Biochemistry 1992,31, 7463-7476
7463
Two-Dimensional NMR Studies of the Zinc Finger Motif Solution Structures and Dynamics of Mutant ZFY Domains Containing Aromatic Substitutions in the Hydrophobic Core+,$ Xiuqi Q i a d and Michael A. Weiss*.%vIl Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, and Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts 021 14 Received January 28, 1992; Revised Manuscript Received May 18, 1992
ABSTRACT: Solution structures of mutant Zn fingers containing aromatic substitutions in the hydrophobic core are determined by 2D-NMR spectroscopy and distance-geometry/simulatedannealing (DG/SA). The wild-type domain (designated ZFY-6) is derived from the human male-associated protein ZFY and represents a sequence motif (Cys-X2-Cys-X-Ar-X,-Leu-X2-His-X4-His) that differs from the consensus ( C ~ S - X ~ , ~ - C ~ S - X ~ - P ~ ~ - X ~ - L ~ the U - location X ~ - H (“aromatic ~ S - X ~ - swap”) H ~ ~ ~and ~ ~ diversity (Ar = tyrosine, phenylalanKor histidine) of the central aromatic residue (underlined). In a given ZFY domain the choice of a particular aromatic residue is invariant among vertebrates, suggesting that alternative “swapped” aromatic residues are functionally inequivalent. 2D-NMR studies of analogues containing tyrosine, phenylalanine, or histidine at the swapped site yield the following results. (i) The three DG/SA structures each retain the BBa motif and exhibit similar staggered-horizontal packing between the variant aromatic residue and the proximal histidine in the hydrophobic core. (ii) Thestructures and stabilities of the tyrosine and phenylalanine analogues are essentially identical, differing only by local exposure of polar (Tyrp-OH) or nonpolar (Phep-H) surfaces. (iii) The dynamic stability of the histidine analogue is reduced as indicated by more rapid proteindeuterium exchange of hydrogen bonds related to secondary structure and amide-sulfur coordination (slowly exchanging amide resonances in D20) and by more extensiveaveraging of main-chain dihedral angles ( 3 J coupling a ~ ~ constants). An aspartic acid in the putative DNA recognition surface, whose configuration is well-defined as a possible helix N-cap in the tyrosine and phenylalanine analogues, exhibits multiple weak main-chain contacts in the NOESY spectrum of the histidine analogue; such NOES are geometrically inconsistent and so provide complementary evidence for structural fluctuations. (iv) Because the three DG ensembles have similar apparent precision, the finding of reduced dynamic stability in the histidine analogue emphasizes the importance of experiments that directly probe fluctuations a t several time scales. Our results provide insight into the design of biological metal-binding sites and the relationship of protein sequence to structure and dynamics.
The classical Zn finger motif (X3-Cys-Xz-Cys-X3-Phe-Xs-Insight into the informationalcontent of this template (Bowie Leu-X2-His-X4-His-X4) defines a highly conserved class of et al., 1990) may be obtained by analysis of patterns of eukaryotic nucleic acid binding proteins involved in the conservation and divergence among Zn finger sequences. regulation of gene expression (Klug & Rhodes, 1987). Single In previous studies we have investigated the implications Zn fingers exhibit metal-dependent folding (Frankel et al., of sequence variations among Zn finger domains for ther1987) as globular minidomains (Parraga et al., 1988). 