Conserved thermodynamic contributions of backbone hydrogen bonds in a protein fold

doi:10.1073/pnas.0508121103

Conserved thermodynamic contributions of backbone hydrogen bonds in a protein fold

Department of Chemistry, Duke University, Durham, NC 27708

Edited by Harold A. Scheraga, Cornell University, Ithaca, NY, and approved November 18, 2005 (received for review September 19, 2005)

Abstract

Backbone–backbone hydrogen-bonding interactions are a ubiquitous and highly conserved structural feature of proteins that adopt the same fold (i.e., have the same overall backbone topology). This work addresses the question of whether or not this structural conservation is also reflected as a thermodynamic conservation. Reported here is a comparative thermodynamic analysis of backbone hydrogen bonds in two proteins that adopt the same fold but are unrelated at the primary amino acid sequence level. With amide-to-ester bond mutations introduced by total chemical synthesis methods, the thermodynamic consequences of backbone–backbone hydrogen-bond deletions at five different structurally equivalent positions throughout the β-α-α fold of Arc repressor and CopG were assessed. The ester bond-containing analogues all folded into native-like three-dimensional structures that were destabilized from 2.5 to 6.0 kcal/(mol dimer) compared with wild-type controls. Remarkably, the five paired analogues with amide-to-ester bond mutations at structurally equivalent positions were destabilized to exactly the same degree, regardless of the degree to which the mutation site was buried in the structure. The results are interpreted as evidence that the thermodynamics of backbone–backbone hydrogen-bonding interactions in a protein fold are conserved.

Footnotes

^‡To whom correspondence should be addressed. E-mail: michael.c.fitzgerald{at}duke.edu
↵ ^†Present address: Department of Chemistry, University of New Mexico, Albuquerque, NM 87131.
Author contributions: M.W., T.E.W., and M.C.F. designed research; M.W. and T.E.W. performed research; M.W., T.E.W., and M.C.F. analyzed data; and M.W., T.E.W., and M.C.F. wrote the paper.
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
↵ § All Arc* analogues are identical to wild-type Arc repressor except that they contain a Pro-to-Leu mutation at position 8, the effects of which are described in the text, and the Arc** analogue contains the Pro-to-Leu mutation at position 8 and an Asp-to-Ala mutation at position 20. All CopG* analogues are identical to wild-type CopG except that they contain Glu-to-Ala and amide-to-ester bond mutations at position 11, the effects of which are described in the text. The CopG** analogue includes the CopG* mutations noted above as well as a Glu-to-Ala mutation at position 15. In all parenthetical notations, the O refers to the ester bond, the second letter refers to the one-letter code of the amino acid (e.g., A, alanine; L, leucine; I, isoleucine; V, valine) that is C-terminal to the ester bond, and the number refers to the position of this C-terminal amino acid in the protein’s polypeptide chain. For example, (OL12)Arc* means that there is an ester-bond mutation N-terminal to the Leu at position 12 in the 53-aa polypeptide chain of Arc*.
↵ ¶ Two additional CopG analogues were also chemically synthesized and subject to thermodynamic analysis. The analogues included (KOA3)CopG* that contained both Lys-to-Ala and ester-bond mutations at the third amino acid position in CopG*’s polypeptide chain (see the β-b position in Fig. 2 D), and (KA3)CopG* that contained just the Lys-to-Ala mutation. The Ala mutation facilitated the incorporation of the ester bond and had minimal impact on the protein’s thermodynamic stability (as determined in GdmCl-induced equilibrium unfolding experiments) or on the protein’s three-dimensional structure (as determined by far-UV CD spectroscopy) (see Figs. 3 and 4). The ester bond effectively eliminated the outermost two backbone–backbone hydrogen bonds in the intersubunit β-sheet region of CopG* and generated a subunit interface with the identical number of hydrogen bonds as an ester bond-containing Arc analogue (POL8)Arc [or (OL8)Arc*] on which we have previously reported (7).
Abbreviations:

Gdm,

guanidinium;

TMAO,

trimethylamine N-oxide.