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Backbone solution structures of proteins using residual dipolar couplings: Application to a novel structural genomics target

Journal of Structural and Functional Genomics volume 5, pages 241–254 (2005)Cite this article

Abstract

Structural genomics (or proteomics) activities are critically dependent on the availability of high-throughput structure determination methodology. Development of such methodology has been a particular challenge for NMR based structure determination because of the demands for isotopic labeling of proteins and the requirements for very long data acquisition times. We present here a methodology that gains efficiency from a focus on determination of backbone structures of proteins as opposed to full structures with all sidechains in place. This focus is appropriate given the presumption that many protein structures in the future will be built using computational methods that start from representative fold family structures and replace as many as 70% of the sidechains in the course of structure determination. The methodology we present is based primarily on residual dipolar couplings (RDCs), readily accessible NMR observables that constrain the orientation of backbone fragments irrespective of separation in space. A new software tool is described for the assembly of backbone fragments under RDC constraints and an application to a structural genomics target is presented. The target is an 8.7 kDa protein from Pyrococcus furiosus, PF1061, that was previously not well annotated, and had a nearest structurally characterized neighbor with only 33% sequence identity. The structure produced shows structural similarity to this sequence homologue, but also shows similarity to other proteins, which suggests a functional role in sulfur transfer. Given the backbone structure and a possible functional link this should be an ideal target for development of modeling methods.

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Abbreviations

c12E5:

pentaethylene glycol monododecyl ether

CTAB:

hexadecyltrimethylammonium bromide

DSS:

2,2-dimethyl-2-silapentane-5-sulfonic acid

DTT:

dithiothreitol

RDC:

residual dipolar coupling

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Affiliations

  1. Southeast Collaboratory for Structural Genomics, University of Georgia, 30602, Athens, GA, USA

    H. Valafar, K. L. Mayer & P. D. LeBlond

  2. Institut de Biologie Structurale, 41 rue Jules Horowitz, F-38027, Grenoble Cedex 01, France

    C. M. Bougault

  3. Department of Biochemistry and Molecular Biology, University of Georgia, 30602, Athens, GA, USA

    F. E. Jenney Jr, P. S. Brereton & M. W. W. Adams

  4. Complex Carbohydrate Research Center, University of Georgia, 30602, Athens, GA, USA

    J. H. Prestegard

Authors
  1. H. Valafar
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  2. K. L. Mayer
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  3. C. M. Bougault
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  4. P. D. LeBlond
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  5. F. E. Jenney Jr
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  6. P. S. Brereton
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  7. M. W. W. Adams
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  8. J. H. Prestegard
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Correspondence to J. H. Prestegard.

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Valafar, H., Mayer, K.L., Bougault, C.M. et al. Backbone solution structures of proteins using residual dipolar couplings: Application to a novel structural genomics target. J Struct Func Genom 5, 241–254 (2005). https://doi.org/10.1007/s10969-005-4899-5

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  • DOI: https://doi.org/10.1007/s10969-005-4899-5

Key words

  • NMR
  • partial alignment
  • Pyrococcus furiosus
  • RDC
  • structure determination