2.5

2.0

1.5

PPm 1.0

0.5

6(1H)

Figure 6. The folded Overbodenhausen spectrum (600 MHz) (pulse sequence of Figure 5 ) of the hinge peptide derivative. The C 6 H / C bcross peaks of the proline residues and the C"H/C"resonanceofAla7arenot folded. TheThr'C"H/C",ProC"H/C",andCysC*H/ C" resonances are folded once, and the Thr' [email protected]/COis folded twice from the low field part of the spectrum, while the C'H/[email protected] C y s signals are folded one time and the C$H/C# and C1H/C7 proline resonances are folded two times from the high-field part of the spectrum. One-time folded signals are framed in the spectrum, two-times folded signals are indicated by a double frame. The letters H and L a t the boxes indicate the high-field or low-field nonfolded resonance position in reference to the actual sweep width.

of van der Waals interactions and electrostatic contributions. A further distance restraint function is added to include the NOE distance information. This restraint function switches from harmonic to linear behavior when the deviation from the upper or lower boundaries is greater than 10%. The parameters of the force field are optimized specially for simulations of proteins and nucleic acids. For all calculations, the full GROMOS force field was used. To constrain all bond lengths in order to allow an integration time step of 2 s, the SHAKE algorithm32was used for the MD runs with

-

A dielectric permittivity a relative tolerance of of 1 was used for all calculations. The velocities, given to the atoms initially, were taken from a Maxwellian distribution.

RESULTS AND DISCUSSION Assignment of 'H and

13C

Resonances

Complete proton and carbon assignments (see T a bles I and 11) were achieved from TOCSY,".'2

KESSLER ET AL.

1194

Table I Proton NMR Data of the Hinge Peptide Derivative in DMSO at 300 K. Proton Assignments (&Reference DMSO = 2.5 ppm), NH Temperature Gradients -Ab/AT, and 3J(C"H/CBH) Coupling Constants

Amino Acid

- A&/AT

(mb/K)

NH

C"H

Thr'

5.8

7.58

4.31

3.86

cys2

4.9

4.84

-

7.98 -

-

-

-

-

-

-

4.48 4.31

2.93pr('S 2.72pr" 2.14~"'s 1.85pr" 2.02"'" 1.79"'"' 3.03pro S 2.69""' 2.02pro s 1.79"'" 1.17

pro3 Pro4 cys5

Pro6 ~

1

~

7

Pro8

-

-

-

-

4.8

4.68

-

8.28 -

-

-

4.31

-

-

-

-

7.1 -

8.03 7.20" 6.85"

4.48 4.18

-

C"H/C"H'

-

' '

'

2,OOP'U

s

1.79pr"'

C7H/C7H'

C*H/C*H'

3J

0.99

1.11 (CH3tBu) 1.88 (COCH,)

-

-

-

-

-

1.98 1.87 1.92 1.87

3.77 3.65 3.52 3.52

-

-

-

-

-

1.86 1.86

3.62 3.60

3.7 10.2 8.5 4.5 8.5 4.5 5.0 10.0 8.5 4.5

-

-

-

1.90 1.85

3.52 3.52

8.5 3.5

C terminal NH2.

NOESY,6s'4 ROESY,15,16HMQC,l7-'' and HMQC with TOCSY transfer spectra,20 and a modified "Overbodenhausen" All nmr spectra show a single signal set that was observed before in one-dimensional ( 1 D ) nmr spectra5 representing only one of the parallel peptide chains, which accounts for Cz symmetry of the dimeric peptide. Our nmr spectra prove the existence of only one isomer with exclusively trans prolines ( t h e C8 and C y chemical shift values of prolines (Table 11) give evidence for trans peptide bond a t these residues).35 Very small additional peaks could be observed for T h r ' and Ala7 of a second conformation, probably represented by one cis proline residue. T h e adoption of a n almost uniform conformation is in contrast to

the behavior of the hinge peptide4 (deprotected N and C termini), which adopts a t least three different conformations. Problems arose with the assignment of the proline resonances. Four proline residues with similar surroundings in the molecule led to severe overlap of resonances. Not even the spin patterns of four individual proline residues could be identified from simple TOCSY or HMQC spectra. While the proton assignments of the residual amino acids obtained by homonuclear nmr experiments could easily be checked by the HMQC with TOCSY transfer, the low F1 resolution of this experiment as routinely run prevented the identification of the four proline spin systems.

Table I1 Carbon Assignments of the Hinge Peptide Derivative in DMSO at 3 0 0 K

Amino Acid

co

C"

C"

Thr'

169.8 169.1 (COCHd 167.5 170.2 171.5h 168.4' 170.8h 168.9' 173.6

57.00

66.90

cys2 Pro3 Pro4 cys5 Pro6 ~

1

Pro' a

~

7

49.50 57.59 59.31 50.00 58.31 46.00 59.38

38.76 27.71 29.15 38.26 28.90 16.70 29.15

C7

Cd

-

27.80 (tBuCH,) 22.30 (COCH,)

24.40 24.36"

46.74 46.31

8.80

-

-

-

24.19"

46.65

-

-

24.36"

46.31

The assignment is ambiguous because of strong signal overlap even in the folded Overbodenhausen experiment. The assignments of the Pro4 and Cyss CO resonance as well as those of the Pro6 and Ala7 CO signals may be exchanged.

