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PEDS Advance Access published online on February 1, 2006
Protein Engineering Design and Selection, doi:10.1093/protein/gzj017
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© The Author 2006. Published by Oxford University Press. All rights reserved
Received January 5, 2006
Accepted January 6, 2006

Article

The relationship between chain connectivity and domain stability in the equilibrium and kinetic folding mechanisms of dihydrofolate reductase from E.coli

Anna-Karin E. Svensson 1, Jill A. Zitzewitz 2, C.Robert Matthews 2 *, and Virginia F. Smith 3

1 Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA; The Huck Institute of Life Sciences, Pennsylvania State University, University Park, PA 16802, USA
2 Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA
3 Chemistry Department, United States Naval Academy, 572 Holloway Road, Annapolis, MD 21402, USA

* To whom correspondence should be addressed.
C.Robert Matthews, E-mail: c.robert.matthews{at}umassmed.edu


  Abstract

The role of domains in defining the equilibrium and kinetic folding properties of dihydrofolate reductase (DHFR) from Escherichia coli was probed by examining the thermodynamic and kinetic properties of a set of variants in which the chain connectivity in the discontinuous loop domain (DLD) and the adenosine-binding domain (ABD) was altered by permutation. To test the concept that chain cleavage can selectively destabilize the domain in which the N- and C-termini are resident, permutations were introduced at one position within the ABD, one within the DLD and one at a boundary between the domains. The results demonstrated that a continuous ABD is required for a stable thermal intermediate and a continuous DLD is required for a stable urea intermediate. The permutation at the domain interface had both a thermal and urea intermediate. Strikingly, the observable kinetic folding responses of all three permuted proteins were very similar to the wild-type protein. These results demonstrate a crucial role for stable domains in defining the energy surface for the equilibrium folding reaction of DHFR. If domain connectivity affects the kinetic mechanism, the effects must occur in the sub-millisecond time range.

Keywords: circular permutation; equilibrium intermediate; protein folding mechanisms; thermal denaturation; urea denaturation.
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