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Dynamic properties of proteins from NMR spectroscopy
Dynamic properties of proteins from NMR spectroscopy Arthur G. Palmer, III C o l u m b i a University, N e w York, USA Two-dimensional proton-detected heteronuclear nuclear magnetic resonance spectroscopy has been used to measure 13C and 15N spin-relaxation rate constants for several proteins. Generalized order parameters and effective internal correlation times have been calculated from the relaxation data to characterize intramolecular motions that are more rapid than overall rotational diffusion. These studies provide detailed descriptions of the magnitudes and timescales of fluctuations in protein molecules. Current Opinion in Biotechnology 1993, 4:385-391
Introduction Nuclear magnetic spin relaxation has long b e e n recognized as a source of information concerning the intramolecular dynamic properties of proteins in solution [1]. Historically, the low sensitivities and low natural abundances of 13C and 15N nuclei as well as the limited resolution of one-dimensional nuclear magnetic resonance (NMR) spectroscopy have impeded studies of spin relaxation in biological macromolecules. These constraints have b e e n alleviated, and investigations of spin relaxation in proteins invigorated, b y two-dimensional proton-detected heteronuclear NMR spectroscopy [2] and biosynthetic methods for isotopic enrichment of proteins [3,4]. Identification of functional consequences of the dynamic properties of proteins has b e c o m e feasible as the requisite spectroscopic techniques have matured. This review surveys two-dimensional proton-detected NMR techniques for measurements of 13C and 15N spin relaxation, applications of the techniques to determine intramolecular dynamic properties of multiple atomic sites in proteins and protein domains, and comParisons of results from NMR spectroscopy and molecular dynamics simulations. For brevity, studies of protein dynamics using h o m o n u c l e a r 1H NMR spectroscopy are not reviewed.
Experimental techniques In the m o s t c o m m o n experimental design [5-7], two-dimensional proton-detected heteronuclear NMR spectroscopy is used to measure spin-lattice relaxation rate constants (R1) , spin-spin relaxation rate constants (R2) and steady-state heteronuclear nuclear Overhauser effect (nOe) enhancements for the 13C or 15N nuclei in a protein. Physical parameters incorportating the amplitudes a n d timescales of the intramolecular motions of the protein are calculated from the relaxation data using the model-free formalism pioneered by Lipari and
Szabo [8,9]. For 15N relaxation studies, proteins can be uniformly enriched to > 90 % isotopic abundance. Unenriched, random fractionally enriched or specifically labeled proteins are required for 13C relaxation studies to minimize 13C-13C dipolar and scalar coupling interactions.
Pulse sequencesfor spin relaxation experiments Typical pulse sequences applicable to X nuclei (where X = 13C or 15N) with a single directly bonded proton, such as backbone amide groups or methine moieties, are illustrated in Fig. 1. The pulse sequences for R 1 (Fig. la) and R2 (Fig. lb) measurements comprise: an initial polarization transfer step (from the proton spin to the X spin); a relaxation period, T; an evolution period, h; a reverse polarization transfer step (from the X spin to the proton spin); and the acquisition period, t 2. The relaxation period in Fig. l(a) constitutes an inversion recovery experiment. The relaxation period in Fig. l(b) consists of a Carr-Purcell-Meiboom-Gill (CPMG) spinecho sequence. Peak intensities are measured from spectra recorded for different values of T, and firstorder rate constants are extracted from the resulting time series. For nOe measurements (Fig. lc), the initial polarization transfer is omitted and the duration of the relaxation period is fixed. The n O e is determined from the ratio of p e a k intensities in spectra recorded with and without saturation of the proton resonances during the relaxation period. The original experiments [5,10-13] have b e e n modified to minimize the effects of evolution under the heteronuclear scalar coupling Hamiltonian during CPMG experiments [14,15",16"] and cross-correlation b e t w e e n dipolar and chemical shift anisotropy interactions [15",16",17"].
A proton-detected Tip experiment that substitutes a continuous spin-locking radiofrequency field for the CPMG spin-echo sequence in the relaxation period of Fig. l ( b ) has b e e n presented as an alternative m e t h o d
Abbreviations CPMG--Carr-PurcelI-Meiboom-Gill; NMR--nuclear magnetic resonance; nOe--nuclear Overhauser effect; Rl--spin-lattice relaxation rate constant; R2--spin-spin relaxation rate constant; S2--square of the generalized order parameter.
© Current Biology Ltd 0958-1669
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Fig. 1. Pulse schemes used to measure (a) R1, (b) R2 and (c) nOe with indirect proton detection. Thin and thick bars represent 90 ° and 180 ° radiofrequency pulses, respectively. Pulse phases are indicated above the bars; 180 ° pulses without phase designations are applied with y phase. The basic phase cycling is as follows: 01 = (x, - x , x, -x), 02 = (Y, Y, -Y, -Y) and receiver (x, - x , - x , x). Pulses labeled SL are high-power spin-lock purge pulses used for water suppression. Decoupling during the relaxation period of (a) and during the initial delay of (c) can be performed using trains of high-power pulses or broadband decoupling sequences. Broadband decoupling sequences are applied during acquisition. The value of "~ is set to 1/4JxH, (where JXH is the one-bond heteronuclear scalar coupling constant). The value of 8 is set to - 0.5 ms. For (a) and (b), the delay, T, is varied parametrically in a series of two-dimensional experiments; for (c), pairs of spectra are acquired with and without proton saturation.
for measuring R 2 [14,18]. The d e p e n d e n c e o f Tlp on the amplitude of the spin-locking field potentially can provide information on dynamic processes slower than overall rotational diffusion [19]. Broadband proton decoupling during a constant-time evolution period has b e e n utilized to improve water suppression in pulse sequences for measuring the n O e [20"]. In addition, the sensitivities of the experiments h a v e been increased b y using pulse sequences that transfer orthogonal magnetization c o m p o n e n t s from X nuclei to protons [21", 22"]. The pulse sequences shown in Fig. 1 are not applicable to nuclei with m o r e than one directly attached proton, such as 13C spins in methylene a n d methyl groups [11]. Modified pulse sequences have b e e n developed for measurements of methyl 13C spin relaxation [23",24]. Proton-detected pulse sequences for measurements of methylene 13C spin relaxation h a v e not b e e n reported, as yet.
characteristic frequencies: J(0), J(~)tt-- COx),J(~x), J(ml-I), and J(mH+mX), in which mH and COx are the Larmor frequencies of the 1H and X nuclei, respectively [25]. The spectral density is determined b y the overall rotational diffusion of the protein and by the intramolecular m o tions of the X - H b o n d vector that occur on timescales faster than the overall rotational diffusion. The spectral density does not contain information on internal m o tions slower than rotational diffusion; however, systematic increases in R 2 from exchange broadening provide qualitative evidence of motions on slower time scales [5,6]. In the model-free formalism [8,9], J(m) is a p p r o x imated as:
J(~)=(2/5)[s2"g m/(1 +~2"cm2)+(1- S2)'~/(1+~2"~2)]
(1)
Interpretation of spin-relaxation parameters
w h e r e ~m is the isotropic overall rotational correlation
The relaxation parameters, R1, R 2 and nOe are functions of the values of the spectral density, J(m), at five
time of the protein, S2 is the square of the generalized order parameter, "1:e is the effective internal correlation
Dynamic properties of proteins from NMR spectroscopy Palmer 387 time for internal motions and "c=(1/'Cm+l/'Q)- 1. Equation (1) has b e e n extended to include internal motions on two resolvable timescales [26] and anisotropic overall rotational diffusion [8,27.,,28.]. The accuracy of the motional parameters calculated from the relaxation data using the model-free formalism has b e e n discussed [8,9,26,29]. The squares of the order parameters measure the motional restriction of a X-H b o n d vector in a molecular reference frame and range from unity for vectors with fixed orientations to zero for vectors with isotropic orientational distributions. For example, if motion of a X - H bond vector is modeled as restricted diffusion in a cone, then S2 is given by:
S2 = [cos00(1 + cos00) / 2]2
(2)
and the amplitude of motion is characterized b y the cone semiangle, 00 [8]. For simplicity, S2 will b e referred to as the order parameter in the following discussion. An alternative strategy for determining the five characteristic values of J(c0) directly from relaxation data without recourse to the model-free formalism has b e e n described [30",31"]. In this approach, R1 and R 2 a r e measured as described above, and rate constants for relaxation of heteronuclear antiphase coherence, relaxation of two-spin longitudinal order and cross-relaxation b e t w e e n X and proton spins are measured using recently developed pulse sequences (in practice, the proton spin-lattice relaxation rate constant must also b e measured) [31"]. The values of J(c0) at the characteristic frequencies are determined from the relaxation data by inversion of a linear system of equations. Values of the spectral density functions for N - H b o n d vectors in eglin c determined by this approach are similar--to values calculated from model-free parameters using Equation (1); although, some significant differences have b e e n observed [30"'].
Applications to proteins In the period from 1989 to 1991, the model-free dynamic parameters of three proteins and protein domains were determined using two-dimensional protondetected NMR spectroscopy [5-7]. At least twelve experimental studies have b e e n reported since the beginning of 1992 [21",27"',28",30"',31",32"',33"',34",35",36"', 37",38"',39"]. In addition, 15N R2 values have b e e n reported for an antibody Fv fragment [40] and for a peptide complexed with an antibody Fab fragment [41].