ANALYSIS OF A IgGl HINGE PEPTIDE DERIVATIVE

We decided to record a n inverse shift correlation with high resolution in F1 to spread the overlap in the carbon dimension for the proline resonances. For this purpose the Overbodenhausen experiment2i.25 with two I N E P T transfers ['H "'C ( t ,) -+ 'H ( t 2 )] was chosen. The spectra obtained by this sequence do not suffer from unwanted passive proton-proton couplings and unfavorable relaxation behavior of multiple quantum c ~ h e r e n c e s The .~~ latter effect is a disadvantage of the HMQC whereas single quantum coherences are used in the tl domain of the Overbodenhausen experiment. The result is a smaller line width in F1 in the latter experiment in comparison to the HMQC. T o achieve a sufficient resolution in F1, extensive folding was used in a small spectral range. For this purpose we performed a modification (Figure 5 ) ,which uses the insertion of a T 13C pulse directly after tl and a delay (see caption of Figure 5 ) to allow a correct phasing of all resonances including the one and two times folded resonances.23The obtained digital resolution in F1 (6.6 H z / p t ) approaches the resolution of a 1D 13C spectrum (200 ppm, 16K: 3.2 Hz a t 600 MHz) . For the interpretation of such folded spectra the knowledge of a 1D spectrum or a nonfolded spectrum for comparison is necessary to identify the (one or more) folded signals. Signals are folded according to the T P P I Z 6method, which was used here to obtain phase sensitive spectra. Figure 6, in comparison t o Figure 4, clearly demonstrates the improved resolution. Especially in the region of the C 6 H resonances, the reduced signal overlap becomes obvious. While in the usual HMQC (Figure 4 ) no differentiation a t all is possible between the C*resonances of the proline residues, in the folded Overbodenhausen spectrum (Figure 6 ) three individual resonances a t 46.74, 46.65, and 46.31 can be distinguished. The resonances a t 46.91 still represent two proline residues. T h e situation is similar for the C" and resonances, which overlap pairwise. Unambiguous identification of the proline spin systems was finally achieved by evaluation of sequential NOE and COLOC connectivities (see below). Hence it was possible to complete the carbon assignments (Table I1 1 . -+

1195

T h e NOE connectivity between the C 6 H proton of each proline to the preceeding amino acid gives evidence for the position of each individual proline in the peptide chain. T h e Pro3 C"H/Pro4 C 6 H cross peak overlaps with the Ala7 C"H/Pro' C * H NOE signal, but the Pro' C"H/NH, cross peaks unambigously identifies the Pro' spin system. The whole spin system of Pro' is found in the TOCSY and in the HMQC with TOCSY transfer. The assignment of the Pro' NH, resonances were confirmed by exchange peaks in the ROESY spectrum. T h e chemical shifts of the carbonyl carbons determined by the HMBCS experimentz7," are included in Table 11. The Pro' could easily be assigned by the connectivities to its own C "H proton and the NH2 protons. T h e resonances a t 168.4 and 171.5 ppm represent the Cys5 and Pro4 carbonyls, respectively. Due to the lack of some long-range connectivities in the spectrum the assignment of the two latter resonances is interchangeable, but without influence on the sequence information. A similar case is the Ala7 CO/Pro6 CO resonance pair (see Table 11), but due to a Pro6 CO/Pro6CC"Hcross peak of low intensity we assigned Pro"C0 to the resonance a t 170.8 ppm. The Pro3 carbonyl carbon shows connectivities to its own C"H and to the C"H of Pro4. The completely assignment of all proline 'H resonances for the four proline residues was straightforward. Their assignment is a requirement for a n appropriate conformational analysis. If some resonances are not assigned, the interpretation of NOES in a spectral region that contains unassigned resonances is ambiguous. Although the assignment of the Pro4 resonances was difficult to achieve, we succeeded in finding all prolines in the trans conformation. Cis/trans isomerism of only one proline residue would lead to two conformers, four proline residues could result in a maximal number of 24 = 16 different conformers. In the case of the nonprotected hinge peptide derivative, cisltrans isomerism was observed for Pro6 and Pro', which therefore prevented a structural determination. Hence in this case the protected hinge peptide derivative exclusively contains trans peptide bonds allowing a realistic assignment procedure and unambiguous NOE crosspeak evaluation, which is necessary for conformational analysis.

Sequential Assignments

The part of the NOESY spectrum that was used for sequential assignment is shown in Figure 3. Starting a t the T h r 'NH/COCH3 cross peak, one can follow the sequential NOE cross peaks along the arrowmarked lines to the final Pro' C"H/NH2 cross peak.

Extraction of the Conformationally Relevant NMR Parameters

For the determination of the solution structure of the hinge peptide derivative we ( a ) integrated a

1196

KESSLER ET AL.

