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Sequential Assignments (sequential + assignment)
Selected AbstractsSolid-State NMR of Matrix Metalloproteinase 12: An Approach Complementary to Solution NMRCHEMBIOCHEM, Issue 5 2007Stéphane Balayssac Dr. Data transfer. The solid-state proton-driven spin diffusion (PDSD) and J -decoupled PDSD NMR spectra of the microcrystalline catalytic domain of matrix metalloproteinase 12 (MMP-12, 17 kDa) have been recorded. It is shown that such spectra can be largely assigned in a few days by using the available liquid-state assignment and validated with an independent sequential assignment based on 3D NCACX and NCOCX PDSD experiments. This demonstrates how quickly the liquid-state assignment of comparably large protein can be transferred to the solid state. [source] Improved pulse sequences for pure exchange solid-state NMR spectroscopyMAGNETIC RESONANCE IN CHEMISTRY, Issue 2 2004Thomas Vosegaard Abstract Spin-exchange experiments are useful for improving the resolution and establishment of sequential assignments in solid-state NMR spectra of uniformly 15N-labeled proteins oriented macroscopically in phospholipid bilayers. To exploit this advantage fully, it is crucial that the diagonal peaks in the two-dimensional exchange spectra are suppressed. This may be accomplished using the recent pure-exchange (PUREX) experiments, which, however, suffer from up to a threefold reduction of the cross-peak intensity relative to experiments without diagonal-peak suppression. This loss in sensitivity may severely hamper the applicability for the study of membrane proteins. In this paper, we present a two-dimensional exchange experiment (iPUREX) which improves the PUREX sensitivity by 50%. The performance of iPUREX is demonstrated experimentally by proton-mediated 15N,15N spin-exchange experiments for a 15N-labeled N -acetyl- L -valyl- L -leucine dipeptide. The relevance of exchange experiments with diagonal-peak suppression for large, uniformly 15N-labeled membrane proteins in oriented phospholipid bilayers is demonstrated numerically for the G-protein coupled receptor rhodopsin. Copyright © 2004 John Wiley & Sons, Ltd. [source] NMR studies on the basic pancreatic trypsin inhibitor,MAGNETIC RESONANCE IN CHEMISTRY, Issue S1 2003G. Wagner Abstract In 1973, the state of protein NMR was in its infancy and the tremendous developments that have happened since were not foreseeable. This article describes developments as they happened in the laboratory of Professor Kurt Wüthrich while the author was a graduate student and postdoctoral fellow during the time period from 1973 to 1987. This is a subjective view and deals primarily with the developments in which the author was involved. This includes the characterization of the dynamics of aromatic side chains in basic pancreatic trypsin inhibitor, the development of strategies for sequential assignments in proteins, and the first attempts of calculating protein structures. Copyright © 2003 John Wiley & Sons, Ltd. [source] Rapid mass spectrometric analysis of 15N-Leu incorporation fidelity during preparation of specifically labeled NMR samplesPROTEIN SCIENCE, Issue 9 2008Stephanie M.E. Truhlar Abstract Advances in NMR spectroscopy have enabled the study of larger proteins that typically have significant overlap in their spectra. Specific 15N-amino acid incorporation is a powerful tool for reducing spectral overlap and attaining reliable sequential assignments. However, scrambling of the label during protein expression is a common problem. We describe a rapid method to evaluate the fidelity of specific 15N-amino acid incorporation. The selectively labeled protein is proteolyzed, and the resulting peptides are analyzed using MALDI mass spectrometry. The 15N incorporation is determined by analyzing the isotopic abundance of the peptides in the mass spectra using the program DEX. This analysis determined that expression with a 10-fold excess of unlabeled amino acids relative to the 15N-amino acid prevents the scrambling of the 15N label that is observed when equimolar amounts are used. MALDI TOF-TOF MS/MS data provide additional information that shows where the "extra" 15N labels are incorporated, which can be useful in confirming ambiguous assignments. The described procedure provides a rapid technique to monitor the fidelity of selective labeling that does not require a lot of protein. These advantages make it an ideal way of determining optimal expression conditions for selectively labeled NMR samples. [source] Simultaneous assignment and structure determination of a membrane protein from NMR orientational restraintsPROTEIN SCIENCE, Issue 3 2003Francesca M. Marassi Abstract A solid-state NMR approach for simultaneous resonance assignment and three-dimensional structure determination of a membrane protein in lipid bilayers is described. The approach is based on the scattering, hence the descriptor "shotgun," of 15N-labeled amino acids throughout the protein sequence (and the resulting NMR spectra). The samples are obtained by protein expression in bacteria grown on media in which one type of amino acid is labeled and the others are not. Shotgun NMR short-circuits the laborious and time-consuming process of obtaining complete sequential assignments prior to the calculation of a protein structure from the NMR data by taking advantage of the orientational information inherent to the spectra of aligned proteins. As a result, it is possible to simultaneously assign resonances and measure orientational restraints for structure determination. A total of five two-dimensional 1H/15N PISEMA (polarization inversion spin exchange at the magic angle) spectra, from one uniformly and four selectively 15N-labeled samples, were sufficient to determine the structure of the membrane-bound form of the 50-residue major pVIII coat protein of fd filamentous bacteriophage. Pisa (polarity index slat angle) wheels are an essential element in the process, which starts with the simultaneous assignment of resonances and the assembly of isolated polypeptide segments, and culminates in the complete three-dimensional structure of the protein with atomic resolution. The principles are also applicable to weakly aligned proteins studied by solution NMR spectroscopy. [The structure we determined for the membrane-bound form of the Fd bacteriophage pVIII coat protein has been deposited in the Protein Data Bank as PDB file 1MZT.] [source] Recombinant decorsin: Dynamics of the RGD recognition sitePROTEIN SCIENCE, Issue 8 2000Andrzej M. Krezel Abstract Decorsin is an antagonist of integrin ,IIb,3 and a potent platelet aggregation inhibitor. A synthetic gene encoding decorsin, originally isolated from the leech Macrobdella decora, was designed, constructed, and expressed in Escherichia coli. The synthetic gene was fused to the stII signal sequence and expressed under the transcriptional control of the E. coli alkaline phosphatase promoter. The protein was purified by size-exclusion filtration of the periplasmic contents followed by reversed-phase high-performance liquid chromatography. Purified recombinant decorsin was found to be indistinguishable from leech-derived decorsin based on amino acid composition, mass spectral analysis, and biological activity assays. Complete sequential assignments of 1H and proton bound 13C resonances were established. Stereospecific assignments of 21 of 25 nondegenerate ,-methylene groups were determined. The RGD adhesion site recognized by integrin receptors was found at the apex of a most exposed hairpin loop. The dynamic behavior of decorsin was analyzed using several independent NMR parameters. Although the loop containing the RGD sequence is the most flexible one in decorsin, the conformation of the RGD site itself is more restricted than in other proteins with similar activities. [source] | ||||||||||