Characteristic dynamic properties of proteins A remarkably consistent picture is obtained of the b a c k b o n e dynamics of folded proteins ranging in size from a single zinc-finger peptide (25 residues) [7] to the glucose p e r m e a s e IIA domain (162 residues) [35"]. As an example, b a c k b o n e 15N order parameters for
calbindin D 9 k , a small helical protein that binds two calcium ions, are s h o w n in Fig. 2. Relaxation parameters for 13C or 15N spins in regular (x-helices or ~sheets are described accurately by Equation (1) with S2 - 0 . 8 - 0 . 9 and '~e < 50ps; using Equation (2), the cone semiangle for the X - H b o n d vector ranges from - 15-22 degrees. Thus, mobility within elements of regular secondary structure is highly restricted. Lower order parameters and longer effective correlation times are observed frequently for 13C or 15N spins near the amino and carboxyl termini, in loops and in irregular secondary structures. Additionally, internal motions on m o r e than one timescale may contribute significantly to spin relaxation of these nuclei [6,8,9,26]. Regions of proteins that have low-order parameters or large exchange broadening contributions to R 2 a r e often correlated, albeit imperfectly, with regions that have high amide proton exchange rates, are poorly defined in NMR solution structures or have large crystallographic B-factors. Dynamic parameters have b e e n reported for the valine ]3, leucine y, aromatic and methyl 13C spins in a zincfinger peptide [7,37"], the leucine 8 methyl 13C spins in staphylococcal nuclease [32"'] and the tryptophan indole 15N spins in thioredoxin [38"']. Order parameters for different side-chain b o n d vectors are highly variable and indicate the importance of local interactions in restricting the mobility of amino acid side chains.
Dynamic aspects of protein function Very recent studies have correlated biological function with the intramolecular dynamics of proteins; however, establishment of causality remains an important objective. The following section summarizes some of the m o s t significant findings obtained in the past year from two-dimensional heteronuclear NMR spectroscopy. Barbato et al. [27"q have determined the b a c k b o n e dynamics of Drosophila calmodulin using 15N relaxation measurements. The rotational correlation times are found to be inconsistent with predictions from hydrodynamic calculations for rigid models of calmodulin, and the central helix in calmodulin is s h o w n to have a high degree of mobility, with order parameters -0.5-0.6. The results are interpreted in support of a model for the structure of the protein in solution in which a flexible central helix facilitates binding to target proteins. In another report, the internal dynamics of the leucine side chain methyl groups of staphylococcal nuclease h a v e been characterized b y 13C spin relaxation using newly developed pulse sequences for accurate measurements of 13C spin relaxation in methyl groups [32"']. The amplitudes of the internal motions of the leucine side chains are found to be larger than suggested by X-ray crystallographic studies or nOeSY spectra. The order parameters increase for methyl groups in regions of the protein near the ligand-binding sites u p o n complexing with Ca 2+ and thymidine 3',5'-bisphosphate. These results suggest that ligation increases the local rigidity of the protein.
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Fig. 2. Generalized order parameters for the backbone amide nitrogens of calcium-loaded calbindin D9k. The order parameters were calculated from relaxation rate constants and nOes measured at a magnetic field strength of 11.74 T [21"]. The protein contains two EF-hand domains. Each domain contains an ion-binding site flanked by two cuhelices. Residues 36-45 form a loop between the two domains. A representative error bar is shown for residue 2.
Powers et al. [33"'] have reported the backbone dynamics of ribonuclease H domain of the human immunodeficiency virus reverse transcriptase determined using 15N relaxation measurements. Ribonuclease H displays extensive and complex internal dynamics; nearly 90 % of the N-H bond vectors exhibit motions o n more than one timescale. Low-order parameters are observed for the carboxyl terminus, the loop between p-strands [~1 and ~2, and the loop b e t w e e n helix cxB and p-strand ~4. The high mobility of the carboxy-terminal region of the protein may contribute to the reduced enzymatic activity of the isolated ribonuclease H domain. Backbone 15N dynamics of apo calbindin D9k , (Cd2+)1 -calbindin D9k and (Ca2+)2-calbindin D9k have also been compared [21",36"']. Binding of Cd 2+ or Ca 2+ to the carboxy-terminal loop significantly increases the rigidity of the loop; smaller changes in order parameters are observed for helix III and the carboxyl terminus. The observed changes in the dynamic properties of the protein u p o n ion binding may contribute entropically to cooperativity. The backbone and tryptophan side-chain dynamics of oxidized and reduced thioredoxin have b e e n determined by 15N relaxation [38"q. Internal motions on picosecond to nanosecond timescales are similar for both forms of the protein; however, reduction of the active-site disulfide bridge increases the dynamic mo-
bility of residues 73-75 o n microsecond t o millisecond timescales. Increased mobility of the active site in the reduced form of the protein may facilitate binding to other proteins. Van Mierlo et al. [39"q have used 15N relaxation to determine the backbone dynamics of an analog of the folding intermediate of bovine pancreatic trypsin inhibitor that contains only the disulfide b o n d between Cys30 and Cys51. The amino-terminal 15 residues and the residues from 37-41 are very flexible with order parameters < 0.5; order parameters for the hydrophobic cole of the molecule are - 0 . 7 . The results suggest that formation of non-native disulfide bridges during folding of the protein is a consequence of the flexibility of the regions of the protein containing Cys5, Cys14 and Cys38. Comparisons
with theoretical
results
Order parameters calculated from molecular dynamics simulations have been compared with order parameters measured by two-dimensional proton-detected NMR spectroscopy for interleukin-l~ [42"-], calbindin D9k [43"] and a zinc-finger peptide [44-]. Order parameters have also been calculated from normal mode and Langevin mode analyses of the zinc-finger peptide. Theoretical calculations reproduce the general features
Dynamic properties of proteinsfrom NMR spectroscopyPalmer 389 of the experimental results; however, internal motions for some b o n d vectors are sampled inadequately during the simulations. The molecular basis for the fast and slow dynamic processes observed experimentally for interleukin 1~ can be inferred from the molecular dynamics simulation.
Nuclear Magnetic R e s o n a n c e Spectra of P r o t e i n s . Q Rev Biophys 1990, 23:1-38. 5.
KAY LE, TORCHIA DA, BAX A: B a c k b o n e D y n a m i c s o f Prot e i n s as Studied b y 15Nitrogen Inverse Detected Hete r o n u c l e a r NMR Spectroscopy: Application to S t a p h y l o c o c c a l N u d e a s e . Biochemistry 1989, 28:8972~97%
6.
CLORE GM, DRISCOLL PC, WINGFIELD PT, GRONENBORN AM: A n a l y s i s o f the B a c k b o n e D y n a m i c s o f I n t e r l e u k i n - l ~ using T w o - D i m e n s i o n a l Inverse Detected Heteronuclear 15N-1H NMR S p e c t r o s c o p y . Biochemistry 1990, 29:7387-7401.
7.
PALMERAG, RANCE M, WRIGHT PE: I n t r a m o l e c t d a r Mot i o n s of a Z i n c F i n g e r D N A - B i n d i n g D o m a i n f r o m X f i n Characterized by Proton-Detected Natural Abundance 13C H e t e r o n u c l e a r NMR S p e c t r o s c o p y . J Am Chem Soc 1991, 113:4371-4380.
8.
LIPARI G, SZABO A: Model-Free A p p r o a c h to the Interp r e t a t i o n of N u c l e a r M a g n e t i c R e s o n a n c e R e l a x a t i o n i n M a c r o m o l e c u l e s . I. T h e o r y and Range o f Validity. J Am Chem Soc 1982, 104:4546-4559.
9.
LIPAm G, SZABO A: M o d e l - F r e e A p p r o a c h to the Interpretation of Nuclear Magnetic R e s o n a n c e Relaxation i n M a c r o m o l e c u l e s . II. A n a l y s i s o f E x p e r i m e n t a l Restilts. J Am Chem Soc 1982, 104:4559-4570.
10.
KAY LE, JUE TL, BANGERTER B, DEMOU PC: S e n s i t i v i t y Enh a n c e m e n t o f 13C T1 M e a s u r e m e n t s v i a P o l a r i z a t i o n Transfer. J Magn Resort 1987, 73:558-564.
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SKLENARV, TORCHIA D, BAX A: Measurement o f C a r b o n -
Conclusion Recent studies of spin relaxation by two-dimensional proton-detected NMR spectroscopy provide detailed characterizations, and are beginning to elucidate the biological consequences, of the dynamic behavior of proteins. The characterization of side-chain dynamics, the investigation of motional processes slower than overall rotational diffusion, the assessment of the effects of mutations and the comparison of proteins in different functional states will all be the focus of experimental studies of spin relaxation in proteins over the next few years. The results of such investigations can be expected to illuminate the critical relationships among biological activities, structures and the dynamics of proteins.
Note added in proof Proton-detected heteronuclear spin-relaxation measurements recently have b e e n applied to additional protein systems of particular biological interest, including a DNA-binding domain [45"], an antibody fragment [46"] and a receptor-ligand complex [47"']. In addition, novel interpretations of chemical exchange broadening contributions to spin-spin relaxation have been proposed [46",48"]. In a significant advance, Szyperski et al. [49"'] have recently used the d e p e n d e n c e of Tip on the strength of the spin-locking field to characterize millisecond conformational rate processes in bovine pancreatic trypsin inhibitor.
Acknowledgements I thank Drs Nuria Assa-Munt a n d Richard A Friedman for helpful discussions.
References and recommended reading Papers of particular interest, published within the annual period of review, have b e e n highlighted as: of special interest •. of outstanding interest 1.
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13 L o n g i t u d i n a l R e l a x a t i o n u s i n g 1H D e t e c t i o n . J Magn Reson 1987, 73:375-379. 12.
NIRMAIANR, WAGNER G: Measurement o f 13C R e l a x a t i o n T i m e s i n P r o t e i n s b y T w o - D i m e n s i o n a l Heteronuclear 1H-13C Correlation S p e c t r o s c o p y . J Am Chem Soc 1988, 110:7557-7558.
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NIRMALANR, WAGNER G: Measurement o f 13C S p i n - S p i n R e l a x a t i o n T i m e s b y T w o - D i m e n s i o n a l Heteronuclear 1H-13C Correlation S p e c t r o s c o p y . J Magn Reson 1989, 82:659-661.