Table I11 Comparison of Experimental (NOESY) and Calculated (MD) H,H-Distances of HINloo (Structure in uacuo) and HINlooWA (Conformation in Water) with Distances in picometer. ppper

Proton Pair Thr' N H Thr' N H Thr' N H Thr' C"H Cys' N H Cys' N H Cys' N H Cys' N H Cys' N H Cys' N H Cys2 N H Cys2 C"H Cys2 C"H Cys' C"H Cys2 CPHPro s Pro3 C"H Pro3 C"H Pro3 C4HPr" Cys5 N H Cys' N H Cys' N H Cys' N H Cys' N H Cys' N H Cys5 N H Cys' C"H Cys' C"H Cys5 C"H Cys' C"H Cys5 CBHPr0 Cys5 COHHPrO s Pro6 C"H Ala' N H Ala' N H Ala' N H Pro8 C"H Pro8 C"H Pro' C"H Pro' C"H Thr' N H Thr' N H Thrg N H Thr' C"H Cys'" N H Cys" N H Cys'" N H Cys'" N H Cys" N H Cys" N H C Y S 'N ~H Cys" C"H Cys" C"H

~HINIOO

Thr' COCH3 Cys' N H Thr' COH Thr' CpH Thr' C"H Thr' CPH Cys' C"H Cys' C'Hpr" Cys2 COHHPr" s Pro3 C"H Pro3 C*H2" Cys' C6HHPr" Cys2 CPHPru s Pro3 C*H2" Pro3 C*H2' Pro3 C"HHPr0 pro3 COHHPr" s Pro4 C6H2" Pro4 C"H Pro4 C'Hp'" pro4 COHPrO S

Cys5 C"H Cys' CPHP'" Cys5 CflHPrOs Pro6 C6H2" Pro6 C"H Cys5 CBHP'" Cys5 CfiHHPrOs Pro6 C*H2" Pro6 C6H2" Pro6 C*H2" pro6 COHP'O

S

Pro6 C"H Pro6 C'HHPro Pro' C6H2" Pro' NH, Pro' NH2 Pro8 CBHPr" pro8 CPHP'O

pwerh

S

Thrg COCH3 Cys'" N H Thrg C4H Thrg CpH Thrg CeH Thrg CpH Cys" C"H Cys10 Ci(HPr0 R Cyslo COHHPr" s Pro" C"H Pro" CbH2" Cys'o C6H ' HPrO Cyslo CPHHPro S

400 278 351 270 253 395 311 263 316 489 495 292 265 352 433 257 250 414 240 293 335 313 266 314 479 430 299 274 416 469 372 235 254 369 467 316 433 338 303 400 278 35 1 270 253 395 311 263 316 489 495 292 265

280 243 315 232 214 359 278 232 285 359 337 261 233 202 264 225 219 218 203 263 298 273 234 278 315 372 266 240 295 335 248 200 221 312 330 286 343 301 272 280 243 315 232 214 359 278 232 285 359 337 261 233

276 381 341 241 225 439 292 225 350 500 462 297 261 202 380 275 239 413 217 429 407 291 223 335 455 446 293 274 208 480 404 24 1 229 361 485 254 363 305 241 281 346 336 243 259 459 293 216 344 494 428 295 258

rHINIOOWA

278 295 371 245 252 453 293 260 279 487 446 253 295 199 452 272 24 1 413 194 363 387 296 275 271 48 1 442 255 296 229 44 1 417 239 203 400 489 227 350 305 241 279 296 372 247 253 456 294 222 337 495 449 288 271

ANALYSIS OF A IgGl HINGE PEPTIDE DERIVATIVE

1197

Table I11 (Continued) Proton Pair Cys'" C"H Cysl(l [email protected] Pro" C"H Pro" C"H pro'1 COHPK s Cys','' N H Cys':' N H Cys':' N H Cys':' N H CYS'~ NH Cys'" N H Cys'" N H Cys':' C"H Cys':' C"H Cys'" C " H Cys'" C"H Cysl:3 CPHPI"R Cysl? COHprO s ProI4 C"H AlalS N H AlaI5 N H AlaIs N H Pro16 C"H ProI6 C"H Pro16 C"H Pro1fiC"H

,.upper

352 433 257 250 414 240 293 335 313 266 314 479 430 299 274 416 469 372 235 254 369 467 316 433 338 303

S,wer h

rHIN100

rHINlOOWA

202 264 225 219 281 203 263 298 273 234 278 315 372 266 240 295 335 248 200 221 312 330 286 343 301 272

209 426 273 238 372 239 295 360 294 227 353 491 412 300 248 234 414 272 241 208 375 460 250 368 305 242

229 407 276 240 376 207 314 360 295 253 267 481 445 252 293 223 449 428 238 213 381 468 258 357 277 239

Increased by 90 pm for MD run, because of the lack of diastereotopic assignment. The experimental values rupper and pwe' were extracted from different sides of the diagonal in the NOESY spectrum. a

NOESY spectrum, ( b ) determined coupling constants from a n E. COSY spectrum, and ( c ) established temperature coefficients of the N H protons. The 'H- 'H distances for structure refinement via restrained MD were obtained from NOE cross-peak intensities in the 600-MHz NOESY spectrum. When applying the two-spin approximation, the cross-peak intensities derived from NOESY spectra with short mixing times directly yield interproton distances. It is possible to calibrate the intensities against known distances such as that between two geminal methylene protons. According to the severe overlap of the proline methylene resonances, the choice of the reference peak was difficult. So we chose the overlapping C"H/[email protected]' cross peak of both Cys2 and Cys". Therefore, the integrated intensity, which is the sum of two cross peaks, correlating geminal protons, is divided by two, before the intensity is used to calculate the distances of all NOEs. Seventy-eight distances, 39 for each peptide chain were evaluated (Table 111).

