Biochemistry 1992, 31, 1647-1651 1647 Kitamura, S., & Sturtevant, J. M. (1989) Biochemistry 28, Matsumura, ‘ w* (lg8’) Science 243, Matthews, B. W., Nicholson, H., & Becktel, W. J. (1987) Privalov, P. L. (1980) Pure Appl. Chem. 52, 479-497. Remington, S. J., Anderson, W. F., Owen, J.,, Ten Eyck, L. F., Grainger, C. T., & Matthews, B. W. (1978) J. Mol. Biol. 118, 81-98. 3788-3792. Scheraga, H. A. (1978) Pure Appl. Chem. 50, 315. Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, Suzuki, Y. (1989) Proc. Jpn. Acad., Ser. B 65, 146-148. Tidor, B., & Karplus, M. (1991) Biochemistry 30,3217-3228. Weaver, L. H., & Matthews, B. W. (1987) J. Mol. Biol. 193, 792-794. 2236-2240. Proc. Natl. Acad. Sci. U.S.A. 84, 6663-6667. 189-199. The Chemical Shift Index: A Fast and Simple Method for the Assignment of Protein Secondary Structure through NMR Spectroscopy+ D. S. Wishart,*.*,s B. D. Sykes,a and F. M. Richards$ Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 0651 1, and MRC Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada Received August 20, 1991; Revised Manuscript Received November 4, 1991 ABSTRACT: Previous studies by Wishart et al. [Wishart, D. S., Sykes, B. D., & Richards, F. M. (1991) J. Mol. Biol. (in press)] have demonstrated that ‘H NMR chemical shifts are strongly dependent on the character and nature of protein secondary structure. In particular, it has been found that the ‘H NMR chemical shift of the a-CH proton of all 20 naturally occurring amino acids experiences an upfield shift (with respect to the random coil value) when in a helical configuration and a comparable downfield shift when in a @-strand extended configuration. On the basis of these observations, a technique is described for rapidly and quantitatively determining the identity, extent, and location of secondary structural elements in proteins based on the simple inspection of the a-CH *H resonance assignments. A number of examples are provided to demonstrate both the simplicity and the accuracy of the technique. This new method is found to be almost as accurate as the more traditional NOE-based methods of determining secondary structure and could prove to be particularly useful in light of the recent development of sequential assignment techniques which are now almost NOE-independent [Ikura, M., Kay, L. E., & Bax, A. (1990) Biochemistry 29, 4659-46671, We suggest that this new procedure should not necessarily be seen as a substitute to existing rigorous methods for secondary structure determination but, rather, should be viewed as a complement to these approaches. For more than 20 years NMR spectroscopists have been attempting to apply chemical shift information to conforma- tional problems of biological significance. Early efforts in this regard were first begun by Sternlicht and Wilson (1967) and Markley et al. (1967). Both groups were interested in studying the chemical shift tendencies of a-CH ‘H NMR’ resonances in amino acid homopolymers, particularly with respect to the systematic changes in proton chemical shifts that were asso- ciated with helix formation and helix disruption in these compounds. Subsequent studies by Clayden and Williams (1982) and Dalgamo et al. (1983), based on accumulated data from naturally occurring proteins, suggested that reasonably strong conformationally dependent chemical shift tendencies existed in &strands as well as in a-helices and that these trends were not confined to certain homopolymers or to selected solvent conditions. More recent work by Szilagyi and Jar- detzky (1989) and Wishart et al. (1991) have confirmed these early observations by placing them on a more solid statistical basis. In fact, these workers have clearly demonstrated that ‘Financial support by the MRC Group in Protein Structure and Function and by the National Institutes of Health (NIH Grant GM- 22778) is gratefully acknowledged. * To whom correspondence should be addressed. f Yale University. *University of Alberta. 0006-2960/92/0431- 1647%03.00/0 a strong relationship exists between a-CH ‘H NMR chemical shifts and protein secondary structure for all 20 amino acids. Some of these observations have already begun to be put to use. Pastore and Saudek (1990) have recently described a useful method for displaying chemical shift information which permits the qualitative identification of secondary structure in proteins. This procedure is based on plotting “smoothed” chemical shift differences (with respect to random coil values) versus protein sequence and using the resulting curve to approximate the location and identity of secondary structures. However, because of the qualitative nature of these plots it is often difficult to identify the exact limits as well as the true identity of all significant secondary structures in the protein of interest. We describe a new method for secondary structure deter- mination based on chemical shift propensity that was developed quite independently of Pastore and Saudek‘s work. This particular technique can be used for the precise identification of protein secondary structure from chemical shift information alone. It is fast, simple, and accurate and can be used either Abbreviations: BPTI, bovine pancreatic trypsin inhibitor; CSI, chemical shift index; DSS, 2,2-dimethyl-2-silapentane-5-sulfonic acid; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; TMS, tetramethylsilane; TSP, sodium 3-(trimethylsilyl)propionate. 0 1992 American Chemical Societv1648 Biochemistry, Vol. 31, No. 6, 1992 Table I: Summary of Peptides and Proteins Used in This Analysis Wishart et al. protein (no. of residues) source conditions reference a-bungarotoxin Bungarus pH 4.0, 35 OC Basus (1988) a-neurotoxin Dryopteris pH 4.2, 36 OC Labhardt (1988) anaphylotoxin bovine pH 2.3, 10 “C Zuiderwig (1988) antennapedia Drosophila pH 4.3, 20 OC Billeter (1990) arc repressor phage P22 pH 5.4, 50 OC Breg (1989) histidine protein Escherichia pH 6.5, 30 OC Wittekind (1990) (74) multicinctus (60) poly lepsis C5a (75) homeo (68) melanogaster (53) (85) coli histone H5 chicken DH 3.7. 25 OC Zarbock (1986) ., pH 5.2, 40 OC Clore (1989) (77) interleukin 8 human lac Repressor E. coli pH 6.9, 18 OC Zuiderwig (1983) plastocyanin spinach pH 7.3, 35 OC