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PENGJW, THANABALV, WAGNER G: I m p r o v e d A c c u r a c y of Heteronuclear Transverse R e l a x a t i o n T i m e M e a s u r e m e n t s in Macromolecules: El|mlqation o f A n t i p h a s e Contributions. J Magn Reson 1991, 95:421-427.
15.
KAY LE, NICHOLSON LK, DELAGIO F, BAX A, TORCHIA DA: P u l s e Sequences for Removal o f t h e Effects o f Cross Correlation b e t w e e n Dipolar and C h e m i c a l - S h i f t Anisotropy Relaxation Mechanisms on the Measurem e n t o f Heteronuclear T 1 and T 2 V a l u e s i n P r o t e i n s . J Magn Reson 1992, 97:359-375. See [16"]. 16.
PALMERACT, SKELTON NJ, CHAZIN WJ, WRIGHT PE, RANCE M: S u p p r e s s i o n of t h e Effects o f Cross-Correlation bet w e e n Dipolar and A n i s o t r o p i c C h e m i c a l Shift Relaxa t i o n M e c h a n i s m s in t h e Measurement o f S p i n - S p i n R e l a x a t i o n Rates. Molec Phys 1992, 75:699-711. This paper and [15"], discuss effects of the heteronuclear scalar coupling interaction and cross-correlation between dipolar and chemical shift anisotropy relaxation m e c h a n i s m s on the m e a s u r e m e n t of s p i n - s p i n relaxation rate constants using CPMG sequences. Methods are presented that allow accurate m e a s u r e m e n t s to be performed. 17.
BOYDJ, HOMMELU, CAMPBELLID: Influence o f C r o s s - C o r r e l a t i o n b e t w e e n D i p o l a r and A n i s o t r o p i c C h e m i c a l Shift R e l a x a t i o n M e c h a n i s m s u p o n L o n g i t u d i n a l Relaxation Rates o f 15N i n M a c r o m o l e c u l e s . Chem Phys Lett 1990, 175:477-482. T h e effects of cross-correlation b e t w e e n dipolar a n d chemical shift anisotropy relaxation m e c h a n i s m s on the m e a s u r e m e n t of spin-lattice relaxation rate constants are discussed. Methods are pres e n t e d that allow accurate m e a s u r e m e n t s to be performed.
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PENGy ~ , THANABALV, WAGNER G: 2D H e t e r o n u d e a r NMR M e a s u r e m e n t s of S p i n - L a t t i c e R e l a x a t i o n T i m e s i n the Rotating Frame of X N u c l e i in H e t e r o n u d e a r ItX S p i n Systems. J Magn Reson 1991, 94:82-100.
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DEVEtLELLC, MORGANRE, STRANGEJH: Studies o f C h e m i c a l E x c h a n g e b y N u c l e a r M a g n e t i z a t i o n R e l a x a t i o n i n the Rotating F r a m e . Mol Phys 1970, 18:553-559.
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NEUHAUS D, VAN MIERLO CPM: Measurement of Hete r o n u c l e a r hOe E n h a n c e m e n t s in Biological Macrom o l e c u l e s . A C o n v e n i e n t P u l s e Sequence for Use w i t h A q u e o u s Solutions. J Magn Reson 1992, 100:221-228. The heteronuclear h O e is determined from tile peak intensities in spectra acquired either with or without saturation of the proton spins. Efficient water suppression can b e difficult to achieve in the control spectrum acquired without proton saturation. Water suppression can be improved if broadband proton decoupling is applied during the evolution period of a constant time heterocorrelation experiment. KORDELJ, SKELTON NJ, AKKE M, PALMER AG, CHAZ1N WJ: B a c k b o n e D y n a m i c s o f C a l c i u m - L o a d e d C a l b i n d i n D9k Studied b y Two-Di~aensional Proton-Detected NMR S p e c t r o s c o p y . Biochemistry 1992, 31:4856-4866. Backbone dynamics of (Ca2+)2-calbindin D9k are determined by 15N relaxation using modified pulse s e q u e n c e s that improve the sensitivity of t h e NMR experiments by detecting orthogonal magnetization components. Low-order parameters are observed for the linker loop b e t w e e n the two domains of the protein. Average-order parameters for the four helices of the protein are indistinguishable; however, effective internal correlation times are significantly different. 21.
22.
SKELTON NJ, PALMER AG, AKKE M, KORDEL J, RANCE M, CHAZIN WJ: P r a c t i c a l A s p e c t s of T w o - D i m e n s i o n a l Proton-Detected 15N S p i n Relaxation Measurements. J Magn Reson 1993, in press. Optimized pulse sequences for m e a s u r i n g 15N spin relaxation using two-dimensional proton-detected NMR spectroscopy are presented. Factors affecting the precision a n d accuracy of the relaxation-rate parameters are discussed. KAY LE, BULLTE, NICHOLSON LK, GRIESINGERC, SCHWALBEH, BAX A, TORCHIA DA: The M e a s u r e m e n t of t I e t e r o n u d e a r T r a n s v e r s e Relaxation T i m e s i n AX 3 S p i n S y s t e m s v i a P o l a r i z a t i o n T r a n s f e r T e c h n i q u e s . J Magn Resort 1993, 100:538-558. Various m e t h o d s of measuring s p i n - s p i n relaxation rate constants for methyl 13C spins are evaluated. Pulse sequences that compensate for the multiple-spin effects of the scalar coupling interaction and for dipolar cross-relaxation during polarization transfer sequences are presented. 23. •.
24.
PALMERAG, WRIGHT PE, RANCE M: M e a s u r e m e n t of Rel a x a t i o n T i m e Constants for Methyl Groups b y ProtonD e t e c t e d Iteteronuclear NMR Spectroscopy. Chem Phys Lett 1991, 185:41-46.
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27. ..
BARBATOG, IKURA M, KAy LE, PASTOR RW, BAX A: Backb o n e Dy!t~tmlc8 o f C a l m o d u l i n Studied b y 15N Relaxa t i o n u s i n g Inverse D e t e c t e d T w o - D i m e n s i o n a l NMR Spectroscopy: the Central H e l i x is Flexible. Biochemistry 1992, 31:5269-5278 The b a c k b o n e dynamics of Drosophila caLmodulin are determined using 15N relaxation measurements. Measurements support a flexible tether model, rather than a rigid model, for the structure of calmodulin in solution. 28.
SCHNEIDERDM, DELLWO MJ, WAND AJ: Fast I n t e r n a l MainC h a i n D y n a m i c s o f H u m a n U b i q u i t i n . Biochemistry 1992, 31:3645-3652.
The b a c k b o n e dynamics of ubiquitin are determined using 15N relaxation. A correlation is observed b e t w e e n the pattern of h y d r o g e n bonding in the molecule and the values of the 15N order paramaters. 29.
DELLWOMJ, WAND AJ: S y s t e m a t i c Bias i n t h e Model-Free A n a l y s i s o f Heteronuclear Relaxation. J Magn Resort 1991, 91:505-516.
30. ..
PENG JW, WAGNER G: Mapping o f the Spectral Densities o f N - H Bond Motions i n E g l i n c u s i n g I-Iete r o n u c l e a r R e l a x a t i o n E x p e r i m e n t s . Biochemistry 1992, 31:8571~8586. Values of t h e spectral density for N - H b o n d vectors in eglin c are determined b y analysis of an e x t e n d e d set of relaxation rate constants without u s e of model-free formalisms. Values of J(0) and J(m N) varied significantly with the amino acid sequence of the protein; other values of the spectral density w e r e independent of the sequence. The results generally agree with spectral densities calculated from a model-fi'ee analysis of the same relaxation data, although s o m e significant differences were observed. 31.
PENG JW, WAGNER G: Mapping Spectral D e n s i t y F u n c t i o n s u s i n g Heteronuclear NMR Relaxation Measuremerits. J Magn Reson 1992, 98:308-332. A strategy is outlined for determining the values of the spectral density function directly from relaxation data without utilizing the model-free formalism. Pulse s e q u e n c e s are presented for m e a s u r i n g the rate constants for relaxation o f 15N antiphase coherence, relaxation of two-spin order, cross-relaxation b e t w e e n 15N a n d 1H spins and longitudinal proton relaxation. 32: •.
N1CHOLSON LK, KAY LE, BALDISSERI DM, ARANGO J, YOUNG PE, TORCHIA DA: D y n a m i c s o f Methyl Groups i n Prot e i n s as Studied by P r o t o n Detected 13C NMR Spectro s c o p y . Application to t h e Leucine Residues o f Staphyl o c o c c a l Nuclease. Biochemistry 1992, 31:5253-5263. Internal d y n a m i c s of the leucine side chain methyl groups of staphylococcal n u c l e a s e are characterized using newly d e v e l o p e d pulse sequences optimized for m e a s u r e m e n t s of 13C spin relaxation in methyl groups. 33. •.
POWERSR, CLONE GM, STAHL SJ, WINGFIELD PT, GRONENBORN A: A n a l y s i s of the B a c k b o n e D y n a m i c s o f t h e Rib o n u c l e a s e H D o m a i n o f the H u m a n I m m u n o d e f i c i e n c y Virus Reverse Transcriptase u s i n g 15N Relaxation Measurements. Biochemistry 1992, 31:9150-9157. The b a c k b o n e dynamics of the ribonuclease H domain of the h u m a n immunodeficiency virus reverse transcriptase are determined using 15N relaxation measurements. 34.
REDFIELD C, BOYD J, SMITH LJ, SMITH RAG, DOBSON CM: L o o p M o b i l i t y i n a Four-Helix-Bundle Protein: 15N NMR Relaxation M e a s u r e m e n t s o n H u m a n I n t e r l e u k i n 4. Biochemistry 1992, 31:10431-10437. The b a c k b o n e dynamics of interleukin-4, w h i c h is a four helix b u n dle protein, are determined by 15N relaxation. Order parameters for the helices are - 0.9. Average-order parameters for the AB a n d CD loops are < 0.8; in contrast, order parameters for the BC loop are similar to order parameters for t h e helices. 35.
