Leucine

ABSTRACT Studies by one-dimensional NMR are reported on the interconversion of folded and unfolded forms of the GCN4 leucine zipper in neutral saline buffer. The peptide bears 99% ^sup 13^C^sup alpha^ labels at three sites: V9, Li 2, and G31. Time-domain ^sup 13^C^sup alpha^-NMR spectra are interpreted by global Bayesian lineshape analysis to extract the rate constants for both unfolding and folding as functions of temperature in the range 47-71 deg C. The data are well fit by the assumption that the same rate constants apply at each labeled site, confirming that only two conformational states need be considered. Results show that 1) both processes require a free energy of activation; 2) unfolding is kinetically enthalpy-opposed and entropy-driven, while folding is the opposite; and 3) the transition state dimer ensemble averages ~40% helical. The activation parameters for unfolding, derived from NMR data at the elevated temperatures where both conformations are populated, lead to estimates of the rate constant at low temperatures (5-15 deg C) that agree with extant values determined by stopped-flow CD via dilution from denaturing media. However, the corresponding estimated values for the folding rate constant are larger by two to three orders of magnitude than those obtained by stopped flow. We propose that this apparent disagreement is caused by the necessity, in the stopped-flow experiment, for initiation of new helices as the highly denaturant-unfolded molecule adjusts to the newly created benign solvent conditions. This must reduce the success rate of collisions in producing the folded molecule. In the NMR determinations, however, the unfolded chains always have a small, but essential, helix content that makes such initiation unnecessary. Support for this hypothesis is adduced from recent extant experiments on the helix-coil transition in single-- chain helical peptides and from demonstration that the folding rate constants for coiled coils, as obtained by stopped flow, are influenced by the nature of the denaturant used.

INTRODUCTION

The leucine zipper of the GCN4 transcription factor (called here GCN4-1z) has become one of the most often used models for examining questions concerning the thermodynamics and kinetics of protein folding. Its popularity stems both from its structural simplicity and from our relatively reliable understanding of the relationship between amino acid sequence and structure in proteins of its type (Crick, 1953; McLachlan and Stewart, 1975).

Structurally, GCN4-lz is a coiled coil in benign media; it consists of two 33-residue right-handed a-helical chains, arranged side by side in parallel and register, and with a slight negative supertwist (O'Shea et al., 1989, 1991). Such structures are known to result from sequences with a pseudo-heptad repeat, the amino acids of which are designated abcdefg, and in which residues a and d are hydrophobic and e and g oppositely charged (McLachlan and Stewart, 1975; Lupas, 1996). The resulting amphipathicity of the helices leads to helix-helix interactions that direct parallel, registered dimerization. GCN4-lz is also a leucine zipper, a type of coiled coil in which the d residue positions are predominantly occupied by leucines.

Development of the Bayesian techniques was supported by the National Institutes of Health Grant NS-35912 and by a license agreement with Varian Associates. Mass spectrometry was provided by the Washington University Mass Spectrometry Resource, a National Institutes of Health Research Resource (Grant P41RR0954). Peptide syntheses were performed at the Albert Einstein College of Medicine and were supported in part by a grant from the Mathers Foundation. One of us (A.H.) acknowledges the continuing support of the Luftmensch Society.

REFERENCES

Bilsel, O., and C. R. Matthews. 2000. Barriers in protein folding reactions. Adv. Protein Sci. 53:153-207.

Bretthorst, G. L. 1990a. Bayesian analysis. I. Parameter estimation using quadrature NMR models. J. Magn. Reson. 88:533-551.

Bretthorst, G. L. 1990b. Bayesian analysis. II. Model selection. J. Magn. Reson. 88:552-570.

Bretthorst, G. L. 1990c. Bayesian analysis. III. Applications to NMR signal detection, model selection, and parameter estimation. J. Magn. Reson. 88:571-595.

Bretthorst, G. L. 1997. Bayesian Analysis Software Package User Guide. Pub. No. 87-190172-00. Rev. A0197, Varian Associates, Palo Alto, CA. Carrington, A., and A. D. McLachlan. 1967. Introduction to Magnetic Resonance, Chaps. 11 and 12. Harper and Row, New York.

Clarke, D. T., A. J. Doig, B. J. Stapely, and G. R. Jones. 1999. The a-helix folds on the millisecond time scale. Proc. Natl. Acad. Sci. USA. 96: 7232-7237.

Crick, F. 1953. The Fourier transform of a coiled coil. Acta Crystallogr. 6:689-697.

d'Avignon, D. A., G. L. Bretthorst, M. E. Holtzer, and A. Holtzer. 1998. Site-specific thermodynamics and kinetics of a coiled-coil transition by spin inversion transfer NMR. Biophys. J. 74:3190-3197.

d'Avignon, D. A., G. L. Bretthorst, M. E. Holtzer, and A. Holtzer. 1999. Thermodynamics and kinetics of a folded-folded' transition at valine 9 of a GCN4-like leucine zipper. Biophys. J. 76:2752-2759.

Edelhoch, H. 1967. Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry. 6:1948-1954.

Gilks, W. R., S. Richardson, and D. J. Spiegelhalter. 1996. Markov Chain Monte Carlo in Practice. Chapman & Hall, London.

