Dennis A. Torchia

Comparison of different modes of two-dimensional reverse-correlation NMR for the study of proteins

Different two-dimensional NMR schemes for generating ‘H-detected ‘H-“N and ‘H13C correlation spectra are compared. It is shown that the resolution in the dimension that represents the “C or “N chemical shift depends on the type of correlation scheme used. For “N NMR studies of proteins, it is found that experiments that involve “N single-quantum coherence offer improved resolution compared to multiple-quantum correlation experiments, mainly because the ‘H- ‘H dipolar broadening of the multiplequantum coherence is stronger than the heteronuclear dipolar broadening of “N, but also because ofthe presence ofunresolved Jsplittings in the F, dimension ofthe multiplequantum correlation spectra. For 13C, the heteronuclear dipolar interaction is much larger and the ‘H-13C multiple-quantum relaxation is slower than the “C transverse relaxation; however, because of the presence of ‘H- ‘H Jcouplings in the F, dimension of such spectra, in practice the multiplequantum type correlation experiments often offer no gain or even a small loss in resolution, compared to experiments that use transverse 13C magnetization during the evolution period. A modified pulse scheme that increases F, resolution by elimination of scalar relaxation of the second kind is proposed. Experiments for the proteins calmodulin, uniformly enriched with “N, and staphylococcal nuclease, labeled with 13C in the Ca position of all Pro residues are demonstrated.

Pulse sequences for removal of the effects of cross correlation between dipolar and chemical-shift anisotropy relaxation mechanisms on the measurement of heteronuclear T1 and T2 values in proteins

The effects of cross correlation between dipolar and chemical-shift anisotropy relaxation interactions on the measurement of heteroatom T1 and T2 relaxation times in proteins is considered. It is shown that such effects can produce errors of approximately 25% in the measurement of 15N transverse relaxation times at a field strength of 11.8 T. Cross correlation has a less significant effect on the measurement of 15N spin-lattice relaxation rates and for proteins the errors in T1 decrease as a function of increasing molecular weight. Nevertheless, for T1 measurements at 11.8 T errors of approximately 15 and 5% are calculated for proteins with correlation times, τc, of 5 and 9 ns, respectively. Pulse sequences which eliminate dipolar and chemical-shift anisotropy cross-correlation effects are described. These sequences are used to make more accurate measurements of 15N T1 and T2 values of staphylococcal nuclease and to determine errors in these parameters that result when cross correlations are present.

Protein dynamics from NMR

This review surveys recent investigations of conformational fluctuations of proteins in solution using NMR techniques. Advances in experimental methods have provided more accurate means of characterizing fast and slow internal motions as well as overall diffusion. The information obtained from NMR dynamics experiments provides insights into specific structural changes or configurational energetics associated with function. A variety of applications illustrate that studies of protein dynamics provide insights into protein–protein interactions, target recognition, ligand binding, and enzyme function.

Spectral density function mapping using 15N relaxation data exclusively

SummaryA method is presented for the determination of values of the spectral density function, J(ω), describing the dynamics of amide bond vectors from 15N relaxation parameters alone. Assuming that the spectral density is given by the sum of Lorentzian functions, the approach allows values of J(ω) to be obtained at ω=0, ωN and 0.870ωH, where ωN and ωH are Larmor frequencies of nitrogen and proton nuclei, respectively, from measurements of 15N T1, T2 and 1H−15N steady-state NOE values at a single spectrometer frequency. Alternatively, when measurements are performed at two different spectrometer frequencies of i and j MHz, J(ω) can be mapped at ω=0, ωiN, ωjN, 0.870ωiH and 0.870ωjH, where ωiN, for example, is the 15N Larmor frequency for a spectrometer operating at i MHz. Additionally, measurements made at two different spectrometer frequencies enable contributions to trasverse relaxation from motions on millisecond-microsecond time scales to be evaluated and permit assessment of whether a description of the internal dynamics is consistent with a correlation function consisting of a sum of exponentials. No assumptions about the specific form of the spectral density function describing the dynamics of the 15N−NH bond vector are necessary, provided that dJ(ω)/dω is relatively constant between ω=ωH+ωN to ω=ωH−ωN. Simulations demonstrate that the method is accurate for a wide range of protein motions and correlation times, and experimental data establish the validity of the methodology. Results are presented for a folded and an unfolded form of the N-terminal SH3 domain of the protein drk.

