Konstantin Pervushin

Polarization transfer by cross-correlated relaxation in solution NMR with very large molecules

In common multidimensional NMR experiments for studies of biological macromolecules in solution, magnetization transfers via spin-spin couplings [insensitive nuclei enhanced by polarization transfer (INEPT)] are key elements of the pulse schemes. For molecular weights beyond 100,000, transverse relaxation during the transfer time may become a limiting factor. This paper presents a transfer technique for work with big molecules, cross relaxation-enhanced polarization transfer (CRINEPT), which largely reduces the size limitation of INEPT transfers with the use of cross-correlated relaxation-induced polarization transfer. The rate of polarization transfer by cross-correlated relaxation is proportional to the rotational correlation time, so that it becomes a highly efficient transfer mechanism for solution NMR with very high molecular weights. As a first implementation, [15N,1H]-correlation experiments were designed that make use of cross-correlation between dipole-dipole coupling and chemical shift anisotropy of the 15N---1H-moieties for both CRINEPT and transverse relaxation-optimized spectroscopy (TROSY). When compared with INEPT-based [15N,1H]-TROSY, these experiments yielded up to 3-fold signal enhancement for amide groups of a 110,000-Da protein in aqueous solution at 4 degrees C. CRINEPT opens avenues for solution NMR with supramolecular structures such as membrane proteins solubilized in micelles or lipid vesicles, proteins attached to nucleic acid fragments, or oligomeric proteins.

Single Transition-to-single Transition Polarization Transfer (ST2-PT) in [15N,1H]-TROSY

AbstractThis paper describes the use of single transition-to-single transition polarization transfer (ST2-PT) in transverse relaxation-optimized spectroscopy (TROSY), where it affords a

Impact of Transverse Relaxation Optimized Spectroscopy (TROSY) on NMR as a technique in structural biology

\sqrt 2

Improved sensitivity and coherence selection for [15N,1H]-TROSY elements in triple resonance experiments

sensitivity enhancement for kinetically stable amide 15N-1H groups in proteins. Additional, conventional improvements of [15N,1H]-TROSY include that signal loss for kinetically labile 15N-1H groups due to saturation transfer from the solvent water is suppressed with the ‘water flip back’ technique and that the number of phase steps is reduced to two, which is attractive for the use of [15N,1H]-TROSY as an element in more complex NMR schemes. Finally, we show that the impact of the inclusion of the 15N steady-state magnetization (Pervushin et al., 1998) on the signal-to-noise ratio achieved with [15N,1H]-TROSY exceeds by up to two-fold the gain expected from the gyromagnetic ratios of 1H and 15N.

NMR Structure of the Heme Chaperone Ccme Reveals a Novel Functional Motif

TROSY and CRINEPT are new techniques for solution NMR studies of molecular and supramolecular structures. They allow the collection of high-resolution spectra of structures with molecular weights >100 kDa, significantly extending the range of macromolecular systems that can be studied by NMR in solution. TROSY has already been used to map protein-protein interfaces, to conduct structural studies on membrane proteins and to study nucleic acid conformations in multimolecular assemblies. These techniques will help us to investigate the conformational states of individual macromolecular components and will support de novo protein structure determination in large supramolecular structures.

TROSY NMR with partially deuterated proteins

1. Transverse relaxation and the molecular size limit in liquid state NMR 161 2. TROSY: how does it work? 163 2.1 Transverse relaxation in coupled spin systems 163 2.2 The TROSY effect, relaxation due to remote protons and 2 H isotope labeling 165 3. Direct heteronuclear chemical shift correlations 168 3.1 Single-Quantum [ 15 N, 1 H]-TROSY 168 3.2 Zero-Quantum [ 15 N, 1 H]-TROSY 171 3.3 Single-Quantum TROSY with aromatic 13 C– 1 H moieties 176 4. Resonance assignment and NOE spectroscopy of large biomolecules 180 4.1 TROSY-based triple resonance experiments for 13 C, 15 N and 1 H N backbone resonance assignment in uniformly 2 H, 13 C, 15 N labeled proteins 180 4.2 TROSY-type NOE spectroscopy 186 5. Scalar coupling across hydrogen bonds observed by TROSY 187 6. The use of TROSY for measurements of residual dipolar coupling constants 190 7. Conclusions 191 8. Acknowledgements 191 9. References 191 The application of nuclear magnetic resonance (NMR) spectroscopy for structure determination of proteins and nucleic acids (Wuthrich, 1986) with molecular mass exceeding 30 kDa is largely constrained by two factors, fast transverse relaxation of spins of interest and complexity of NMR spectra, both of which increase with increasing molecular size (Wagner, 1993b; Clore & Gronenborn, 1997, 1998b; Kay & Gardner, 1997). The good news is that neither of these factors represent a fundamental limit for the application of NMR techniques to protein structure determination in solution (Clore & Gronenborn, 1998a; Wuthrich, 1998; Wider & Wuthrich, 1999). In fact, in the past few years the size limitations imposed by these factors have been pushed up to 50–70 kDa by the use of 13 C, 15 N and 2 H isotope labeling combined with selective reprotonation of individual chemical groups in conjunction with the use of triple-resonance experiments (Bax, 1994; Gardner et al . 1997; Gardner & Kay, 1998) and heteronuclear-resolved NMR (Fesik & Zuiderweg, 1988; Marion et al . 1989a; Otting & Wuthrich, 1990). Among the largest biomolecules whose 3D structure was solved by NMR are the 44 kDa trimeric ectodomain of simian immunodeficiency virus (SIV) gp41 (Caffrey et al . 1998) and 40–60 kDa particles of the elongation initiation factor 4E solubilized in CHAPS micelles (Matsuo et al . 1997; McGuire et al . 1998).

Residual Structure in Islet Amyloid Polypeptide Mediates Its Interactions with Soluble Insulin

Membrane proteins are usually solubilized in polar solvents by incorporation into micelles. Even for small membrane proteins these mixed micelles have rather large molecular masses, typically beyond 50 000 Da. The NMR technique TROSY (transverse relaxation‐optimized spectroscopy) has been developed for studies of structures of this size in solution. In this paper, strategies for the use of TROSY‐based NMR experiments with membrane proteins are discussed and illustrated with results obtained with the Escherichia coli integral membrane proteins OmpX and OmpA in mixed micelles with the detergent dihexanoylphosphatidylcholine (DHPC). For OmpX, complete sequence‐specific NMR assignments have been obtained for the polypeptide backbone. The 13C chemical shifts and nuclear Overhauser effect data then resulted in the identification of the regular secondary structure elements of OmpX/DHPC in solution, and in the collection of an input of conformational constraints for the computation of the global fold of the protein. For OmpA, the NMR assignments are so far limited to about 80% of the polypeptide chain, indicating different dynamic properties of the reconstituted OmpA β‐barrel from those of OmpX. Overall, the present data demonstrate that relaxation‐optimized NMR techniques open novel avenues for studies of structure, function and dynamics of integral membrane proteins.

Probing the rotor subunit interface of the ATP synthase from Ilyobacter tartaricus

In experiments with proteins of molecular weights around 100 kDa the implementation of [15N,1H]-TROSY-elements in [15N]-constant-time triple resonance experiments yields sensitivity enhancements of one to two orders of magnitude. An additional gain of 10 to 20% may be obtained with the use of ‘sensitivity enhancement elements’. This paper describes a novel sensitivity enhancement scheme which is based on concatenation of the 13 Cα → 15N magnetization transfer with the ST2-PT element, and which enables proper TROSY selection of the 15N multiplet components.