2Dmodynamic stability2 (Weiss et al., 1990; Weiss & KeutNMR’ structures of isolated domains (Lee et al., 1989a,b; mann, 1990) andstructure (Kochoyanetal., 1991a-d; Jasanoff Klevit et al., 1990; Omichinski et al., 1990) are essentially et al., 1992). These investigations have focused on the ZFYrelated family of putative transcription factors (Page et al., identical to those of corresponding domains in the crystal 1987). This family is encoded by the sex chromosomes of structure of a protein-DNA complex containingthree fingers placental mammals (Schneider-Gadicke et al., 1989); ho(Pavletich & Pabo, 1991). TheisolatedZn finger thus provides mologues are also observed among marsupialmammals, birds, a model for investigation of folding rules in a simple sequence and reptiles (DiLella et al., 1990). ZFY is the prototype of template (the Boa motif; Berg, 1988; Gibson et al., 1988). a class of variant Zn finger proteins defined by an alternating pattern of finger and linker sequences (Page et al., 1987; Nif Supported by grants from the National Institutes of Health and the American Cancer Society to M.A.W. etfeldet al., 1989;Weisset al., 1990). Whereas odd-numbered 3 The coordinates of the NMR structures and tables of NMR-based ZFY domains follow the classical Zn finger consensus, evenrestraints have been deposited in the Brookhaven Protein Data Bank. numbered domains follow the pattern X3-Cys-Xz-Cys-X-Ar* Address correspondenceto this author at the Department of Biological X,-Leu-Xz-His-X4-His-X4, which differs in the placement Chemistry and Molecular Pharmacology, Harvard Medical School. (“swap”)ofthecentralaromaticresidue (Ar)andin thespacing 5 Harvard Medical School. Massachusetts General Hospital. between histidines (His-X,-His). The solution structure of I Abbreviations: CD, circular dichroism;DG, distancegeometry; DQFa representative even domain (ZFY-6) retains the &?a
COSY, double-quantum-filteredcorrelatedspectroscopy;NMR, nuclear magnetic resonance; NOE, nuclear Overhauser enhancement;NOESY, NOE spectroscopy;rms, root mean square;ROESY, rotatingframe Overhauser spectroscopy; rp-HPLC, reverse-phase high-performanceliquid chromatography; SA, simulated annealing;SD, standard deviation;UV, ultraviolet.
0006-2960/92/043 1-7463$03.00/0
*
In this paper the term stability is used in two senses. Thermodynamic stability refers to a free-energydifferencebetween the folded and unfolded states; dynamicstability refersto structuralfluctuationswithin the folded state.
0 1992 American Chemical Society
7464 Biochemistry, Vol. 31, No. 33, I992
Qian and Weiss
Domain 6 chicken
Zfb
KPYQ C
VKTF
-H SKE
human
ZFY
KPYQ C
IKTF H SKE
human
ZFX
KPYQ C
VKTF H SKE
mouse
Zfy-1
KPYE C
IKTF H SKE
mouse
Zfy-2
KPYE C
IKTX H SKE
Domain 8
1
chicken Zfb
KTHQ C
11s H TKD
human ZFY
KTHQ C
VIS\
human ZFX
KTHQ C
IISV H TKD
RTHQ C
IISG H TKA
H TKD
given aromatic residue is invariant (e.g., Tyr in domain 6 in man, mouse, and bird; Figure 1). What structural similarities underlie the relaxed sequence requirement at the swapped site, and what structural differences account for invariance among individual domains? To address these questions, we have determined the 2D-NMR structures of representative Zn finger peptides containing phenylalanine, tyrosine, and histidine at the swapped position. The overall &?CY motif is retained in each case. These analogues nevertheless exhibit local and nonlocal differences whose comparative analysis provides insight into the design of biological metal-binding sites.