Spin-lattice relaxation in solids

Abstract Spin-lattice relaxation times ( T 1 ) contain information about molecular motion. We present a formalism for extracting this information from T 1 values measured for static and spinning polycrystalline solids. General equations are developed for orientation dependent line positions (frequencies) and values of T 1 . The correlation functions in the general T 1 expressions are evaluated for various jump and diffusive models of motion, assuming an axially symmetric coupling mechanism. Explicit expressions for dipolar and quadrupolar values of T 1 are presented for two- and three-site jumps, and for continuous diffusion about a symmetry axis. It is shown that T 1 measurements can readily distinguish between three-site jump and continuous diffusion models of methyl reorientation on the basis of the different angular dependence calculated for T 1 in these two models. In general, T 1 is axially asymmetric for the three-site jump model, resulting in nonexponential relaxation for the spectral lines in the axially symmetric powder pattern.

An alternative 3D NMR technique for correlating backbone 15N with side chain Hβ resonances in larger proteins

Values of ’ 5N‘H spin-spin coupling constants can be measured in a quantitative manner by recording ‘H‘H TOCSY /HOHAHA or NOESY spectra of a 15N-enriched protein in the absence of 15N decoupling (I ). Although this technique is elegant and potentially very powerful, the absence of “N decoupling halves the signal-to-noise ratio and may make the small displacements of broad resonances which contain the long-range JNH information difficult to measure. Moreover, for larger proteins the TOCSY / HOHAHA cross peaks frequently are too weak to be detected at all. As recently shown by Chary et al. (2), measurement of cross-peak intensity in a 3D spectrum that correlates amide proton and “N chemical shifts with those of the intraresidue HP resonances provides qualitative information on the size of the threebond 15N-HP J coupling. This yields information about the side-chain torsion angle x1 and aids with the stereospecific assignment of nonequivalent methylene protons. In their experiment, net magnetization is transferred from the amide proton via its “N to the side-chain HP protons, which are detected during the acquisition period, t3, of the 3D experiment. At the beginning of t3, there is no net magnetization and only after rephasing with respect to the “N nucleus takes place can the magnetization be detected. In larger proteins, the line widths of HP methylene protons are among the largest in the protein because of their strong geminal dipolar interactions, which are superimposed on large and often unresolvable geminal and vicinal ‘H‘H J couplings. Here we propose a simple modification of this experiment that removes this dependence on the HP linewidth and which therefore can be applied to proteins of a substantially larger size. Instead of using net magnetization transfer, the new technique relies on the “out-and-back” magnetization-transfer approach previously used in a series of other 3D triple-resonance techniques (3-6). The pulse scheme for the new experiment is sketched in Fig. 1A. Because the main use of this experiment is to correlate amide proton (HN) and nitrogen resonances with side-chain HP proton signals, we name the experiment HNHB. Briefly, the magnetization transfer goes as follows: Magnetization is transferred from the amide proton to the amide nitrogen using an INEPT transfer. During the subsequent evolution period, t,, the antiphase magnetization evolves with the chemical shift of the 15N

Atomic resolution dynamics on the surface of amyloid β protofibrils probed by solution NMR

Exchange dynamics between molecules free in solution and bound to the surface of a large supramolecular structure, a polymer, a membrane or solid support are important in many phenomena in biology and materials science. Here we present a novel and generally applicable solution NMR technique, known as dark-state exchange saturation transfer (DEST), to probe such exchange phenomena with atomic resolution. This is illustrated by the exchange reaction between amyloid-β (Aβ) monomers and polydisperse, NMR-invisible (‘dark’) protofibrils, a process of significant interest because the accumulation of toxic, aggregated forms of Aβ, from small oligomers to very large assemblies, has been implicated in the aetiology of Alzheimer’s disease. The 15N-DEST experiment imprints with single-residue-resolution dynamic information on the protofibril-bound species in the form of 15N transverse relaxation rates (15N-R2) and exchange kinetics between monomers and protofibrils onto the easily observed two-dimensional 1H–15N correlation spectrum of the monomer. The exchanging species on the protofibril surface comprise an ensemble of sparsely populated states where each residue is either tethered to (through other residues) or in direct contact with the surface. The first eight residues exist predominantly in a mobile tethered state, whereas the largely hydrophobic central region and part of the carboxy (C)-terminal hydrophobic region are in direct contact with the protofibril surface for a significant proportion of the time. The C-terminal residues of both Aβ40 and Aβ42 display lower affinity for the protofibril surface, indicating that they are likely to be surface exposed rather than buried as in structures of Aβ fibrils, and might therefore comprise the critical nucleus for fibril formation. The values, however, are significantly larger for the C-terminal residues of Aβ42 than Aβ40, which might explain the former’s higher propensity for rapid aggregation and fibril formation.

