title: Effects of excluded volume upon protein stability in covalently cross-linked proteins with variable linker lengths.
abstract: To explore the effects of molecular crowding and excluded volume upon protein stability, we used a series of cross-linking reagents with nine different single-cysteine mutants of staphylococcal nuclease to make covalently linked dimers. These cross-linkers ranged in length from 10.5 to 21.3 A, compelling separations which would normally be found only in the most concentrated protein solutions. The stabilities of the dimeric proteins and monomeric controls were determined by guanidine hydrochloride and thermal denaturation. Dimers with short linkers tend to exhibit pronounced three-state denaturation behavior, as opposed to the two-state behavior of the monomeric controls. Increasing linker length leads to less pronounced three-state behavior. The three-state behavior is interpreted in a three-state model where cross-linked native protein dimer, N-N, interconverts in a two-state transition with a dimer where one protein subunit is denatured, N-D. The remaining native protein in turn can denature in another two-state transition to a state, D-D, in which both tethered proteins are denatured. Three-state behavior is best explained by excluded volume effects in the denatured state. For many dimers, linkers longer than 17 A removed most three-state character. This sets a limit on the flexibility and size of the denatured state. Notably, in contradiction to theoretical predictions, these cross-linked dimers were not stabilized. The failure of these predictions is possibly due to neglect of the alteration in hydrophobic exposure that accompanies any significant reduction in the conformational space of the denatured state.
authors.affiliation: Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701-1021, USA.
authors.forename: Yun Ho
authors.initials: YH
authors.lastname: Kim
authors.forename: Wesley E
authors.initials: WE
authors.lastname: Stites
mesh_terms: D003432:Cross-Linking Reagents / Q000737:chemistry; D019791:Guanidine; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D017711:Nonlinear Dynamics; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding
chemical_list: D003432:Cross-Linking Reagents; D008836:Micrococcal Nuclease; D019791:Guanidine