MATERIALS AND METHODS
Peptide Synthesis. The synthesis and characterization of ZFY-6 (KTYQCQYCEYRSADSSNLKTHIKTKHSKEK Figure 2A) have previozsly been described (Kochoyan IISG H TKA RTHQ C mouse Zjy-2 et al., 1991a). This peptide is derived from finger 6 of the putative DNA-binding exon of the human ZFY gene (exon residues 162-191; Page et al., 1987). Analogues ZFY-phe Domain 10 (substitution TyrlOPhe; underlined above) and ZFY-his KLHQ C ILSG H TKD chicken Zfb (substitution TyrlOHis) were synthesized by the solid-phase procedure (Barany & Merrifield, 1979; Steward & Young, ILSV n TKD human ZFY KMHQ C 1984) using F-mocmethodology (Chang & Meienhofer, 1978; ILSV H TKD human ZFX KMHQ C Atherton et al., 1978); the initial residue was linked to a palkoxybenzyl alcohol resin to provide a C-terminal carboxylic ILSA H TKN mouse Zfy-1 KMHQ C acid. Peptides were reduced and purified by rp-HPLC as described (Weiss et al., 1990; Kochoyan et al., 1991a). The ILSA H TKN mouse Zfy-2 KMHQ C reduced peptideZn2+complexeswere lyophilized and stored FIGURE1: Sequences of even-numbered ZFY domains (fingers 6 , in an anaerobic N2 chamber (Coy Laboratories, Inc.). 8, and 10) demonstrating variation in the identity of the central Circular Dichroism. CD spectra were obtained with an aromatic residue (F, Y , or H; underlined and boldface) but invariance among vertebrate species within individualdomains. Conserved Aviv spectropolarimeter at a peptide concentration of 90 r M cysteines and histidines, presumed to coordinate Zn*+, are boxed. in a 1-mm path-length cuvette as described (Weiss et al., Arrows indicate conserved glutamine in the &sheet (GIn4 in the 1990). CD spectra were obtained in 50 mM Tris-HC1buffer peptide numbering scheme), and dots indicate proposed N-cap ascontaining 0.1 mM ZnCl2; to obtain unfolding curves, the partic acid or asparagine (Asp14 or Asnl4 in the peptide numbering scheme). Previous pH unfolding studies have demonstrated that the buffer pH was adjusted in the range 1-9 with 1% acetic acid. stability of human domain 8 is significantly less than that of domain Buffers were deoxygenated with N2 prior to dissolution of 6 or 10 (Weiss et al., 1990). Sequences are obtained from DiLella peptide. et al. (1990). NMR. ZFY-phe (12 mg) and ZFY-his (14 mg) were dissolved in 0.7 mL of NMR buffer containing 50 mM deustructure (Kochoyan et al., 1991a,b). 2D-NMR studies of terated Tris-HC1 (pH or pD 6.0; direct meter reading) and chimeric (even-odd) analogues of ZFY-6 demonstrate, how5 mM ZnCl2. Two-dimensional experiments were obtained ever, that distinguishing features of even sequences are in 99.98% D20 and 90% H2O/ 10% DzO at 500 MHz and 25 associated with alternative internal architectures and surface OC. Double-quantum-filteredcorrelation spectroscopy(DQFtopologies (Kochoyan et al., 1991c,d). Implications of such COSY), total correlation spectroscopy (TOCSY; mixing times structural variation for mechanisms of DNA binding are not 30 and 55 ms), nuclear Overhauserenhancement spectroscopy understood and are of interest in relation to nonstandard models (NOESY; mixing times 100 and 200 ms), and rotating-frame of Zn finger-DNA recognition (Fairall et al., 1986;Churchill Overhauser enhancement spectroscopy (ROESY; mixing time etal., 1990;Berg, 1990;Kochoyanetal., 1991a;Klevit, 1991). 50 ms) experiments were performed by the pure-phase method The present study is motivated by a striking difference (States et al., 1982). The data were processed with a between the evolutionary history of odd- (consensus) and combination of exponential and shifted sine-bell window even-numbered (nonconsensus) ZFY domains. Among functions in both dimensions. The sweep width was 7000 Hz odd ZFY domains (and in the majority of other conin each dimension; 350 t l increments were obtained. The sensus Zn finger sequences; Gibson et al., 1988) the observed 350 X 4K complex data matrices were zero-filled to central aromatic residue is invariant as phenylalanine 4K X 4K (NOESY, ROESY, and TOCSY) or 4K X 8K (X3-Cys-X2-Cys-X,-Phe-X,-Leu-X2-His-X3-His-X4), Such ~ constants were measured on (DQF-COSY). 3 J a coupling invariance may haveximple chemical basis: the Phe side the basis of the structure of “fingerprint” cross peaks in the chain participates in an internal aromatio-aromatic interaction DQF-COSY spectrum in H20; distortions in apparent mul(“edge to face”; Burley & Petsko, 1985, 1988) in which the tiplet separation arising from finite line widths were corrected para proton packs against the center of the proximal histidine by spectral simulation as described (Redfield & Dobson, 1990; ring (Kochoyan et al., 1991c; Mortishire-Smith et al., 1992). Smith et al., 1991). Chemical shifts are measured in parts In contrast to the invariance of the consensus phenylalanine, per million (ppm) relative to H20 (4.78 ppm at 25 “C). the “swapped” aromatic residue (Ar) among even-numbered NMR-Derived Restraints. NOE and J-coupling (dihedral ZFY domains may be phenylalanine, tyrosine, or histidine angle) restraints were used for molecular modeling as (Figure 1; DiLella et al., 1990). In a particular domain a mouse
Zfy-1
.1
-
Biochemistry, Vol. 31, No. 33, 1992 7465
2D-NMR Studies of the Zn Finger Motif B.