Holtzer, M. E., D. L. Crimmins, and A. Holtzer. 1995. Structural stability of short subsequences of the tropomyosin chain. Biopolymers. 35: 125-136.

Holtzer, M. E., and A. Holtzer. 1992. a-Helix to random coil transitions: determination of peptide concentration from the CD at the isodichroic point. Biopolymers. 32:1675-1677.

Holtzer, M. E., E. G. Lovett, D. A. d'Avignon, and A. Holtzer. 1997. Thermal unfolding in a GCN4-like leucine zipper: '3C"-NMR chemical shifts and local unfolding equilibria. Biophys. J. 73:1031-1041.

Kenar, K. T., B. Garcia-Moreno, and E. Freire. 1995. A calorimetric characterization of the salt dependence of the stability of the GCN4 leucine zipper. Protein Sci. 4:1934-1938.

Lovett, E. G., D. A. d'Avignon, M. E. Holtzer, E. H. Braswell, D. Zhu, and A. Holtzer. 1996. Observation via one-dimensional ^sup 13^C^sup alpha^ NMR of local conformational substates in thermal unfolding equilibria of a synthetic analog of the GCN4 leucine zipper. Proc. Natl. Acad. Sci. USA. 93: 1781-1785.

Lupas, A. 1996. Coiled coils: new structures and new functions. TIBS 21:375-382.

McConnell, H. M. 1958. Reaction rates by nuclear magnetic resonance. J. Chem Phys. 28:430-431.

McLachlan, A. D., and M. Stewart. 1975. Tropomyosin coiled-coil interactions. Evidence for an unstaggered structure. J. MoL BioL 98:293-304. Mo, J., M. E. Holtzer, and A. Holtzer. 1991. Kinetics of self-assembly of

aa-tropomyosin coiled coils from unfolded chains. Proc. Natl. Acad. Sci. USA. 88:916-920.

Moran, L. B., J. P. Schneider, A. Kentsis, G. A. Reddy, and T. R. Sosnick. 1999. Transition state heterogeneity in GCN4 coiled coil folding studied by using multisite mutations and crosslinking. Proc. Natl. Acad. Sci. USA. 96:10699-10704.

Myers, J. K., and T. G. Oas. 1999. Reinterpretation of GCN4-pl folding kinetics: partial helix formation precedes dimerization in coiled coil folding. J. Mol, Biol. 289:205-209.

O'Shea, E. K., J. D. Klemm, P. S. Kim, and T. Alber. 1991. X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil. Science. 254:539-544.

O'Shea, E. K., R. Rutkowski, and P. S. Kim. 1989. Evidence that the leucine zipper is a coiled coil. Science. 243:538-542.

Poland, D., and H. A. Scheraga. 1970. Theory of the Helix-Coil Transition in Biopolymers. Academic Press, New York.

Potekhin, S. A., and P. L. Privalov. 1982. Co-operative blocks in tropomyosin. J. Mol. Biol. 159:519-535.

Rudin, M., and A. Sauter. 1992. Measurement of reaction rates in vivo using magnetization transfer techniques. In NMR Basic Principles and Progress. P. Diehl, E. Fluck, H. Gunther, R. Kosfeld, and J. Seelig, editors. Springer Verlag, Berlin.

Schwarz, G. 1965. On the kinetics of the helix-coil transition of polypeptides in solution. J. Mol. Biol. 11:64-77.

Sosnick, T. R., S. Jackson, R. R. Wilk, W. Englander, and W. F. DeGrado. 1996. The role of helix formation in the folding of a fully a-helical coiled coil. Proteins: Struct., Funct., Genet. 24:427-432.

Zitzewitz, J. A., 0. Bilsel, J. Luo, B. E. Jones, and C. R. Matthews. 1995. Probing the folding mechanism of a leucine zipper peptide by stopped-- flow circular dichroism spectroscopy, Biochemistry. 34:12812-12819.

Zitzewitz, 1. A., B. Ibarra-Molero, D. R. Fishel, K. L. Terry, and C. R. Matthews. 2000. Preformed secondary structure drives the association reaction of GCN4-pl, a model coiled-coil system. J. Mol. Biol. 296: 1105-1116.

Marilyn Emerson Holtzer,* G. Larry Bretthorst,* D. Andre d'Avignon,* Ruth Hogue Angeletti,^ Lisa Mints,^ and Alfred Holtzer*

*Department of Chemistry, Washington University, St. Louis, Missouri 63130-4899; and ^Laboratory for Macromolecular Analysis and Proteomics, Albert Einstein College of Medicine, Bronx, New York 10461 USA

Received for publication 10 July 2000 and in final form 27 November 2000.

Address reprint requests to Dr. Alfred Holtzer, Dept. of Chemistry, Washington University, One Brookings Dr., Campus Box 1134, St. Louis, MO 63130-4899. Tel.: 314-935-6572; Fax: 314-935-4481; E-mail: holtzer@ wuchem.wustl.edu.

(c)2001 by the Biophysical Society

0006-3495/01/02/939/13 $2.00

Copyright Biophysical Society Feb 2001
Provided by ProQuest Information and Learning Company. All rights Reserved