Due to the spectroscopic identity of both chains, a NOESY cross peak can arise from intra- and/or interchain proximities. We started our calculations with the neglect of interchain contributions of the NOE intensities. It turned out that the resulting structure could not be twisted or changed to a conformation in which protons are close enough to cause interchain NOEs. T o exclude definitely the involvement of interchain NOE contributions, it would be necessary to synthesize the double-chain hinge peptide derivative with one of the two peptide chains in fully deuterated form, a methodology recently reported by 0. Jardetzky (personal communication) for trp -repressor. The Jna coupling constants of the proline and cysteine residues were extracted from the E. COSY spectrum (Table I ) . The prochiral assignments of the [email protected] (Table I ) were achieved by combined analysis of the Jnflcoupling constants and the N H / C @ Hand C e H / C @ HNOE connectivities as described by Wagner e t al.37Additional evaluation of

1198

KESSLER ET AL.

COLOC cross-peak intensities2',2R,"8was not required. We checked the C O / C d H and CO/[email protected]' cross-peak intensities in the HMBCS experiment and confirmed the obtained stereospecific assignments. The N H temperature gradients (Table I ) were determined from ID 600-MHz nmr spectra a t 300, 305, 310, 315, 320, and 325 K. MD Simulations

Initially we would like to give a short overview of the general procedure for our investigation. The simulation was started with a model compound to get a reasonable core conformation of the hinge peptide derivative. A random extended conformation (upper part of Figure 7A) serves as a starting structure for a restrained MD run over 100 ps in in uacuo. The resulting structure COREloo (Figure 7B) was elongated by the amino acid sequences in the Fa,, and F, direction, and subsequently simulated over 100 ps in in uucuo to give HIN,oo(Figure 7 C ) . Based upon the result of the in uucuo calculation, we built a starting conformation for a simulation (Figure 7D) in water. Averaging over a 60-ps restrained MD calculation results in the final conformation HINlooWA (Figure 7E) that will be discussed in detail. Modeling a Starting Structure

To get a reasonable starting structure for the subsequent refinement, one can use distance geometry39 to create a set of structures that fit the experimental data. Alternatively, one can use the method of simulated annealing40x41or one can build a n absolute random structure interactively. We constructed a model compound that represents only the core of the hinge peptide: [Ac-CysPro-Pro-Cys-NH212.The simulation of the model compound by restrained MD using the intraannular NOES should result in a conformation that is free of distortions and strain caused by nonbonded interactions with the N- and C-terminal peptide residues: Ac-Thr ( OtBu) and Pro-Ala-Pro-NH2. For modeling the core region, we used two different sets of coordinates: ( a ) a randomly extended conformation (upper part of Figure 7A ) and ( b ) the x-ray structure XRAY (lower part of Figure 7A) of the IgG1.R The randomly built conformation (upper part of Figure 7A) was interactively constructed with the standard residues from the INSIGHT residue library. First a n extended chain with all peptide bonds in trans configuration was built. Two of these chains were connected in parallel alignment via two disul-

fide bridges. To remove the strain caused by defining the interchain bonds, the molecule was allowed to relax by energy minimization for 200 steps. These two structures were simulated independently in in uacuo using 34 intraannular distance restraints. After only 10 ps of restrained MD a t 300 K, the conformations of both molecules turned out to he almost identical, indicating a convergence to a common conformation. For the further simulation the conformation derived from manually model building was used. So we are sure that structural features of the x-ray conformation do not determine the simulation and the resulting conformation. Based on the conformation CORElooin Figure 7B that was obtained after 100 ps MD simulation of the manually constructed conformation (upper structure in Figure 7 A ) , we built up the complete hinge peptide derivative by elongating the peptide chains in the Far,and F, direction, now being sure about a n appropriate core conformation, which is not determined by nonbonded interactions with the C and N termini. In Vacuo Simulations

The in uacuo calculations were performed with the following protocol: First the starting structure was minimized with steepest descents4' t o remove any strain caused by covalent distorsions during model building. The resulting structure serves as a starting structure for a restrained MD run over 2 ps a t 1000 K, further 3 ps a t 500 K, and 5 ps a t 300 K using all distance restraints. T h e resulting conformation was then minimized with the method of conjugate gradient^.^^ This high-energy calculation is necessary to make sure that the starting structure does not remain in a local energy minimum. T h e value of the distance restraining force constant was kDR = 4000 kJmol-' nm-2, which allows violations of a restraint of about 25 pm a t 300 K during MD runs. The cutoff radius for the nonbonded interactions was set to 10 nm to include all possible interactions. After the hightemperature simulation, a restrained MD run was started to calculate a trajectory over 90 ps. The reference temperature was 300 K and the system was coupled t o a heat bath with a time constant of 0.1 ps. T h e restraining force constant was lowered t o kDK= 1000 kJmol-' nm112to allow more flexibility. Every picosecond, 10 structures were stored in order t o get, a reasonable ensemble of conformations for averaging. During the first 30 ps the system was allowed t o equilibrate. The following 60 ps were taken for analysis. Averaging over that period and minimizing the averaged structure with conjugate

ANALYSIS OF A IgGl HINGE PEPTIDE DERIVATIYE

-

1199

-

A A c/ - -

f-

C E Figure 7. Schematic representation of the calculation procedure. ( A ) A random extended conformation serves as starting structure (upper part) and the coordinate set of the x-ray structure XRAY (lower part) was also tested as starting structure afterwards. ( B ) Model peptide after 100-ps restrained MD in in uacuo CORE,,,,,. ( C ) Hinge peptide derivative after 100-ps restrained MD in in uacuo HIN,,,,,. ( D ) Starting structure for the calculation in water. ( E ) Final structure after 100 ps restrained MD in water HINl,,own.