A.
C 30
.I‘lri!L
fit z5 ’ 6.5 I
1
9.5
I
I
8.5
FIGURE 2: (A) Schematic representation of the sequence of ZFY-6. Positions of mutations TyrlOPhe and TyrlOHis are indicated by outer circles (heavily shaded); cysteines and histidines presumed to coordinate Zn2+and conserved framework leucine are indicated (lightly shaded). (B) Amide-aromatic region of the ‘H-NMR spectra of ZFY analogues in H20: (a) ZFY-6 (Kochoyan et al., 1991a), (b) ZFY-phe, and (c) Z FY his.
-
previously described (Kochoyan et al., 1991a,b). NOEs were classifiedasstrong( 8.0 H Z
FIGURE 4: Summary of sequential and medium-range NOEs for ZFY-phe (A) and ZFY-his (B). Symbols (daN,d", etc.) are as defined by Wuthrich (1986). Slowly exchanging amide resonances are indicated by closed boxes in the upper panel; these are observed in the spectrum of ZFY-phe at 25 OC and of ZFY-his at 4 OC. Relative intensity of NOESis indicated schematically by the thickness of the line. Large and small 3JaNvalces are indicated by closed and open circles, respectively; XI values are indicated for residues for which stereospecific assignment of B resonances was obtained.
his also exhibits a weak NOE (T2-yCH3/R1 l-H7) that is not seen in the native domain, which may represent a transient contact between flexible side chains as discussed below. (ii) Whereas both ortho and meta resonances of TyrlO and F12 exhibit NOES to His21 and Leu1 8, such NOES are observed toHislO Ha but not HislO-H,. Such side-specificNOEs define a preferred orientation of the imidazole ring in the hydrophobic core and demonstrate hindered rotation about the C& bond axis. (iii) No NOEs are observed between His10 and Serl2. The distinct NOE pattern of His10 may in part reflect the different placement of protons on an imidazole ring and in part differences in side-chain position (part 11). (iv) In ZFY6 and ZFY-phe the side-chain configuration of Asp14 is welldefined by stereospecific assignment of the /3 resonances and medium-strength NOEs to the amide resonance of Asnl7. As discussed below, this configuration is consistentwith formation of an N-cap hydrogen bond between the Asp14 y-carboxylate and the Asnl7 amide proton. In the spectrum of ZFY-his the /3 resonances are degenerate and exhibit weak multiple NOES to the amide resonances of residues 15, 16, and 17. The dynamic implications of this observation are considered in part 111.
(ZZ)Solution Structures of ZFY-phe and ZFY- his Twenty DG/SA calculations were initiated for each analogue; of these, 16 converged for ZFY-phe and 15 converged for ZFY-his. Models of ZFY-phe (Figure 6B) were obtained on the basis of 176 NOE restraints (76 sequential, 45 medium range, and 50 long range) and 24 dihedral restraints; models of ZFY-his (Figure 6C) were obtained on the basis of 187 NOE restraints (89 sequential, 47 medium range, and 45 long range) and 23 dihedral restraints (supplementary material). Analogous restraints were previously obtained for the parent peptide (Kochoyan et al., 1991a,b). ThemaximumNOErestraint violationin theZFYphe ensemble was 0.14 A and in the ZFY-his ensemble was 0.10 A. The most consistent DG/SA model of ZFY-phe contained two NOE violations between 0.05 and 0.06 A and 13 NOE violations less than 0.05 A. The most consistent DG/SA model of ZFY-his contained one NOE violation of 0.06 A and seven NOE violations less than 0.05 A. For each analogue the distributions of backbone dihedral angles (4, J / ) cluster in the expected regions of the Ramachandran plot as shown in the supplementary material. Statistical parameters
Biochemistry, Vol. 31, No. 33, 1992 7469
2D-NMR Studies of the Zn Finger Motif
6.0
Y 7 ortho
.