Flap opening and dimer-interface flexibility in the free and inhibitor-bound HIV protease, and their implications for function

BACKGROUND (1)H and (15)N transverse relaxation measurements on perdeuterated proteins are ideally suited for detecting backbone conformational fluctuations on the millisecond-microsecond timescale. The identification of conformational exchange on this timescale by measuring the relaxation of both (1)H and (15)N holds great promise for the elucidation of functionally relevant conformational changes in proteins. RESULTS We measured the transverse (1)H and (15)N relaxation rates of backbone amides of HIV-1 protease in its free and inhibitor-bound forms. An analysis of these rates, obtained as a function of the effective rotating frame field, provided information about the timescale of structural fluctuations in several regions of the protein. The flaps that cover the active site of the inhibitor-bound protein undergo significant changes of backbone (φ,psi) angles, on the 100 micros timescale, in the free protein. In addition, the intermonomer beta-sheet interface of the bound form, which from protease structure studies appears to be rigid, was found to fluctuate on the millisecond timescale. CONCLUSIONS We present a working model of the flap-opening mechanism in free HIV-1 protease which involves a transition from a semi-open to an open conformation that is facilitated by interaction of the Phe53 ring with the substrate. We also identify a surprising fluctuation of the beta-sheet intermonomer interface that suggests a structural requirement for maturation of the protease. Thus, slow conformational fluctuations identified by (1)H and (15)N transverse relaxation measurements can be related to the biological functions of proteins.

Flexibility and function in HIV-1 protease

HIV protease is a homodimeric protein whose activity is essential to viral function. We have investigated the molecular dynamics of the HIV protease, thought to be important for proteinase function, bound to high affinity inhibitors using NMR techniques. Analysis of 15N spin relaxation parameters, of all but 13 backbone amide sites, reveals the presence of significant internal motions of the protein backbone. In particular, the flaps that cover the proteins active site of the protein have terminal loops that undergo large amplitude motions on the ps to ns time scale, while the tips of the flaps undergo a conformational exchange on the μs time scale. This enforces the idea that the flaps of the proteinase are flexible structures that facilitate function by permitting substrate access to and product release from the active site of the enzyme.

Rapid structural fluctuations of the free HIV protease flaps in solution: relationship to crystal structures and comparison with predictions of dynamics calculations.

Crystal structures have shown that the HIV‐1 protease flaps, domains that control access to the active site, are closed when the active site is occupied by a ligand. Although flap structures ranging from closed to semi‐open are observed in the free protease, crystal structures reveal that even the semi‐open flaps block access to the active site, indicating that the flaps are mobile in solution. The goals of this paper are to characterize the secondary structure and fast (sub‐ns) dynamics of the flaps of the free protease in solution, to relate these results to X‐ray structures and to compare them with predictions of dynamics calculations. To this end we have obtained nearly complete backbone and many sidechain signal assignments of a fully active free‐protease construct that is stabilized against autoproteolysis by three point mutations. The secondary structure of this protein was characterized using the chemical shift index, measurements of 3hJNC′ couplings across hydrogen bonds, and NOESY connectivities. Analysis of these measurements indicates that the protease secondary structure becomes irregular near the flap tips, residues 49–53. Model‐free analysis of 15N relaxation parameters, T1, T2 (T1ρ) and 15N‐{1H} NOE, shows that residues in the flap tips are flexible on the sub‐ns time scale, in contrast with previous observations on the inhibitor‐bound protease. These results are compared with theoretical predictions of flap dynamics and the possible biological significance of the sub‐ns time scale dynamics of the flap tips is discussed.