STONE MJ, FKIRBROTHER WJ, PALMER AG, REIZER J, SKIER M_H, WRIGHT PE: The B a c k b o n e D y n a m i c s o f t h e Bacillus s u b t i l t s Glucose P e r m e a s e RA D o m a i n D e t e r m i n e d f r o m 15N NMR Relaxation Measurements. Biochemistry 1992, 31:4394-4406. The b a c k b o n e dynamics of the IIA domain of the glucose p e r m e a s e of Bacillus subtilis are determined from 15N relaxation. Residues with higher mobility t h a n average are located in the a m i n o terminus, the loop from residues 25-41, and the region from residues 146-149. The loop from residues 25-41 is adjacent to the active site of the protein and contains highly conserved residues. 36. ..
AKKEM, KORDELJ, SKELTON NJ, PALMERAG, CHAZIN WJ: Elfects o f I o n Binding o n the B a c k b o n e D y n a m i c s i n C a l b i n d i n Dgk D e t e r m i n e d b y 15N NMR Relaxation. Biochemistry 1993, in press. Backbone 15N dynamics of apo calbindin Dgk , (Cd2+)l-Calbindin D9k and (Ca2+)2-calbindin Dgk are compared. Implications of the results for cooperativity of Ca 2+ binding by calbindin are discussed.
D y n a m i c properties of proteins from NMR s p e c t r o s c o p y Palmer 37.
PALMERAG, t-IOCHSTRASSERR, MILLARDP, RANCE M, WRIGHT PE: Characterization o f Ami~to Acid Side Chain Dynamics i n a Z i n c F i n g e r Peptide u s i n g 13C NMR Spectr-
o s c o p y and Time-Resolved Fluorescence Spectroscopy. J Am Chem Soc 1993, in press. The dynamics of the side-chain phenylalanine a n d tyrosine aromatic rings a n d of the alanine, leucine and valine methyl groups in a single zinc-finger peptide are determined by 13C relaxation. The results for the tyrosine ring are compared with order parameters determ i n e d from time-resolved fluorescence polarization anisotropy decay. Mobility differences for different side chains reflect the packing of hydrophobic side chains in the domain. 38.
STONE MJ, CHANDRASEKHARK, HOLMGREN A, WRIGHT PE, D Y S O NHJ: C o m p a r i s o n o f Backbone and T r y p t o p h a n S i d e - C h a i n D y n a m i c s o f Reduced and O x i d i z e d Esc h e r i c h i a coil T h i o r e d o x i n using 15N NM_R Relaxation Measurements. Biochemistry 1993, 32:426-435. Differences between the backbone and tryptophan side-chain dynamics of oxidized a n d reduced thioredoxin are determined by 15N relaxation and discussed. •.
39. •.
VAN MIERLO CPM, DARBY NJ, KEELER J, NEUHAUS D, CREIGHTONTE: P a r t i a l l y Folded Conformation o f the ( 3 0 - 5 1 ) I n t e r m e d i a t e i n the Disulphide Folding Pathw a y o f B o v i n e P a n c r e a t i c T r y p s i n I n h i b i t o r . J Mol Biol 1993, 229:1125-1146. Backbone dynamics of an analog of the folding intermediate o f bovine pancreatic trypsin inhibitor that contains only the disulfide b o n d b e t w e e n Cys30 and Cys51 are determined by 15N relaxation. 40.
41.
BERGLUNDH, KOVACS H, DAHLMAN--WRIGHTK, GUSTAFSSON J-A, HARD T: Backbone D y n a m i c s o f t h e Glucocortic o l d Receptor DNA-Binding Domain. Biochemistry 1992, 31:12001-12011. The backbone motions within the DNA-binding domain of the glucocorticoid receptor are investigated using 15N spin-relaxation measurements. Average-order parameters are similar for the different domains of the protein. The results suggest that the second zinc domain is not disordered in the u n c o m p l e x e d state of the protein. 46.
CONSTANTINEKL, FRIEDRICHS MS, GOLDFARB V, JEFFREY PD, SHERIFF S, MUELLERL: C h a r a c t e r i z a t i o n o f the Backbone D y n a m i c s o f a n A n t i - D i g o x i n A n t i b o d y V L Domain b y Inverse Detected 1H-15N NMR: Comparisons w i t h XR a y Data for the Fab. Proteins 1993, 15:290-311. The backbone dynamics of a recombinant anti-digoxin antibody V L d o m a i n are characterized by measurements of 15N spin relaxation and 1H--2H exchange rates. For several residues, chemical exchange contributions to the 15N s p i n - s p i n relaxation are attributed to motions of nearby aromatic rings. The results indicate enhanced flexibility in the turns, hypervariable loops, a n d portions of ]3-strands A, B a n d G. 47.
LEPRE CA, CHENG J-W,
Structure o f the Antibody Combining Site as Studied b y 1 H J 5 N Shift Correlation NMR Spectroscopy. Bio-
•.
Receptor-Bound Ligand by Heteronuclear NMR: FK506 Bound to FKBP-12. J A m Chem Soc 1993, 115:4929-4930.
chemistry 1992, 31:2464-2468.
13C relaxation m e a s u r e m e n t s are u s e d to characterize the dynamics of the i m m u n o s u p p r e s s a n t drug FK506 in complex with the protein FKBP-12. The results suggest that structural rigidity of particular functional groups m a y be required for activity of the drug.
TSANGP, RANCE M, FIESER TM, OSTRESHJM, HOUGHTEN I:{A, LERNER RA, WRIGHT PE: Conformation and Dynamics o f
CHANDRASEKHARI, CLORE GM, SZABO A, GRONENBORN A M , BROOKS BR: A 500 ps M o l e o d a r D y n a m i c s S i m u l a t i o n S t u d y o f I n t e r l e u k i n - l ~ i n Water: Correlation
w i t h Nuclear Magnetic Resonance Spectroscopy a n d C r y s t a l l o g r a p h y . J Mol Biol 1992, 226:239-250. A 500 p s molecular dynamics simulation of interteukin-1 ~ in water is performed. Order parameters a n d B factors calculated from the trajectory reproduce the values obtained from experimental investigations by NMR,spectroscopy and X-ray crystallography, respectively. Transitions of hydrogen b o n d e d N - H groups between well defined states observed in the trajectory m a y correspond to slow internal m o tions detected by NMR spectroscopy. 43.
K~3RDELJ, TELEMAN O: B a c k b o n e D y n a m i c s o f C a l b i n d i n D9k: Comparison o f M o l e c u l a r Dynamics Simulations a n d 15N NMR Relaxation Measurements. J A m Chem Soc 1992, 114:4934-4936. A series o f - 120 ps molecular dynamics simulations are u s e d to calculate order parameters for the N - H bond vectors in calbindin D9k. The simulations reproduce the general trends evident in order parameters determined from NMR spectroscopy. 44.
45.
TAKAHASHIH, SUZUKI E, SHIMADA I, ARATA Y: D y n a m i c a l
a n Fab-Bound Peptide b y Isotope-Edited NMR Spectroscopy. Biochemistry 1992, 31:3862-3871. 42. •.
Normal m o d e analysis, Langevin mode analysis a n d - 1 0 0 ps molecular dynamics simulations of a 25 residue zinc-finger peptide are performed. Order parameters a n d effective internal correlation times are calculated for the N-H, C - H and H - H b o n d vectors. Order parameters for C-H b o n d vectors calculated from solvated molecular dynamics simulations agree with experimental results from NMR spectroscopy at most carbon sites.
PALMERAG, CASE DA: M o l e c u l a r D y n a m i c s A n a l y s i s o f NMR R e l a x a t i o n i n a Z i n c - F i n g e r Peptide. J A m Chem Soc 1992, 114:9059-9067.
MOORE JiM: D y n a m i c s
of
a
Proton-detected
48.
GRASBERGERBL, GRONENBORN AM, CLORE GM: A n a l y s i s o f
the Backbone D y n a m i c s o f I n t e r l e u k i n - 8 b y 15N Relaxation M e a s u r e m e n t s . J Mol Biol 1993, 230:364-372. Backbone dynamics of interleukin-8 are determined using 15N relaxation measurements. Chemical exchange broadening contributions to s p i n - s p i n relaxation are suggested to arise from concerted motions of regions of secondary structu~:e in the protein. 49. •.
SZYPERSKIT, LUGINBUHLP, OTTING G, GCrNTERTP, WfLrrHRICH K: P r o t e i n D y n a m i c s Studied b y Rotating Frame 15N S p i n R e l a x a t i o n T i m e s . J Biomolec NMR 1993, 3:151-164. Conformational rate processes slower than overall rotational diffusion are investigated for bovine pancreatic inhibitor by measuring 15N rotating frame-relaxation rate constants as a function of spinlock power. A local rate process with a correlation time of approximately 1.3 ms is identified and attributed to isomerization o f the Cys1442ys38 disuifide bond. This work is the first application of field d e p e n d e n c e of Tlp to characterize conformational dynamics in proteins.
AG Palmer III, Department of Biochemistry a n d Molecular Biophysics, Columbia University, 630 West 168th Street, New York, New York 10032, USA.