FlOmeto Y 3 ortho H21 ( 8 )
-
D
*e
e
0
H21 (0
H26(0
O
1 Y7ortho YSortho
- 0
o
t
1
--
-
0
I
I
I
I
I
I
I
I
I
I
_
7.8
-
--H26(r) I
I
I
I
I
I
I
I
I
I
.
FIGURE 5 : Long-range NOEs observed in the NOESY spectra of ZFY-phe (A) and ZFY-his (B). The region shown contains contacts between aromatic and aliphatic protons (conditions: 25 OC, mixing time 200 ms, pD 6.0 in D20, direct meter reading). Table 111: Chemical Shift Difference of ZFY-phe and ZFY-6 at 25 'C (>0.1 ppm) residue R11 D14 N17 H21
NH
CaH
chemical shifts CBH
others C6H-0.31,-0.31
-0.11 0.1 -0.28
-0.16 -0.15,-0.13
~~
Table IV: Chemical Shift Difference of ZFY-his and ZFY-6 at 25 OC (>0.1 uum) residue
NH
T2 Y3 44
S15
N17 L18 H21 K23
chemical shifts CBH
others
0.10 -0.17 -0.11
c5
46 E9 R11 s12 A13 D14
CaH
-0.12 0.10,0.14 0.44 0.18 0.29 -0.67 -0.29 0.45 0.17
-0.10 0.10 -0.37 -0.42 0.13 0.12
CYH -0.22 CYH 0.10 CdH-0.28, -0.28
0.22 0.1 0.20,0.56 -0.17, -0.23 0.13,0.29
C*H 0.13
0.16
describing the DG/SA ensembles are provided in Table V. The overall average of main-chain rms differences (calculated on the basis of pairwise alignment of the main-chain atoms of residues 2-27) between ensembles is similar to those within a single ensemble (0.5-0.6 A). Each analogueretains the 88a motif and folds as a globular minidomain as in canonical Zn fingers (Lee et al., 1989a,b; Omichinski et al., 1990; Klevit et al., 1990). In Figure 6 are shown ensembles of 12 DG/SA structures for ZFY-6, ZFYphe, and ZFY-his (panels A X , respectively) with selected side chains in the hydrophobic core (Arlo, Serl2, Leul8, His21, and His26). In Figure 6D is shown a superposition of backbone configurations of ZFY-6 (red) and ZFY-phe
(green); the ensembles are aligned according to the backbone atoms of residues 21-26 (i.e., the invariant H S H metalbinding site). A corresponding alignment of ZFY-6 (red) and ZFY-his (blue) is given in panel E. In each hydrophobic core displaced-horizontal stacking (Burley & Petsko, 1988) is observed between the swapped aromatic residue (Ar 10) and proximal histidine (His21); the C-terminalhelix partitions between an a-helical portion (residues 15-20) and the nonstandard HX4H helix with characteristic bifurcating hydrogen bonds (residues 21-27; Table VI). These features, which distinguish even (nonconsensus) and odd (consensus) ZFY domains, were previously described in the refined structure of ZFY-6 (Kochoyan et al., 1991b). Hydrogen bonds corresponding to slowly exchanging amide resonances are indicated by asterisks in Table VI. In each analogue bidentate hydrogen bonds are possible but not established between the Gln4 side-chain amino group and the main-chain carbonyl