grudicnts4 leads to a conformation HINloo(Figure 7C ) , which represents the equilibrium conformation of the simulated molecule. This in uucuo conformation does not show the C, symmetry that is required from the nmr spectra, showing only a single signal set, representing only one of the parallel aligned peptide chains. A further possibility for the occurrence of only one set of nmr signals would be the presence of two interchanging unsymmetrical conformat ions contributing to the averaged structure t h a t IS screened by nmr.44Due to the conformational constraints of proline and the application of time-independent distance restraints, it was not possible to verify these conformational changes with the simulation technique used. One should focus the

attention on time-dependent simulation models4;' in the future to circumvent such uncertainties. Further, we think that the vacuum-derived conformation is mainly determined by nonbonded interactions, especially in the region of the terminal peptide residues. T h e C-terminal tripeptides Pro-Ala-Pro-NH, are linked by several hydrogen bonds and it seems as if the molecule tries to reach a globular conformation following the tendency to minimize the surface of the molecule, a fact already known from in uucuo calculations."' The hydrogen bonds can clearly be excluded by the above-mentioned temperature gradients of the polar hydrogen atoms (Table I ) . We assume that the nonbonded interactions in the in vacuo simulations are overemphasized in the ab-

1200

KESSLER ET AL.

sence of solvent molecules during MD runs, leading to structures that may be dominated by electrostatic forces. T o account for these effects we have previously reduced the charges a t solvent-exposed N H protons.46 However, this can reduce charge effects only a t experimentally accessible parts of the molecule ( N H temperature gradients). A better approach is the inclusion of solvent molecules.

Simulations in Water To get a more realistic impression of the molecular dynamics, the MD calculations were performed in solution. Solvents with a high dielectric permittivity

show a shielding effect on electrostatic forces. Although the nmr spectra were recorded in DMSO, we performed the simulations in water parameterized and implemented in the GROMOS software package; moreover, the hydrogen-bond acceptor properties should be comparable. It is known that the introduction of a dielectric medium such as water into MD simulations is a far better model than performing MD simulations in in uucuo. Based upon the conformation HIN,,, (Figure 7C), a starting structure for the water simulation was built by extending the tripeptide fragments Pro6-Ala7-Pro8-NH2and Pro ''-Ala'5-Pro16-NH2 t o avoid interactions between both chains. This struc-

Table IV Backbone and Cystine Side-Chain Dihedrals of the Hinge Peptide Core Fragment of Four Different Structures

cys2

*4 0

x1

X2

x3

Pro3

4 $ w

Pro4

4 $ 0

cys5

*4 0

X1 x2

x3

Cys'O

*4 w

XI XZ x3

Pro"

* *4 * 4 W

Pro'2

w

Cys'3

4

w

X1

x 2

x3

-61.2 143.5 172.9 -65.3 -59.8 -106.5 -57.1 139.9 -178.0 -60.6 112.4 -175.2 -83.3 169.1 - 175.3 45.4 103.4 -61.2 143.5 172.9 -64.5 -61.6 -106.5 -57.1 139.9 -178.0 -60.6 112.4 -175.2 -83.3 169.1 -175.3 45.4 103.4

-119.0 110.0 164.0 -131.2 39.1 -106.8 -68.0 113.2 -178.7 -71.0 109.9 175.9 -118.0 132.5 -131.2 42.8 87.8 -125.6 136.7 177.9 -60.9 -100.7 -106.8 -63.8 126.2 177.5 -67.5 113.5 171.0 -99.3 131.9 -

-114.2 55.2 87.8

-121.3 119.8 178.0 -82.0 -52.9 -94.3 -71.1 134.2 168.7 -67.5 97.8 -173.3 -125.6 130.0 171.8 -98.8 -164.8 90.9 -107.8 99.7 179.8 -82.6 36.7 -94.3 -72.8 148.8 177.9 -64.0 151.3 -171.3 -105.2 143.6 -163.5 -65.1 -63.7 90.9

-108.9 96.6 -168.8 -156.1 122.8 - 100.5 -70.6 144.4 172.7 -63.3 116.4 -176.8 -130.1 137.7 -173.8 - 165.3 92.2 91.0 -98.0 106.6 -173.4 -99.7 -124.5 -100.5 -60.4 143.8 - 176.3 -62.8 139.7 172.9 -98.2 119.7 -170.8 -163.1 71.3 91.0

ANALYSIS OF A IgGl HINGE P E P T I D E DERIVATIVE

ture (Figure 7D) was soaked to get a system containing the peptide in a truncated octahedron-like box of 1014 water molecules with a length of 4.045 nm. The solvent shell was allowed to relax by energy minimization while the peptide was fixed by position restraining. Then the whole system was minimized by 200 steps of restrained EM, and a restrained MD run over 100 ps was performed a t 300 K with a restraining force constant kDR = 1000 kJmol-' nm-2. The cutoff radius was set t o 1 nm to avoid interactions with the peptide molecule in the neighboring boxes, because periodic boundary conditions were applied. The first 40 ps served for equilibrating the system and the trajectory from 40 to 100 ps was analyzed leading to a final conformation HIN,oowA of ( Figure 7E) the hinge peptide derivative in water. Structural Interpretation

