NMR data collection and analysis protocol for high-throughput protein structure determination.

A standardized protocol enabling rapid NMR data collection for high-quality protein structure determination is presented that allows one to capitalize on high spectrometer sensitivity: a set of five G-matrix Fourier transform NMR experiments for resonance assignment based on highly resolved 4D and 5D spectral information is acquired in conjunction with a single simultaneous 3D 15N,13C(aliphatic),13C(aromatic)-resolved [1H,1H]-NOESY spectrum providing 1H-1H upper distance limit constraints. The protocol was integrated with methodology for semiautomated data analysis and used to solve eight NMR protein structures of the Northeast Structural Genomics Consortium pipeline. The molecular masses of the hypothetical target proteins ranged from 9 to 20 kDa with an average of approximately 14 kDa. Between 1 and 9 days of instrument time were invested per structure, which is less than approximately 10-25% of the measurement time routinely required to date with conventional approaches. The protocol presented here effectively removes data collection as a bottleneck for high-throughput solution structure determination of proteins up to at least approximately 20 kDa, while concurrently providing spectra that are highly amenable to fast and robust analysis.

[1]  F. Young.  Biochemistry , 1955, The Indian Medical Gazette.

[2]  K. Wüthrich.  NMR of proteins and nucleic acids , 1988 .

[3]  L. Kay,et al.  Simultaneous Acquisition of 15N- and 13C-Edited NOE Spectra of Proteins Dissolved in H2O , 1994 .

[4]  A G Murzin,et al.  SCOP: a structural classification of proteins database for the investigation of sequences and structures. , 1995, Journal of molecular biology.

[5]  C. Sander,et al.  Dali: a network tool for protein structure comparison. , 1995, Trends in biochemical sciences.

[6]  S. Grzesiek,et al.  NMRPipe: A multidimensional spectral processing system based on UNIX pipes , 1995, Journal of biomolecular NMR.

[7]  J. Thornton,et al.  AQUA and PROCHECK-NMR: Programs for checking the quality of protein structures solved by NMR , 1996, Journal of biomolecular NMR.

[8]  Protein NMR Spectroscopy: Principles and Practice.JohnCavanagh, WayneJ. Fairbrother, ArthurG. PalmerIII,andNicholasJ. Skelton. Academic Press, San Diego, 1996. , 1996 .

[9]  M. Billeter,et al.  MOLMOL: a program for display and analysis of macromolecular structures. , 1996, Journal of molecular graphics.

[10]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[11]  K. Wüthrich,et al.  Torsion angle dynamics for NMR structure calculation with the new program DYANA. , 1997, Journal of molecular biology.

[12]  P E Bourne,et al.  Protein structure alignment by incremental combinatorial extension (CE) of the optimal path. , 1998, Protein engineering.

[13]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[14]  Mark J Howard,et al.  Protein NMR spectroscopy , 1998, Current Biology.

[15]  A. Bax,et al.  Protein backbone angle restraints from searching a database for chemical shift and sequence homology , 1999, Journal of biomolecular NMR.

[16]  T. N. Bhat,et al.  The Protein Data Bank , 2000, Nucleic Acids Res..

[17]  D. Richardson,et al.  Exploring steric constraints on protein mutations using MAGE/PROBE , 2000, Protein science : a publication of the Protein Society.

[18]  H N Moseley,et al.  Automatic determination of protein backbone resonance assignments from triple resonance nuclear magnetic resonance data. , 2001, Methods in enzymology.

[19]  T. Szyperski,et al.  Aromatic ring-flipping in supercooled water: implications for NMR-based structural biology of proteins. , 2001, Journal of the American Chemical Society.

[20]  D. Wishart,et al.  An NMR approach to structural proteomics , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[21]  Thomas Szyperski,et al.  Reduced-dimensionality NMR spectroscopy for high-throughput protein resonance assignment , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[22]  Torsten Herrmann,et al.  Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA. , 2002, Journal of molecular biology.

[23]  Ming Luo,et al.  The Southeast Collaboratory for Structural Genomics: a high-throughput gene to structure factory. , 2003, Accounts of chemical research.

[24]  C. Arrowsmith,et al.  1HC and 1HN total NOE correlations in a single 3D NMR experiment. 15N and 13C time-sharing in t1 and t2 dimensions for simultaneous data acquisition , 2003, Journal of biomolecular NMR.

[25]  T. Szyperski,et al.  GFT NMR, a new approach to rapidly obtain precise high-dimensional NMR spectral information. , 2003, Journal of the American Chemical Society.

[26]  John L Markley,et al.  Macromolecular structure determination by NMR spectroscopy. , 2005, Methods of biochemical analysis.

[27]  M. Nilges,et al.  Refinement of protein structures in explicit solvent , 2003, Proteins.

[28]  Thomas Szyperski,et al.  G-matrix Fourier transform NMR spectroscopy for complete protein resonance assignment. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[29]  Gaetano T Montelione,et al.  Automatic target selection for structural genomics on eukaryotes , 2004, Proteins.

[30]  G. Nicastro,et al.  The role of unstructured extensions in the rotational diffusion properties of a globular protein: the example of the titin i27 module. , 2004, Biophysical journal.

[31]  Hunter N. B. Moseley,et al.  Rapid analysis of protein backbone resonance assignments using cryogenic probes, a distributed Linux-based computing architecture, and an integrated set of spectral analysis tools , 2004, Journal of Structural and Functional Genomics.

[32]  T. Szyperski,et al.  GFT NMR Experiments for Polypeptide Backbone and 13Cβ Chemical Shift Assignment , 2004, Journal of biomolecular NMR.

[33]  Robert Powers,et al.  An integrated platform for automated analysis of protein NMR structures. , 2005, Methods in enzymology.

[34]  Thomas Szyperski,et al.  Rapid NMR data collection. , 2005, Methods in enzymology.

[35]  Mark Gerstein,et al.  Robotic cloning and Protein Production Platform of the Northeast Structural Genomics Consortium. , 2005, Methods in enzymology.

[36]  Robert Powers,et al.  Protein NMR recall, precision, and F-measure scores (RPF scores): structure quality assessment measures based on information retrieval statistics. , 2005, Journal of the American Chemical Society.

[37]  Thomas Szyperski,et al.  Resonance assignment of proteins with high shift degeneracy based on 5D spectral information encoded in G2FT NMR experiments. , 2005, Journal of the American Chemical Society.

[38]  A. Palmer,et al.  Protein NMR Spectroscopy: principles and practice, 2nd ed. , 2006 .