oxygens of CysS and Glu9 (Figure 8A). Formation of such hydrogen bonds would rationalize the inequivalent chemical shifts of the two Gln4 Ha resonances (Tables I and 11) and is suggested by strong NOEs from Gln4-HC1and -He* to the HNresonances of CysS and Glu9. Gln4 is an invariant feature of most even-numbered ZFY domains (Figure 1) but is otherwise unusual among consensus Zn fingers (Gibson et al., 1988). Further investigation of possible side-chain hydrogen bonds will require a high-resolution solution structure (Omichinksi et al., 1990). A small shift is observed in PhelO relative to TyrlO or HislO, reflecting input of a tighter restraint to one of the /3 protons of Serl2 in the PhelO DG calculation. This has the effect of decreasing the solvent accessibility of the PhelO para proton (mean normalized solvent accessibility 14%) relative to that of the TyrlO p-OH group (mean normalized solvent accessibility 31%). It is also possible that the detailed configuration of the aromatic rings is influenced by hydrogen bonding between Serl2 yOH and TyrlO p-OH (ZFY-6) or between Serl2 yOH and HislO Na (ZFY-his), which would not be possible in ZFY-phe. Although such TyrlO and HislO hydrgen bonds cannot be directly observed by 'H-NMR, in the majority of TyrlO structures and in the minority of HislO structures they are allowed following rotation of Serl2 yOH.
‘3
I
I
I
‘3
Biochemistry, Vol. 31, No. 33, 1992 7471
2D-NMR Studies of the Zn Finger Motif Table V: Statistical Parameters Describing NMR Ensembles'
ZFY-his ZW-phe ZFY-6T (A) rms deviations (excluding disordered regions)b 0.58 0.56 main chain (A) 0.50 1.53 1.49 side chains (A) 1.42 (B) average NOE restraint violationsC 0.018 upper-bound violations (A) 0.020 0.014 (C) deviations from ideal covalent geometry 0.11 bond lengths (A) 0.10 0.12 bond angles (deg) 3.4 3.2 3.4 In each case the ensembles were calculated assuming S-HN distances of 2.2-2.8 A for the Cys5-Tyr7, CysS-CysB, and Cys8-Arlo sulfur-amide hydrogen bonds (see Materials and Methods). NMR ensembles were aligned in each case according to the Ca positions of residues 3-26; rms values shown correspond to these regions. NOE restraint violations represent rms upper-bound violations; no lower bounds were assumed. A.
Native Zn Finger
B. Phe 10 Analogue 4-
.
01 0
10
20
I
04
30
0
10
Residue
20
30
D. Comparison
20
10
3
Residue
C. HIS 10 Analogue
0
20
I 30
Residue
..
0
10
Residue
FIGURE7: rms deviations shown by residue for the main-chain (m) and side-chain atoms (e) of ZFY-6 (A), ZFY-phe (B), and ZFY-his (C). (D) Comparison of main-chain rms deviations for the three analogues: ZFY-6 (m), ZFY-phe (e),and ZFY-his (0).