The C2 symmetry of the hinge peptide derivative is observed during the time scale of nmr. Averaging over 60 ps of dynamics is too short to obtain an average property, observed by nmr. Nevertheless the conformation HIN10oWA (Figure 8) fulfills the required symmetry quite well. The deviation is shown by the rms value of 83 pm for the superimposed structure HINlooWAand HINlooWA, turned around the twofold axis. T h e conformation HINlooWA accomplishes the 78 distance restraints without remarkable deviations. T h e value of the average restraint violation is 7 pm

1201

and thereby smaller than the uncertainty derived from the integration and calibration of 2D NOESY cross peaks. In contrast to a n earlier i n ~ e s t i g a t i o n , ~ there are no intramolecular hydrogen bonds as unambiguously indicated by the temperature gradients of the backbone N H protons (Table I ) . The backbone dihedrals of the core region (Table IV ) confirm the beginning of a polyproline I1 double helix, as described in the x-ray structure of IgGl XRAY (lower part of Figure 7A) .' Each turn of the double helix consists of three residues and the ideal values of the backbone dihedrals are 4 = -78", 1c/ = 149" and o = 180°.47This helix type is supported by the absence of any proline cis peptide bond, confirmed by the 13C shifts of the proline C p and C' carbons (Table II)."5 The averaged and $ values of all amino acids of the core region are (4) = -65.6", ($) = 141.2" and (4) = -86.1", ($) = 127.2" for XRAY and HINloowA, respectively. Hence, the helical structure of the hinge core as revealed by the x-ray crystallographic study is also retained in solution. In Figure 9 the core region of the x-ray structure and HINlmWA are compared. Whereas the lower region of the core in both structures in Figure 8 are almost identical, there are some minor differences in the upper part. The solution structure turns out to be not exactly C2 symmetrical during the time scale of the calculation. T h e two parallel helices are linked by two disulfide bridges. In HINl-A the disulfide bridges show

P

0

Figure 8 . Stereopair of the hinge peptide derivative HINlooWA after averaging over 60ps restrained MD and EM in water. The polypeptide chain is shown with the residue sequence numbers running from the top to the bottom.

-

1202

KESSLER ET AL

the same chirality and similar side chain dihedrals as in the x-ray structure XRAY (Table IV) . There is a left-handed disulfide conformation between Cys and Cys'" with a dihedral X S S = -100.5". The cysteine side chain dihedrals X1 and X 2 do not fit to the ideal values found for a left-handed spiral conformation with X1 = -60", x2 = -go", X S S = -go", X ; = -go", and x',= -60". In this portion of the molecule the most significant distortions of the symmetry were found. T h e X 2 dihedrals of Cys' and Cys '(' are different, and do not match the symmetry (Table IV), although the side-chain conformations should be well defined because of the diastereotopic assignments of the [email protected] The disulfide bridge between Cys5 and CysI3 adopts a righthanded conformation with a Xss = $91.0" (Figure 8 ) . The side-chain dihedrals XI and x2 do not fit to a typical class of right-handed disulfide conformations like the right-handed hooked c o n f ~ r m a t i o n . ~ ' In a current investigation of disulfide bridges in proteins, Srinivasan e t aLS0found the side-chain dihedral X I of disulfide-linked cysteines to be 180", if both cysteines are located in helices, which is in good agreement with the values of X I = -165.3", x2 = 92.2", xss = 91", X ; = 71.3", and xi = -163.1" in HINloowa (Figure 9 ) . The disulfide bridges in HINlooWA are extended with C"-C" distances of 619 pm (Cys2-Cys'") and 599 pm (Cys'-Cys'.'), which are unexpectedly long compared with the main classes of disulfide conformation^.^^^^^ This can be taken as a proof for the validity of the assumption that the NOEs are only intrachain proximities, because a polyproline I1 double helix does not allow a close proximity of both chains. The N-terminal peptide part Ac-Thr ( OtBu) shows no conformational transitions and adopts a preferred conformation during the trajectory of the MD simulation. There are three sequential NOEs determining its backbone conformation, so that no remarkable flexibility characterizes this part of the molecule. T h e rms fluctuations are 37 and 33 pm for the C" carbons of T h r ' and T h r g , respectively, averaged over the 60 ps of the MD simulation. This is comparable to the flexibility of the core residues. Hence, there is no experimental existence for flexibility in this region. We assume that the reason for this observation is the bulky protecting group OtBu, which constrains the segmental flexibility in the Fa,) direct,ion. With rms fluctuations of 33 and 32 pm for the C" atoms of Pro6 and Pro'4 in the core, the molecular mobility is increasing in the F, direction from the core-bound residues to the terminal residues ProRand Pro" with rms fluctuations of 48 and 49 pm of their C" atoms. T h e simulated confor-

