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

Abstract

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 relaxationenhanced 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. Structure determination of proteins by NMR in solution (1) has so far been limited to the molecular weight range up to Ϸ30 kDa (2), and experiments yielding backbone assignments for 2 H-labeled proteins up to Ϸ60 kDa have been reported (3). In larger molecular species, the standard experimental techniques (4, 5) lead to severe sensitivity loss because of transverse relaxation even when optimal isotope-labeling is used (6). The situation has recently been improved with the introduction of the transverse relaxation-optimized spectroscopy (TROSY) technique, which reduces transverse relaxation during evolution and observation periods in heteronuclear NMR experiments (7,8). For example, for 15 NO 1 H-moieties in proteins, significant reduction of transverse relaxation during the 15 N evolution and amide proton acquisition periods can be achieved at the highest presently available 1 H frequencies, and nearly complete cancellation is expected at 1 H frequencies near 1 GHz (7, 9). TROSY already has been applied for detailed NMR studies of a protein with molecular weight Ͼ100 kDa (8). Theoretical considerations indicate that TROSY will reach its limits at somewhat larger sizes because of rapid transverse relaxation during the insensitive nuclei enhanced by polarization transfer (INEPT)-type (10) magnetization transfers via scalar couplings between the different nuclei. This paper presents a transfer technique for work with very large molecules, cross relaxation-enhanced polarization transfer (CRINEPT), which is based on combining cross-correlated relaxation-induced polarization transfer (11-14) and polarization transfer via scalar couplings (10). To provide a foundation for practical applications of CRINEPT, the performance of the previously little-used cross relaxation-induced polarization transfer (CRIPT) is investigated for 15

Managing the solvent water polarization to obtain improved NMR spectra of large molecular structures

“…For structures of higher molecular weight, NMR studies are more difficult, mainly because of fast transverse spin relaxation due to slow molecular tumbling (Brownian motion). In the last few years, NMR techniques for large molecules have been developed, which improve the quality of the spectra through optimization of transverse relaxation during evolution and acquisition times (TROSY) (Pervushin et al, 1997;Wu¨thrich and Wider, 2003), and through the use of both cross correlated relaxation-induced polarization transfer and relaxation optimization during magnetization transfer steps (CRINEPT) (Riek et al, 1999) in multi-dimensional NMR experiments. Combined with perdeuteration of the non-labile proton positions of the macromolecule (LeMaster, 1994), these techniques have so far enabled studies with molecular sizes up to 870 kDa .…”

Nuclear magnetic resonance spectroscopy with the stringent substrate rhodanese bound to the single‐ring variant SR1 of the E. coli chaperonin GroEL

“…The spectra were processed with the program TOPSPIN (Bruker Biospin, Billerica, MA) and analyzed using the CARA software package. 36 The following parameters were used for the recording and processing of 2D [ 15 26 In these series, the transfer delay, T, was incremented from 0.7 to 6.0 ms in steps of 41 ls. The 15 N-evolution delay, t 1 , was held constant at 2 ls, so that the pulse sequences effectively yielded one-dimensional spectra.…”

Direct measurement of dipole–dipole/CSA cross-correlated relaxation by a constant-time experiment

“…The spectral noise is assumed to be random within ±0.05. Thus, the signal intensity of a TROSY/anti-TROSY measurement is modulated by: (6) where R = λ ± η for up-and downfield relaxation respectively, and c is the random noise within ±0.05.…”

“…In TROSY-type experiments they improve spectral resolution, or enhance sensitivity for experiments involving long transverse 15 N relaxation delays [5]. In the CRIPT/CRINEPT experiment they can be used for enhancing polarization transfer and improving sensitivity [6]. In certain situations they can be used to retrieve significant structural information [7,8].…”