391
Introduction Nuclear magnetic spin relaxation has long b e e n recognized as a source of information concerning the intramolecular dynamic properties of proteins in solution [1]. Historically, the low sensitivities and low natural abundances of 13C and 15N nuclei as well as the limited resolution of one-dimensional nuclear magnetic resonance (NMR) spectroscopy have impeded studies of spin relaxation in biological macromolecules. These constraints have b e e n alleviated, and investigations of spin relaxation in proteins invigorated, b y two-dimensional proton-detected heteronuclear NMR spectroscopy [2] and biosynthetic methods for isotopic enrichment of proteins [3,4]. Identification of functional consequences of the dynamic properties of proteins has b e c o m e feasible as the requisite spectroscopic techniques have matured. This review surveys two-dimensional proton-detected NMR techniques for measurements of 13C and 15N spin relaxation, applications of the techniques to determine intramolecular dynamic properties of multiple atomic sites in proteins and protein domains, and comParisons of results from NMR spectroscopy and molecular dynamics simulations. For brevity, studies of protein dynamics using h o m o n u c l e a r 1H NMR spectroscopy are not reviewed.
Experimental techniques In the m o s t c o m m o n experimental design [5-7], two-dimensional proton-detected heteronuclear NMR spectroscopy is used to measure spin-lattice relaxation rate constants (R1) , spin-spin relaxation rate constants (R2) and steady-state heteronuclear nuclear Overhauser effect (nOe) enhancements for the 13C or 15N nuclei in a protein. Physical parameters incorportating the amplitudes a n d timescales of the intramolecular motions of the protein are calculated from the relaxation data using the model-free formalism pioneered by Lipari and
Szabo [8,9]. For 15N relaxation studies, proteins can be uniformly enriched to > 90 % isotopic abundance. Unenriched, random fractionally enriched or specifically labeled proteins are required for 13C relaxation studies to minimize 13C-13C dipolar and scalar coupling interactions.
Pulse sequencesfor spin relaxation experiments Typical pulse sequences applicable to X nuclei (where X = 13C or 15N) with a single directly bonded proton, such as backbone amide groups or methine moieties, are illustrated in Fig. 1. The pulse sequences for R 1 (Fig. la) and R2 (Fig. lb) measurements comprise: an initial polarization transfer step (from the proton spin to the X spin); a relaxation period, T; an evolution period, h; a reverse polarization transfer step (from the X spin to the proton spin); and the acquisition period, t 2. The relaxation period in Fig. l(a) constitutes an inversion recovery experiment. The relaxation period in Fig. l(b) consists of a Carr-Purcell-Meiboom-Gill (CPMG) spinecho sequence. Peak intensities are measured from spectra recorded for different values of T, and firstorder rate constants are extracted from the resulting time series. For nOe measurements (Fig. lc), the initial polarization transfer is omitted and the duration of the relaxation period is fixed. The n O e is determined from the ratio of p e a k intensities in spectra recorded with and without saturation of the proton resonances during the relaxation period. The original experiments [5,10-13] have b e e n modified to minimize the effects of evolution under the heteronuclear scalar coupling Hamiltonian during CPMG experiments [14,15",16"] and cross-correlation b e t w e e n dipolar and chemical shift anisotropy interactions [15",16",17"].
A proton-detected Tip experiment that substitutes a continuous spin-locking radiofrequency field for the CPMG spin-echo sequence in the relaxation period of Fig. l ( b ) has b e e n presented as an alternative m e t h o d
Abbreviations CPMG--Carr-PurcelI-Meiboom-Gill; NMR--nuclear magnetic resonance; nOe--nuclear Overhauser effect; Rl--spin-lattice relaxation rate constant; R2--spin-spin relaxation rate constant; S2--square of the generalized order parameter.
© Current Biology Ltd 0958-1669
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Fig. 1. Pulse schemes used to measure (a) R1, (b) R2 and (c) nOe with indirect proton detection. Thin and thick bars represent 90 ° and 180 ° radiofrequency pulses, respectively. Pulse phases are indicated above the bars; 180 ° pulses without phase designations are applied with y phase. The basic phase cycling is as follows: 01 = (x, - x , x, -x), 02 = (Y, Y, -Y, -Y) and receiver (x, - x , - x , x). Pulses labeled SL are high-power spin-lock purge pulses used for water suppression. Decoupling during the relaxation period of (a) and during the initial delay of (c) can be performed using trains of high-power pulses or broadband decoupling sequences. Broadband decoupling sequences are applied during acquisition. The value of "~ is set to 1/4JxH, (where JXH is the one-bond heteronuclear scalar coupling constant). The value of 8 is set to - 0.5 ms. For (a) and (b), the delay, T, is varied parametrically in a series of two-dimensional experiments; for (c), pairs of spectra are acquired with and without proton saturation.
for measuring R 2 [14,18]. The d e p e n d e n c e o f Tlp on the amplitude of the spin-locking field potentially can provide information on dynamic processes slower than overall rotational diffusion [19]. Broadband proton decoupling during a constant-time evolution period has b e e n utilized to improve water suppression in pulse sequences for measuring the n O e [20"]. In addition, the sensitivities of the experiments h a v e been increased b y using pulse sequences that transfer orthogonal magnetization c o m p o n e n t s from X nuclei to protons [21", 22"]. The pulse sequences shown in Fig. 1 are not applicable to nuclei with m o r e than one directly attached proton, such as 13C spins in methylene a n d methyl groups [11]. Modified pulse sequences have b e e n developed for measurements of methyl 13C spin relaxation [23",24]. Proton-detected pulse sequences for measurements of methylene 13C spin relaxation h a v e not b e e n reported, as yet.
characteristic frequencies: J(0), J(~)tt-- COx),J(~x), J(ml-I), and J(mH+mX), in which mH and COx are the Larmor frequencies of the 1H and X nuclei, respectively [25]. The spectral density is determined b y the overall rotational diffusion of the protein and by the intramolecular m o tions of the X - H b o n d vector that occur on timescales faster than the overall rotational diffusion. The spectral density does not contain information on internal m o tions slower than rotational diffusion; however, systematic increases in R 2 from exchange broadening provide qualitative evidence of motions on slower time scales [5,6]. In the model-free formalism [8,9], J(m) is a p p r o x imated as:
J(~)=(2/5)[s2"g m/(1 +~2"cm2)+(1- S2)'~/(1+~2"~2)]
(1)
Interpretation of spin-relaxation parameters
w h e r e ~m is the isotropic overall rotational correlation
The relaxation parameters, R1, R 2 and nOe are functions of the values of the spectral density, J(m), at five
time of the protein, S2 is the square of the generalized order parameter, "1:e is the effective internal correlation
Dynamic properties of proteins from NMR spectroscopy Palmer 387 time for internal motions and "c=(1/'Cm+l/'Q)- 1. Equation (1) has b e e n extended to include internal motions on two resolvable timescales [26] and anisotropic overall rotational diffusion [8,27.,,28.]. The accuracy of the motional parameters calculated from the relaxation data using the model-free formalism has b e e n discussed [8,9,26,29]. The squares of the order parameters measure the motional restriction of a X-H b o n d vector in a molecular reference frame and range from unity for vectors with fixed orientations to zero for vectors with isotropic orientational distributions. For example, if motion of a X - H bond vector is modeled as restricted diffusion in a cone, then S2 is given by:
S2 = [cos00(1 + cos00) / 2]2
(2)
and the amplitude of motion is characterized b y the cone semiangle, 00 [8]. For simplicity, S2 will b e referred to as the order parameter in the following discussion. An alternative strategy for determining the five characteristic values of J(c0) directly from relaxation data without recourse to the model-free formalism has b e e n described [30",31"]. In this approach, R1 and R 2 a r e measured as described above, and rate constants for relaxation of heteronuclear antiphase coherence, relaxation of two-spin longitudinal order and cross-relaxation b e t w e e n X and proton spins are measured using recently developed pulse sequences (in practice, the proton spin-lattice relaxation rate constant must also b e measured) [31"]. The values of J(c0) at the characteristic frequencies are determined from the relaxation data by inversion of a linear system of equations. Values of the spectral density functions for N - H b o n d vectors in eglin c determined by this approach are similar--to values calculated from model-free parameters using Equation (1); although, some significant differences have b e e n observed [30"'].
Applications to proteins In the period from 1989 to 1991, the model-free dynamic parameters of three proteins and protein domains were determined using two-dimensional protondetected NMR spectroscopy [5-7]. At least twelve experimental studies have b e e n reported since the beginning of 1992 [21",27"',28",30"',31",32"',33"',34",35",36"', 37",38"',39"]. In addition, 15N R2 values have b e e n reported for an antibody Fv fragment [40] and for a peptide complexed with an antibody Fab fragment [41].
Characteristic dynamic properties of proteins A remarkably consistent picture is obtained of the b a c k b o n e dynamics of folded proteins ranging in size from a single zinc-finger peptide (25 residues) [7] to the glucose p e r m e a s e IIA domain (162 residues) [35"]. As an example, b a c k b o n e 15N order parameters for
calbindin D 9 k , a small helical protein that binds two calcium ions, are s h o w n in Fig. 2. Relaxation parameters for 13C or 15N spins in regular (x-helices or ~sheets are described accurately by Equation (1) with S2 - 0 . 8 - 0 . 9 and '~e < 50ps; using Equation (2), the cone semiangle for the X - H b o n d vector ranges from - 15-22 degrees. Thus, mobility within elements of regular secondary structure is highly restricted. Lower order parameters and longer effective correlation times are observed frequently for 13C or 15N spins near the amino and carboxyl termini, in loops and in irregular secondary structures. Additionally, internal motions on m o r e than one timescale may contribute significantly to spin relaxation of these nuclei [6,8,9,26]. Regions of proteins that have low-order parameters or large exchange broadening contributions to R 2 a r e often correlated, albeit imperfectly, with regions that have high amide proton exchange rates, are poorly defined in NMR solution structures or have large crystallographic B-factors. Dynamic parameters have b e e n reported for the valine ]3, leucine y, aromatic and methyl 13C spins in a zincfinger peptide [7,37"], the leucine 8 methyl 13C spins in staphylococcal nuclease [32"'] and the tryptophan indole 15N spins in thioredoxin [38"']. Order parameters for different side-chain b o n d vectors are highly variable and indicate the importance of local interactions in restricting the mobility of amino acid side chains.