mation of the C-terminal tripeptides is in agreement with a n extended conformation, well surrounded by the solvent. There are no NOEs, which would be indicative of an interaction of both C-terminal ends. This result is in agreement with those of Ito and Arata,' who state that the segment following the core is extended and the presence of the disulfidelinked core is essential in maintaining the extended conformation. In contrast to their work, we do not find any remarkable mobility of the x2 dihedrals of Cyss and Cys". They reported a significant degree of freedom of internal motion involving 4 and x2 of Cys5 and Cys13. In our work the values of 4 of Cyss and CysI3 are oscillating around the values of -130.1" t- 12" and -98.2" k 13" during the dynamics simulation. For the x 2 dihedrals the equilibrium values are 92.2" k 12" for Cyss and 71.3" 8" for Cys':'. In terms of dynamics this will not be a remarkable flexible region. Ito and Aratas assigned the resonances of the cysteine residues comparing the Cys proton chemical shifts as well as their p H dependency from the 1D nmr spectra of two different hinge fragments I and 11. In fragment I1 three amino acids are cleaved off from the N-terminal end. In both hinge fragments they found a larger temperature dependency for the chemical shift of Cys22gthan for CysZz6.They correspond to our C y ~ ~ / l C yand s ' ~ C y ~ ~ / C residues, ys~~ respectively, which we assigned with respect to their position in the peptide chain by NOE and COLOC connectivities (see above). In addition, Ito and Arata" extracted Jnoand Jno.coupling constants for the cysteine residues from 1D 'H-nmr spectra a t 400 MHz. They found one small and one large coupling constant indicating side-chain rotamers with x1 of 180" or -60". This is consistent with our J coupling constants determined from well-separated E. COSY cross peaks. Succeeding in prochiral assignments for the C'H protons (see above), we could differentiate between the two side-chain conformers that are consistent with the homonuclear protonproton coupling constants. In HINlooWA the cysteine residues adopt the side-chain rotamer with sulfur and nitrogen in antiperiplanar position. Deviation from this conformation is observed only for Cys'" (see Table IV with X I = 99.7" ) , but for the pair Cys5 and Cys '"the symmetry requirement from nmr data is fulfilled with x1values of -165.3" and 165.1", respectively (Table IV) .

CONCLUSION T h e hinge region has been suggested to be the main structural part for the segmental flexibility between

ANALYSIS OP A Ig(;l HINGE PEPTIDE DERIVATIVE

A

,J

u

1203

h

Figure 9. Comparison ofthe hinge region (core only) in crystal (XRAY, left) and solution ( H I N I O oright). ~~~,

Fell, and F, portions of the IgGl molecule. So we found the core region af the hinge peptide derivative (Figure 9 ) consisting of two parallei poll\” L ) -proline helices in solution that are linked by a left- and a right-handed SS bond. We do not agree with the assumption that the internal flexibility of two core residues is responsible for regulatory effects. T h e main fiexibility was found in the C-terminal peptide fragments, which are orientated into the solvent. T h e N-terminal exocyclic peptide chains are too short t o allow any statement about the mobility in Fk31> direction. Although the investigated hinge peptide derivative is only a small part of the IgGl molecule, we assume t h a t the main flexibility is not located in the core but in the C terminal parts in the F,. direction. In addition, we assume that the conservation of the native structure in the peptide fragment indicates a stable spatial structure and may be responsible for the high yield of the correct dimer in the air oxidation of the linear bis-cysteinyl-octapeptide.:3

Financial support by the Fond der Chemischen Industrie and the Deutsche Forschungsgemeinschaft is greatfully acknowledged. GM thanks the Fond der Chernischen Industrie for a fellowship. S M thanks G. Kurz for support and P. Schmieder for instrumental help.

REFERENCES 1. Roitt, I. M., Brostoff, J . & Male, D. K. (1985) in I m -

munology, Gower Medical Publishing Ltd., London. 2. Moroder, L. Hovermann, G. & Wunsch, E. (1988) in

Peptide Chemistry, Shiba, T. & Sakabibara, S., Eds., Protein Research Foundation, Osaka, pp. 759. 3. Moroder, L. Hubener, G., Gbhring-Rornani, S., Gijhring, W., Musiol, H.-J. & Wunsch, E. (1990) Tetrahedron 46, 3305. 4. Bovermann, G., Moroder, I,. & Wunsch, E. ( 1988) in l’rocecdings of tl7c 20th European Peptidc Sj’mposium Spptember ‘1-9, J u n g , G. & Rayer, E., Eds., de Gruyter, Berlin, pp. 748. 5. Ito, W. & Arata, W. (1985) Biochemistry 24, 6467. 6. Wuthrich, K. ( 1986) in N M R of Proteiris arid Nuclc.ic Acids, Wiley, New York. 7. Kessler, H., Bats, .J. W., Griesinger, C., Koll, S. Will, M. & Wagner, K. (1988) J . A m . Chern. Soc. 110, 1033. 8. Marquart, M., Deisenhofer, J., Huller, R. & Palm, W. (1980) J . Mol. Biol. 141, 369. 9. Wunsch, E., Moroder, L., Giihring-Romani,S., Musiol, H.-J., Gohring, W. & Boverman, G . (1988) I n t . J . Peptide Protein Kes. 32, 358. 10. Ronai, A. Z., Skekely, J. I., Graf, Id.,Dunai-Kovacs, Z. & Berzeti, J . (1977) FEBS Lett. 76, 91. 11. Braunschweiler, L. & Ernst, R. R. (1983) J . Mugn. Reson. 53, 521. 12. Bax, A,, Byrd, R. A. & Azolos, A. (1985) J . Am. Chem. Soc. 106, 7632. 13. Griesinger, C., Sorensen, 0. W. & Ernst, R. R. (1987) J . Magn. Reson. 75, 474. 14. Jeener, J . , Meier, B. H., Hachmann, P. & Ernst, R. R. (1979) J. Chem. Pys. 71, 4546. 15. Bothner-By, A. A,, Stephens, R. L., Lee, J., Warren, C. D. & Jeanloz, R. W. (1984) J . A m . Chem. SOC. 106, 811. 16. Kessler, H., Griesinger, C., Kerssebaum, R., Wagner, K. & Ernst, R. R. (1987) J . Am. Chem. Soc. 109, 609. 17. Muller, L. (1979) J . Am. Chem. Soc. 101, 4481. 18. Bendall, M. R., Pegg, D. T. & Dodrell, D. M. (1983) J. Magn. Reson. 52, 81.