The presence of a hydrogen bond between Serl2 r O H (as donor) and TyrlO p-OHf (as acceptor) has recently been demonstrated by analysis of the tyrosine Fermi vibrational doublet in the laser Raman spectrum of ZFY-6 (T. Li, M. A. Weiss, and G. J. Thomas, Jr., manuscript in preparation). Aspects of the ZFY-his ensemble are less precise than corresponding features in the other ensembles. The N-terminal hydrogen bond of the 8-sheet (Tyr3 H ~ S e r 1 2( 2 4 ) is not consistently predicted (Table VI), for example, and certain side-chain rms deviations are larger (residues 11, 14, 15, and 16; Figure 7). These reflect differences in the NOESY data (see part I). In the case of AsplCH,j, which in ZF'Y-his exhibits weak NOES to the amide resonances of residues 15, 16,and 17,norestraintswereintrodudas these threecontacts cannot simultaneously be satisfied by a single structure. The configuration of Asp14 in ZFY-6 and ZFY-phe is consistent with formation of an N-cap hydrogen bond (Richardson &
Richardson, 1988) to the amide proton of Asn 17 (Figure 8B). The rms deviation of the His10 side chain (0.9 A) is larger than that of the TyrlO or PhelO side chains (0.7 A); whether this reflects increased mobility or "proton scarcity" is not clear and is discussed further below. (III) Comparative Studies of Protein Dynamics Distinguishing between informational and physical uncertainties in the analysis of dispersion in a DG structure is a general problem in NMR spectroscopy. Imprecision is in part technical. The imidizole ring contains fewer protons, for example, than that of phenylalanine or tyrosine and so provides fewer probes with which to define local structure by NOES (informational uncertainty); other sources of informational uncertainty include chemical shift overlap, coarse interpretation of NOES (as strong, medium, or weak), and incomplete stereospecific assignment of methylene protons. Fluctuations
1412 Biochemistry, Vol. 31, No.33, 1992
Qian and Weiss
Table VI: Predicted Hydrogen Bonds in DG/SA Ensembles'
donor
no. of structures energy distance angle acceptor involved (kcal/mol) ID (deg)lc ZFY-6
3 N.-HN 5N-*HN* 10 OH-HH 12N-.HN* 18N-HN 19 N-HN 20N-HN 21 N-HN 22 N-HN* 23 N-HN* 24 N-.HN* 24N*-HN 25 N*-HN 26N-HN
o=C 12 o=C 10 Or 12 0-c3 -14 o=C 15 o=C 16 0-c 17 o=C 18 o=C 19 0-c 20 -21 o=C 21 0-c 22
3N*-HN o=C 12 5N--HN* 0-c 10 12N-.HN* 0-c 3 18N***HN M 1 4 19N*.*HN o=C 15 20N***HN o=C 16 21N-HN -17 22 N***HN* o=C 18 23 N*-HN* o=C 19 24 N-HN* o=C 20 24N-HN M 2 1 25N-sHN M 2 1 26N-*HN -22
11 13 8 14 6 14 13 13 14 13 11 6 9 13
-0.6 -1.4 -0.1 -0.7 -0.3 -0.6 -1.8 -0.3 -1.4 -1 .o
-0.0 -0.1 -0.1
3.4 3.1 3.0 3.3 3.2 3.1 3.3 3.1 3.0 2.9 3.0 3.3 2.9 3.1
35 31 60 35 45 37 12 45 27 38 45 43 57 49
ZFY-phe 6 -0.4 14 -0.7 11 -1.0 5 -0.2 -0.5 6 5 -0.3 14 -0.2 14 -1.8 14 -0.7 14 -0.6 7 -0.0 11 -0.3 13 -0.1
3.3 3.0 3.4 3.2 3.6 3.8 3.0 3.0 2.9 3.0 3.1 2.9 3.1
43 44 34 50 31 15 47 22 41 44 45
-0.5
55
48
ZFY-his 6 -0.4 3.4 50 3N-HN 0-c 12 43 12 -0.7 3.1 5 N*-HN* 0-c 10 11 -1.1 25 3.4 12N*-HN' o=C 3 43 6 -0.3 3.6 18N*-HN -14 24 10 -1.2 3.3 19N-*HN o=C 15 22 9 -0.7 3.5 20N-*HN OEC 16 41 12 -0.4 3.2 21 N*.-HN o=C 17 28 14 -1.4 3.0 22 N-*HN* OSc 18 40 2.9 14 -0.8 23 N*-HN* o=C 19 12 -1.0 38 3.1 24 N*-HN* 0-c 20 -0.2 55 12 3.0 25 N-HN OIC 21 47 3.2 14 -0.1 26N-HN -22 Presumptive hydrogen bonds are listed only if present in more than 4 out of 14 structures. Carbonyl oxygen acceptors are designated o=C. Distances are between heavy atoms and so differ from those in Table VII. D (deg) is defined as deviation from linearity. Asterisks indicate slowly exchanging amide resonances in D2O.
on a time scale faster than the rotational correlation time (minutes), conformational broadening of resonances in intermediate exchange (milliseconds), and tem~ constants of perature-dependent changes in 3 J a coupling resonances in fast exchange (