Dynamic aspects of protein function Very recent studies have correlated biological function with the intramolecular dynamics of proteins; however, establishment of causality remains an important objective. The following section summarizes some of the m o s t significant findings obtained in the past year from two-dimensional heteronuclear NMR spectroscopy. Barbato et al. [27"q have determined the b a c k b o n e dynamics of Drosophila calmodulin using 15N relaxation measurements. The rotational correlation times are found to be inconsistent with predictions from hydrodynamic calculations for rigid models of calmodulin, and the central helix in calmodulin is s h o w n to have a high degree of mobility, with order parameters -0.5-0.6. The results are interpreted in support of a model for the structure of the protein in solution in which a flexible central helix facilitates binding to target proteins. In another report, the internal dynamics of the leucine side chain methyl groups of staphylococcal nuclease h a v e been characterized b y 13C spin relaxation using newly developed pulse sequences for accurate measurements of 13C spin relaxation in methyl groups [32"']. The amplitudes of the internal motions of the leucine side chains are found to be larger than suggested by X-ray crystallographic studies or nOeSY spectra. The order parameters increase for methyl groups in regions of the protein near the ligand-binding sites u p o n complexing with Ca 2+ and thymidine 3',5'-bisphosphate. These results suggest that ligation increases the local rigidity of the protein.
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Protein engineering
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Fig. 2. Generalized order parameters for the backbone amide nitrogens of calcium-loaded calbindin D9k. The order parameters were calculated from relaxation rate constants and nOes measured at a magnetic field strength of 11.74 T [21"]. The protein contains two EF-hand domains. Each domain contains an ion-binding site flanked by two cuhelices. Residues 36-45 form a loop between the two domains. A representative error bar is shown for residue 2.
Powers et al. [33"'] have reported the backbone dynamics of ribonuclease H domain of the human immunodeficiency virus reverse transcriptase determined using 15N relaxation measurements. Ribonuclease H displays extensive and complex internal dynamics; nearly 90 % of the N-H bond vectors exhibit motions o n more than one timescale. Low-order parameters are observed for the carboxyl terminus, the loop between p-strands [~1 and ~2, and the loop b e t w e e n helix cxB and p-strand ~4. The high mobility of the carboxy-terminal region of the protein may contribute to the reduced enzymatic activity of the isolated ribonuclease H domain. Backbone 15N dynamics of apo calbindin D9k , (Cd2+)1 -calbindin D9k and (Ca2+)2-calbindin D9k have also been compared [21",36"']. Binding of Cd 2+ or Ca 2+ to the carboxy-terminal loop significantly increases the rigidity of the loop; smaller changes in order parameters are observed for helix III and the carboxyl terminus. The observed changes in the dynamic properties of the protein u p o n ion binding may contribute entropically to cooperativity. The backbone and tryptophan side-chain dynamics of oxidized and reduced thioredoxin have b e e n determined by 15N relaxation [38"q. Internal motions on picosecond to nanosecond timescales are similar for both forms of the protein; however, reduction of the active-site disulfide bridge increases the dynamic mo-
bility of residues 73-75 o n microsecond t o millisecond timescales. Increased mobility of the active site in the reduced form of the protein may facilitate binding to other proteins. Van Mierlo et al. [39"q have used 15N relaxation to determine the backbone dynamics of an analog of the folding intermediate of bovine pancreatic trypsin inhibitor that contains only the disulfide b o n d between Cys30 and Cys51. The amino-terminal 15 residues and the residues from 37-41 are very flexible with order parameters < 0.5; order parameters for the hydrophobic cole of the molecule are - 0 . 7 . The results suggest that formation of non-native disulfide bridges during folding of the protein is a consequence of the flexibility of the regions of the protein containing Cys5, Cys14 and Cys38. Comparisons
with theoretical
results
Order parameters calculated from molecular dynamics simulations have been compared with order parameters measured by two-dimensional proton-detected NMR spectroscopy for interleukin-l~ [42"-], calbindin D9k [43"] and a zinc-finger peptide [44-]. Order parameters have also been calculated from normal mode and Langevin mode analyses of the zinc-finger peptide. Theoretical calculations reproduce the general features
Dynamic properties of proteinsfrom NMR spectroscopyPalmer 389 of the experimental results; however, internal motions for some b o n d vectors are sampled inadequately during the simulations. The molecular basis for the fast and slow dynamic processes observed experimentally for interleukin 1~ can be inferred from the molecular dynamics simulation.
Nuclear Magnetic R e s o n a n c e Spectra of P r o t e i n s . Q Rev Biophys 1990, 23:1-38. 5.
KAY LE, TORCHIA DA, BAX A: B a c k b o n e D y n a m i c s o f Prot e i n s as Studied b y 15Nitrogen Inverse Detected Hete r o n u c l e a r NMR Spectroscopy: Application to S t a p h y l o c o c c a l N u d e a s e . Biochemistry 1989, 28:8972~97%
6.
CLORE GM, DRISCOLL PC, WINGFIELD PT, GRONENBORN AM: A n a l y s i s o f the B a c k b o n e D y n a m i c s o f I n t e r l e u k i n - l ~ using T w o - D i m e n s i o n a l Inverse Detected Heteronuclear 15N-1H NMR S p e c t r o s c o p y . Biochemistry 1990, 29:7387-7401.
7.
PALMERAG, RANCE M, WRIGHT PE: I n t r a m o l e c t d a r Mot i o n s of a Z i n c F i n g e r D N A - B i n d i n g D o m a i n f r o m X f i n Characterized by Proton-Detected Natural Abundance 13C H e t e r o n u c l e a r NMR S p e c t r o s c o p y . J Am Chem Soc 1991, 113:4371-4380.
8.
LIPARI G, SZABO A: Model-Free A p p r o a c h to the Interp r e t a t i o n of N u c l e a r M a g n e t i c R e s o n a n c e R e l a x a t i o n i n M a c r o m o l e c u l e s . I. T h e o r y and Range o f Validity. J Am Chem Soc 1982, 104:4546-4559.
9.
LIPAm G, SZABO A: M o d e l - F r e e A p p r o a c h to the Interpretation of Nuclear Magnetic R e s o n a n c e Relaxation i n M a c r o m o l e c u l e s . II. A n a l y s i s o f E x p e r i m e n t a l Restilts. J Am Chem Soc 1982, 104:4559-4570.
10.
KAY LE, JUE TL, BANGERTER B, DEMOU PC: S e n s i t i v i t y Enh a n c e m e n t o f 13C T1 M e a s u r e m e n t s v i a P o l a r i z a t i o n Transfer. J Magn Resort 1987, 73:558-564.
11.
SKLENARV, TORCHIA D, BAX A: Measurement o f C a r b o n -
Conclusion Recent studies of spin relaxation by two-dimensional proton-detected NMR spectroscopy provide detailed characterizations, and are beginning to elucidate the biological consequences, of the dynamic behavior of proteins. The characterization of side-chain dynamics, the investigation of motional processes slower than overall rotational diffusion, the assessment of the effects of mutations and the comparison of proteins in different functional states will all be the focus of experimental studies of spin relaxation in proteins over the next few years. The results of such investigations can be expected to illuminate the critical relationships among biological activities, structures and the dynamics of proteins.
Note added in proof Proton-detected heteronuclear spin-relaxation measurements recently have b e e n applied to additional protein systems of particular biological interest, including a DNA-binding domain [45"], an antibody fragment [46"] and a receptor-ligand complex [47"']. In addition, novel interpretations of chemical exchange broadening contributions to spin-spin relaxation have been proposed [46",48"]. In a significant advance, Szyperski et al. [49"'] have recently used the d e p e n d e n c e of Tip on the strength of the spin-locking field to characterize millisecond conformational rate processes in bovine pancreatic trypsin inhibitor.
Acknowledgements I thank Drs Nuria Assa-Munt a n d Richard A Friedman for helpful discussions.
References and recommended reading Papers of particular interest, published within the annual period of review, have b e e n highlighted as: of special interest •. of outstanding interest 1.
LONDONRE: I n t r a m o l e c u l a r D y n a m i c s o f P r o t e i n s and Peptides as Monitored b y Nuclear Magnetic R e l a x a t i o n Measurements. In Magnetic Resonance in Biology, vol 1. Edited by Cohen JS. New York: Wiley; 1980:1-69.
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BAX A, SPARKS SW, TORCHIA DA: Detection o f I n s e n s i t i v e Nuclei. Methods Enzymol 1989, 176:134-150.
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BOLTONPH: A P r i m e r o n Isotopic L a b e l i n g i n NMR Investigations o f B i o p o l y m e r s . Prog NMR Spectroscopy 1990, 22:423-452.
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MCINTOSHLP, DAHLQUISTFW: Biosynthetic I n c o r p o r a t i o n of 15N a n d 13C for A s s i g n m e n t and Interpretation o f
13 L o n g i t u d i n a l R e l a x a t i o n u s i n g 1H D e t e c t i o n . J Magn Reson 1987, 73:375-379. 12.
NIRMAIANR, WAGNER G: Measurement o f 13C R e l a x a t i o n T i m e s i n P r o t e i n s b y T w o - D i m e n s i o n a l Heteronuclear 1H-13C Correlation S p e c t r o s c o p y . J Am Chem Soc 1988, 110:7557-7558.
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NIRMALANR, WAGNER G: Measurement o f 13C S p i n - S p i n R e l a x a t i o n T i m e s b y T w o - D i m e n s i o n a l Heteronuclear 1H-13C Correlation S p e c t r o s c o p y . J Magn Reson 1989, 82:659-661.
14.
