Trosy Cript and Crinept for Studies of Very Large Structures

With increasing molecular mass, most signal is lost during polarization transfer periods in heteronuclear NMR experiments where TROSY is not active. If the conventional polarization transfer element INEPT (Morris and Freeman 1979; Burum and Ernst 1980) is replaced by CRINEPT (Riek et al. 1999, 2002) and CRIPT (Dalvit 1992), the signal loss can be reduced. Using NMR experiments based on TROSY and CRINEPT or CRIPT, we can now investigate macromolecular systems with molecular masses up to 900 kDa by solution NMR (Fiaux et al. 2002).

A general strategy to assign signals in macromolecules with molecular masses above approximately 150 kDa is not (yet) available and therefore CRINEPT/CRIPT spectroscopy is usually applied to obtain fingerprints of very large structures. As

GroES

GroEL

GroES

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Fig.5.8. NMR measurements of molecular complexes with very high molecular mass using TROSY./4-CAll-atom representation ofthe structure ofthe molecules studied. A GroES, which is a 72-kDa homoheptameric protein (one subunit is shown in darkgrey). B GroES in a complex with unlabeled SRI, with a molecular mass of 470 kDa. SRI is a single-ring variant of GroEL. C GroES in a complex with unlabeled GroEL with a total molecular mass of almost 900 kDa. D-F 2D [15N,1H] correlation spectra of uniformly 2H,15N-labeled GroES in the macromolecular complexes shown in A-C. D 2D [15N,1H]-TROSY spectrum of free GroES. E 2D [15N,1H] cross-correlated relaxation-induced polarization transfer (CRIPT) TROSY spectrum of GroES bound to SRI in the presence of ADP. F 2D [15N,1H]-CRIPT-TROSY spectrum of GroES bound to GroEL. In E and F, the peaks that shifted significantly upon binding to SRI or GroEL are marked with an asterisk. The numbers in D-F indicate the individual assignments ofthe resonances. (Adapted from Fiaux et al. 2002 with permission)

83C=

Fig.5.8. NMR measurements of molecular complexes with very high molecular mass using TROSY./4-CAll-atom representation ofthe structure ofthe molecules studied. A GroES, which is a 72-kDa homoheptameric protein (one subunit is shown in darkgrey). B GroES in a complex with unlabeled SRI, with a molecular mass of 470 kDa. SRI is a single-ring variant of GroEL. C GroES in a complex with unlabeled GroEL with a total molecular mass of almost 900 kDa. D-F 2D [15N,1H] correlation spectra of uniformly 2H,15N-labeled GroES in the macromolecular complexes shown in A-C. D 2D [15N,1H]-TROSY spectrum of free GroES. E 2D [15N,1H] cross-correlated relaxation-induced polarization transfer (CRIPT) TROSY spectrum of GroES bound to SRI in the presence of ADP. F 2D [15N,1H]-CRIPT-TROSY spectrum of GroES bound to GroEL. In E and F, the peaks that shifted significantly upon binding to SRI or GroEL are marked with an asterisk. The numbers in D-F indicate the individual assignments ofthe resonances. (Adapted from Fiaux et al. 2002 with permission)

GroES

GroEL

GroES

kDs with TROSY-based experiments, the preferred current use of CRINEPT is to study relatively short polypeptide chains (up to approximately 100-200 amino acid residues) in large supramolecular assemblies, in complexes with large macromolecules, or in large detergent/lipid micelles. In these large structures, recording of 15N-1H correlation spectra can be very useful for studies of intermolecular interactions and for NMR screening to search for low molecular mass ligands. For information at atomic resolution, sequence-specific resonance assignments are required. In favorable situations, assignment of the amide group chemical shifts may be obtained for smaller subunits in the absence of the whole complex, and these assignments can be transferred to the 15N-1H correlation spectra of the whole complex (Fiaux et al.

2002). Without assignments, 15N-1H correlation spectra still provide, for example, proof of binding events or information about dynamics of the polypeptide backbone. In both cases some of the information may be masked by spectral overlap and selective isotope-labeling techniques, e.g., segmental isotope labeling and selective amino acid labeling (see earlier), can help to simplify the spectra.

tte potential of the CRINEPT and CRIPT experiments has recently been demonstrated for 900- and 500-kDa complexes formed by GroES with GroEL and SRI (SRI is a single-ring variant of GroEL), respectively (Fiaux et al. 2002; Riek et al. 2002) (Fig. 5.8). In this example, the sequence-specific assignments for free GroES (a hep-tamer of 72-kDa molecular mass) were obtained from TROSY triple-resonance experiments with a 2H,13C,15N-labeled sample. Tentative assignments of GroES in the complexes with GroEL and SRI were obtained by transferring assignments of free GroES to the 2D [15N,1H]-CRIPT-TROSY spectra of GroES bound either to GroEL or to SRI. Comparison of the 2D [15N,1H]-CRIPT-TROSY and 2D [15N,1H]-TROSY spectra of GroES in the bound and in free form, respectively, provided structural and dynamical information on the 900-kDa GroEL-GroES complex.

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