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19. Bax, A., Griffey, R. H. & Hawkins, B. L. (1983) J . Magn. Reson. 5 5 , 81. 20. Lerner, L. & Bax, A. ( 1986) J. Magn. Reson. 69,375. 21. Kessler, H., Mronga, S.,Will, M. & Schmidt, U. (1990) Helv. Chim. Acta 73, 25. 22. Shaka, A. J., Barker, P. B. & Freemann, R. (1985) 64, 547-552. 23. Schmieder, P., Zimmer, S. & Kessler, H. (1990) Magn. Reson. Chem., in press. 24. Bax, A., Ikura, M., Kay, L. E., Torchia, D. A. & Tschudin, R. (1990) J. Magn. Reson. 86, 304. 25. Bodenhausen, G. & Ruben, D. J. (1980) Chem. Phys. Lett. 69, 185. 26. Marion, D. & Wuthrich, K. (1983) Biochem. Biophys. Res. Commun. 113, 967. 27. Bermel, W., Griesinger, C. & Wagner, K. (1989) J . Magn. Reson. 83, 223. 28. Kessler, H., Schmieder, P., Kock, M. & Kurz, M. (1990) J. Magn. Reson. 88, 615. 29. Lautz, J., Kessler, H., Boelens, R. Kaptein, R. & van Gunsteren, W. F. ( 1987) Int. J. Peptide Protein Res. 30,404. 30. Van Gunsteren, W. F. & Berendsen, H. J. C. (1990) Angew. Chem. Int. Ed. Engl. 29,992. 31. Karplus, M. & Petsko, G. A. (1990) Nature 347,631. 32. Berendsen, H. J. C. & van Gunsteren, W. F. ( 1984) in Molecular Liquids, Dynamics and Interactions, N A T O AS1 Series, C135, Barnes, A. J., et al., Eds., Reidel, Dordrecht, pp. 475. 33. Kaptein, R., Zuiderweg, E. R. P., Scheek, R. M., Boelens, R. & van Gunsteren, W. F. (1985) J. Mol. Biol. 182, 179. 34. Aqvist, J., van Gunsteren, W. F., Leijonmarck, M. & Tapia, 0. (1985) J. Mol. Biol. 183,461.

35. Kessler, H. (1982) Angew. Chem. Int. Ed. 21, 512. 36. Nonvood, T. J., Boyd, J., Heritage, J. E., Soffe, N. & Campbell, I. D. (1990) J. Magn. Reson. 87,488. 37. Wagner, G., Braun, W., Havel, T. F., Schauman, T., Go, N. & Wuthrich, K. (1987) J . Mol. Biol. 196,611. 38. Kessler, H., Griesinger, C. & Wagner, K. ( 1987) J. A m . Chem. SOC.109, 6927. 39. Crippen, G. M. & Havel, T. F. (1988) in Distance Geometry and Molecular Conformation, John Wiley & Sons, New York. 40. Nilges, M., Clore, G. M. & Gronenborn, A. M. ( 1988) F E B S Lett. 239, 129. 41. Barakat, M. T. & Dean, P. M. (1990) J . Cornput.Aided Mol. Design 4, 295. 42. Wiberg, K. B. (1965) J . A m . Chem. SOC.87, 1070. 43. Williams, J. E., Stang, P. J. & Schleyer, P. v. R. (1968) A n n . Rev. Phys. Chem. 19, 531. 44. Kessler, H., Bats, J. W., Lautz, J. & Muller, A. ( 1989) Liebigs A n n . Chem. 913. 45. Torda, A. E., Scheek, R. M. & van Gunsteren, W. F. (1989) Chem. Phys. Lett. 157, 289. 46. Kessler, H., Bats, J . W., Griesinger, C., Koll, S., Will, M. & Wagner, K. (1988) J. A m . Chem. SOC.110, 1033. 47. Ramachandran, G. N. & Sasisekharan, V. (1968) Adu. Protein Chem. 23, 283. 48. Richardson, J. S. ( 1981) Adu. Protein Chem. 34,167. 49. Srinivasan, N., Sowdhamini, R., Ramakrishnan, C. & Balaram, P. ( 1990) Int. J. Peptide Protein Res. 36, 147.

Received January 24, I991 Accepted May 27, 1991

Conformational analysis of a IgG1 hinge peptide derivative in solution determined by NMR spectroscopy and refined by restrained molecular dynamics simulations.

The hinge region links the antigen binding Fab part to the constant Fc domain in immunoglobulins. For the hinge peptide derivative [AcThr(OtBu)-Cys-Pr...
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