PENGJW, THANABALV, WAGNER G: I m p r o v e d A c c u r a c y of Heteronuclear Transverse R e l a x a t i o n T i m e M e a s u r e m e n t s in Macromolecules: El|mlqation o f A n t i p h a s e Contributions. J Magn Reson 1991, 95:421-427.
15.
KAY LE, NICHOLSON LK, DELAGIO F, BAX A, TORCHIA DA: P u l s e Sequences for Removal o f t h e Effects o f Cross Correlation b e t w e e n Dipolar and C h e m i c a l - S h i f t Anisotropy Relaxation Mechanisms on the Measurem e n t o f Heteronuclear T 1 and T 2 V a l u e s i n P r o t e i n s . J Magn Reson 1992, 97:359-375. See [16"]. 16.
PALMERACT, SKELTON NJ, CHAZIN WJ, WRIGHT PE, RANCE M: S u p p r e s s i o n of t h e Effects o f Cross-Correlation bet w e e n Dipolar and A n i s o t r o p i c C h e m i c a l Shift Relaxa t i o n M e c h a n i s m s in t h e Measurement o f S p i n - S p i n R e l a x a t i o n Rates. Molec Phys 1992, 75:699-711. This paper and [15"], discuss effects of the heteronuclear scalar coupling interaction and cross-correlation between dipolar and chemical shift anisotropy relaxation m e c h a n i s m s on the m e a s u r e m e n t of s p i n - s p i n relaxation rate constants using CPMG sequences. Methods are presented that allow accurate m e a s u r e m e n t s to be performed. 17.
BOYDJ, HOMMELU, CAMPBELLID: Influence o f C r o s s - C o r r e l a t i o n b e t w e e n D i p o l a r and A n i s o t r o p i c C h e m i c a l Shift R e l a x a t i o n M e c h a n i s m s u p o n L o n g i t u d i n a l Relaxation Rates o f 15N i n M a c r o m o l e c u l e s . Chem Phys Lett 1990, 175:477-482. T h e effects of cross-correlation b e t w e e n dipolar a n d chemical shift anisotropy relaxation m e c h a n i s m s on the m e a s u r e m e n t of spin-lattice relaxation rate constants are discussed. Methods are pres e n t e d that allow accurate m e a s u r e m e n t s to be performed.
390
Protein engineering 18.
PENGy ~ , THANABALV, WAGNER G: 2D H e t e r o n u d e a r NMR M e a s u r e m e n t s of S p i n - L a t t i c e R e l a x a t i o n T i m e s i n the Rotating Frame of X N u c l e i in H e t e r o n u d e a r ItX S p i n Systems. J Magn Reson 1991, 94:82-100.
19.
DEVEtLELLC, MORGANRE, STRANGEJH: Studies o f C h e m i c a l E x c h a n g e b y N u c l e a r M a g n e t i z a t i o n R e l a x a t i o n i n the Rotating F r a m e . Mol Phys 1970, 18:553-559.
20.
NEUHAUS D, VAN MIERLO CPM: Measurement of Hete r o n u c l e a r hOe E n h a n c e m e n t s in Biological Macrom o l e c u l e s . A C o n v e n i e n t P u l s e Sequence for Use w i t h A q u e o u s Solutions. J Magn Reson 1992, 100:221-228. The heteronuclear h O e is determined from tile peak intensities in spectra acquired either with or without saturation of the proton spins. Efficient water suppression can b e difficult to achieve in the control spectrum acquired without proton saturation. Water suppression can be improved if broadband proton decoupling is applied during the evolution period of a constant time heterocorrelation experiment. KORDELJ, SKELTON NJ, AKKE M, PALMER AG, CHAZ1N WJ: B a c k b o n e D y n a m i c s o f C a l c i u m - L o a d e d C a l b i n d i n D9k Studied b y Two-Di~aensional Proton-Detected NMR S p e c t r o s c o p y . Biochemistry 1992, 31:4856-4866. Backbone dynamics of (Ca2+)2-calbindin D9k are determined by 15N relaxation using modified pulse s e q u e n c e s that improve the sensitivity of t h e NMR experiments by detecting orthogonal magnetization components. Low-order parameters are observed for the linker loop b e t w e e n the two domains of the protein. Average-order parameters for the four helices of the protein are indistinguishable; however, effective internal correlation times are significantly different. 21.
22.
SKELTON NJ, PALMER AG, AKKE M, KORDEL J, RANCE M, CHAZIN WJ: P r a c t i c a l A s p e c t s of T w o - D i m e n s i o n a l Proton-Detected 15N S p i n Relaxation Measurements. J Magn Reson 1993, in press. Optimized pulse sequences for m e a s u r i n g 15N spin relaxation using two-dimensional proton-detected NMR spectroscopy are presented. Factors affecting the precision a n d accuracy of the relaxation-rate parameters are discussed. KAY LE, BULLTE, NICHOLSON LK, GRIESINGERC, SCHWALBEH, BAX A, TORCHIA DA: The M e a s u r e m e n t of t I e t e r o n u d e a r T r a n s v e r s e Relaxation T i m e s i n AX 3 S p i n S y s t e m s v i a P o l a r i z a t i o n T r a n s f e r T e c h n i q u e s . J Magn Resort 1993, 100:538-558. Various m e t h o d s of measuring s p i n - s p i n relaxation rate constants for methyl 13C spins are evaluated. Pulse sequences that compensate for the multiple-spin effects of the scalar coupling interaction and for dipolar cross-relaxation during polarization transfer sequences are presented. 23. •.
24.
PALMERAG, WRIGHT PE, RANCE M: M e a s u r e m e n t of Rel a x a t i o n T i m e Constants for Methyl Groups b y ProtonD e t e c t e d Iteteronuclear NMR Spectroscopy. Chem Phys Lett 1991, 185:41-46.
25.
ABRAGAM A: Principles of Nuclear Magnetism. Oxford: Clarendon Press; 1961.
26.
CLONE GM, SZABO A, BAX A, KAY LE, DRISCOLL PC, GRONENBOKN AMi D e v i a t i o n s f r o m the Simple Two-Par a m e t e r Model-Free A p p r o a c h to the Interpretation o f N i t r o g e n - 1 5 N u c l e a r M a g n e t i c Relaxation o f Proteins. J Am Chem Soc 1990, 112:4989-4991.
27. ..
BARBATOG, IKURA M, KAy LE, PASTOR RW, BAX A: Backb o n e Dy!t~tmlc8 o f C a l m o d u l i n Studied b y 15N Relaxa t i o n u s i n g Inverse D e t e c t e d T w o - D i m e n s i o n a l NMR Spectroscopy: the Central H e l i x is Flexible. Biochemistry 1992, 31:5269-5278 The b a c k b o n e dynamics of Drosophila caLmodulin are determined using 15N relaxation measurements. Measurements support a flexible tether model, rather than a rigid model, for the structure of calmodulin in solution. 28.
SCHNEIDERDM, DELLWO MJ, WAND AJ: Fast I n t e r n a l MainC h a i n D y n a m i c s o f H u m a n U b i q u i t i n . Biochemistry 1992, 31:3645-3652.
The b a c k b o n e dynamics of ubiquitin are determined using 15N relaxation. A correlation is observed b e t w e e n the pattern of h y d r o g e n bonding in the molecule and the values of the 15N order paramaters. 29.
DELLWOMJ, WAND AJ: S y s t e m a t i c Bias i n t h e Model-Free A n a l y s i s o f Heteronuclear Relaxation. J Magn Resort 1991, 91:505-516.
30. ..
PENG JW, WAGNER G: Mapping o f the Spectral Densities o f N - H Bond Motions i n E g l i n c u s i n g I-Iete r o n u c l e a r R e l a x a t i o n E x p e r i m e n t s . Biochemistry 1992, 31:8571~8586. Values of t h e spectral density for N - H b o n d vectors in eglin c are determined b y analysis of an e x t e n d e d set of relaxation rate constants without u s e of model-free formalisms. Values of J(0) and J(m N) varied significantly with the amino acid sequence of the protein; other values of the spectral density w e r e independent of the sequence. The results generally agree with spectral densities calculated from a model-fi'ee analysis of the same relaxation data, although s o m e significant differences were observed. 31.
PENG JW, WAGNER G: Mapping Spectral D e n s i t y F u n c t i o n s u s i n g Heteronuclear NMR Relaxation Measuremerits. J Magn Reson 1992, 98:308-332. A strategy is outlined for determining the values of the spectral density function directly from relaxation data without utilizing the model-free formalism. Pulse s e q u e n c e s are presented for m e a s u r i n g the rate constants for relaxation o f 15N antiphase coherence, relaxation of two-spin order, cross-relaxation b e t w e e n 15N a n d 1H spins and longitudinal proton relaxation. 32: •.
N1CHOLSON LK, KAY LE, BALDISSERI DM, ARANGO J, YOUNG PE, TORCHIA DA: D y n a m i c s o f Methyl Groups i n Prot e i n s as Studied by P r o t o n Detected 13C NMR Spectro s c o p y . Application to t h e Leucine Residues o f Staphyl o c o c c a l Nuclease. Biochemistry 1992, 31:5253-5263. Internal d y n a m i c s of the leucine side chain methyl groups of staphylococcal n u c l e a s e are characterized using newly d e v e l o p e d pulse sequences optimized for m e a s u r e m e n t s of 13C spin relaxation in methyl groups. 33. •.
POWERSR, CLONE GM, STAHL SJ, WINGFIELD PT, GRONENBORN A: A n a l y s i s of the B a c k b o n e D y n a m i c s o f t h e Rib o n u c l e a s e H D o m a i n o f the H u m a n I m m u n o d e f i c i e n c y Virus Reverse Transcriptase u s i n g 15N Relaxation Measurements. Biochemistry 1992, 31:9150-9157. The b a c k b o n e dynamics of the ribonuclease H domain of the h u m a n immunodeficiency virus reverse transcriptase are determined using 15N relaxation measurements. 34.
REDFIELD C, BOYD J, SMITH LJ, SMITH RAG, DOBSON CM: L o o p M o b i l i t y i n a Four-Helix-Bundle Protein: 15N NMR Relaxation M e a s u r e m e n t s o n H u m a n I n t e r l e u k i n 4. Biochemistry 1992, 31:10431-10437. The b a c k b o n e dynamics of interleukin-4, w h i c h is a four helix b u n dle protein, are determined by 15N relaxation. Order parameters for the helices are - 0.9. Average-order parameters for the AB a n d CD loops are < 0.8; in contrast, order parameters for the BC loop are similar to order parameters for t h e helices. 35.
STONE MJ, FKIRBROTHER WJ, PALMER AG, REIZER J, SKIER M_H, WRIGHT PE: The B a c k b o n e D y n a m i c s o f t h e Bacillus s u b t i l t s Glucose P e r m e a s e RA D o m a i n D e t e r m i n e d f r o m 15N NMR Relaxation Measurements. Biochemistry 1992, 31:4394-4406. The b a c k b o n e dynamics of the IIA domain of the glucose p e r m e a s e of Bacillus subtilis are determined from 15N relaxation. Residues with higher mobility t h a n average are located in the a m i n o terminus, the loop from residues 25-41, and the region from residues 146-149. The loop from residues 25-41 is adjacent to the active site of the protein and contains highly conserved residues. 36. ..
AKKEM, KORDELJ, SKELTON NJ, PALMERAG, CHAZIN WJ: Elfects o f I o n Binding o n the B a c k b o n e D y n a m i c s i n C a l b i n d i n Dgk D e t e r m i n e d b y 15N NMR Relaxation. Biochemistry 1993, in press. Backbone 15N dynamics of apo calbindin Dgk , (Cd2+)l-Calbindin D9k and (Ca2+)2-calbindin Dgk are compared. Implications of the results for cooperativity of Ca 2+ binding by calbindin are discussed.
D y n a m i c properties of proteins from NMR s p e c t r o s c o p y Palmer 37.
PALMERAG, t-IOCHSTRASSERR, MILLARDP, RANCE M, WRIGHT PE: Characterization o f Ami~to Acid Side Chain Dynamics i n a Z i n c F i n g e r Peptide u s i n g 13C NMR Spectr-
o s c o p y and Time-Resolved Fluorescence Spectroscopy. J Am Chem Soc 1993, in press. The dynamics of the side-chain phenylalanine a n d tyrosine aromatic rings a n d of the alanine, leucine and valine methyl groups in a single zinc-finger peptide are determined by 13C relaxation. The results for the tyrosine ring are compared with order parameters determ i n e d from time-resolved fluorescence polarization anisotropy decay. Mobility differences for different side chains reflect the packing of hydrophobic side chains in the domain. 38.
STONE MJ, CHANDRASEKHARK, HOLMGREN A, WRIGHT PE, D Y S O NHJ: C o m p a r i s o n o f Backbone and T r y p t o p h a n S i d e - C h a i n D y n a m i c s o f Reduced and O x i d i z e d Esc h e r i c h i a coil T h i o r e d o x i n using 15N NM_R Relaxation Measurements. Biochemistry 1993, 32:426-435. Differences between the backbone and tryptophan side-chain dynamics of oxidized a n d reduced thioredoxin are determined by 15N relaxation and discussed. •.
39. •.
VAN MIERLO CPM, DARBY NJ, KEELER J, NEUHAUS D, CREIGHTONTE: P a r t i a l l y Folded Conformation o f the ( 3 0 - 5 1 ) I n t e r m e d i a t e i n the Disulphide Folding Pathw a y o f B o v i n e P a n c r e a t i c T r y p s i n I n h i b i t o r . J Mol Biol 1993, 229:1125-1146. Backbone dynamics of an analog of the folding intermediate o f bovine pancreatic trypsin inhibitor that contains only the disulfide b o n d b e t w e e n Cys30 and Cys51 are determined by 15N relaxation. 40.
41.
BERGLUNDH, KOVACS H, DAHLMAN--WRIGHTK, GUSTAFSSON J-A, HARD T: Backbone D y n a m i c s o f t h e Glucocortic o l d Receptor DNA-Binding Domain. Biochemistry 1992, 31:12001-12011. The backbone motions within the DNA-binding domain of the glucocorticoid receptor are investigated using 15N spin-relaxation measurements. Average-order parameters are similar for the different domains of the protein. The results suggest that the second zinc domain is not disordered in the u n c o m p l e x e d state of the protein. 46.
CONSTANTINEKL, FRIEDRICHS MS, GOLDFARB V, JEFFREY PD, SHERIFF S, MUELLERL: C h a r a c t e r i z a t i o n o f the Backbone D y n a m i c s o f a n A n t i - D i g o x i n A n t i b o d y V L Domain b y Inverse Detected 1H-15N NMR: Comparisons w i t h XR a y Data for the Fab. Proteins 1993, 15:290-311. The backbone dynamics of a recombinant anti-digoxin antibody V L d o m a i n are characterized by measurements of 15N spin relaxation and 1H--2H exchange rates. For several residues, chemical exchange contributions to the 15N s p i n - s p i n relaxation are attributed to motions of nearby aromatic rings. The results indicate enhanced flexibility in the turns, hypervariable loops, a n d portions of ]3-strands A, B a n d G. 47.
LEPRE CA, CHENG J-W,
Structure o f the Antibody Combining Site as Studied b y 1 H J 5 N Shift Correlation NMR Spectroscopy. Bio-
•.
Receptor-Bound Ligand by Heteronuclear NMR: FK506 Bound to FKBP-12. J A m Chem Soc 1993, 115:4929-4930.
chemistry 1992, 31:2464-2468.
13C relaxation m e a s u r e m e n t s are u s e d to characterize the dynamics of the i m m u n o s u p p r e s s a n t drug FK506 in complex with the protein FKBP-12. The results suggest that structural rigidity of particular functional groups m a y be required for activity of the drug.
TSANGP, RANCE M, FIESER TM, OSTRESHJM, HOUGHTEN I:{A, LERNER RA, WRIGHT PE: Conformation and Dynamics o f
CHANDRASEKHARI, CLORE GM, SZABO A, GRONENBORN A M , BROOKS BR: A 500 ps M o l e o d a r D y n a m i c s S i m u l a t i o n S t u d y o f I n t e r l e u k i n - l ~ i n Water: Correlation
w i t h Nuclear Magnetic Resonance Spectroscopy a n d C r y s t a l l o g r a p h y . J Mol Biol 1992, 226:239-250. A 500 p s molecular dynamics simulation of interteukin-1 ~ in water is performed. Order parameters a n d B factors calculated from the trajectory reproduce the values obtained from experimental investigations by NMR,spectroscopy and X-ray crystallography, respectively. Transitions of hydrogen b o n d e d N - H groups between well defined states observed in the trajectory m a y correspond to slow internal m o tions detected by NMR spectroscopy. 43.
K~3RDELJ, TELEMAN O: B a c k b o n e D y n a m i c s o f C a l b i n d i n D9k: Comparison o f M o l e c u l a r Dynamics Simulations a n d 15N NMR Relaxation Measurements. J A m Chem Soc 1992, 114:4934-4936. A series o f - 120 ps molecular dynamics simulations are u s e d to calculate order parameters for the N - H bond vectors in calbindin D9k. The simulations reproduce the general trends evident in order parameters determined from NMR spectroscopy. 44.
45.
TAKAHASHIH, SUZUKI E, SHIMADA I, ARATA Y: D y n a m i c a l
a n Fab-Bound Peptide b y Isotope-Edited NMR Spectroscopy. Biochemistry 1992, 31:3862-3871. 42. •.
Normal m o d e analysis, Langevin mode analysis a n d - 1 0 0 ps molecular dynamics simulations of a 25 residue zinc-finger peptide are performed. Order parameters a n d effective internal correlation times are calculated for the N-H, C - H and H - H b o n d vectors. Order parameters for C-H b o n d vectors calculated from solvated molecular dynamics simulations agree with experimental results from NMR spectroscopy at most carbon sites.
PALMERAG, CASE DA: M o l e c u l a r D y n a m i c s A n a l y s i s o f NMR R e l a x a t i o n i n a Z i n c - F i n g e r Peptide. J A m Chem Soc 1992, 114:9059-9067.
MOORE JiM: D y n a m i c s
of
a
Proton-detected
48.
GRASBERGERBL, GRONENBORN AM, CLORE GM: A n a l y s i s o f
the Backbone D y n a m i c s o f I n t e r l e u k i n - 8 b y 15N Relaxation M e a s u r e m e n t s . J Mol Biol 1993, 230:364-372. Backbone dynamics of interleukin-8 are determined using 15N relaxation measurements. Chemical exchange broadening contributions to s p i n - s p i n relaxation are suggested to arise from concerted motions of regions of secondary structu~:e in the protein. 49. •.
SZYPERSKIT, LUGINBUHLP, OTTING G, GCrNTERTP, WfLrrHRICH K: P r o t e i n D y n a m i c s Studied b y Rotating Frame 15N S p i n R e l a x a t i o n T i m e s . J Biomolec NMR 1993, 3:151-164. Conformational rate processes slower than overall rotational diffusion are investigated for bovine pancreatic inhibitor by measuring 15N rotating frame-relaxation rate constants as a function of spinlock power. A local rate process with a correlation time of approximately 1.3 ms is identified and attributed to isomerization o f the Cys1442ys38 disuifide bond. This work is the first application of field d e p e n d e n c e of Tlp to characterize conformational dynamics in proteins.
AG Palmer III, Department of Biochemistry a n d Molecular Biophysics, Columbia University, 630 West 168th Street, New York, New York 10032, USA.
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