While may small proteins can refold spontaneously in vitro, the identification of the folding pathway, and the detection and structural characterization of folding intermediates have been difficult. Recently, attention has turned to the molten globule state and other nonnative equilibrium states of proteins which are thought to be models for kinetic intermediates in protein folding. Fragments of staphylococcal nuclease have been produced which also have properties similar to those of the molten globule, i.e. a somewhat compact structure with some secondary structure but without a defined tertiary structure. Pulsed hydrogen-deuterium exchange during refolding has been used to probe the protection of backbone amide hydrogens from solvent exchange during refolding of a number of proteins and most recently the staphylococcal nuclease Pro 117 yields Gly variant. The extent of exchange for 39 residues is determined by two-dimensional proton NMR after refolding for 5 ms to 10s. Three kinetic phases are inferred. Modest protection of amides in the early refolding intermediate composed to two beta-sheets formed by local sequence interactions is observed after a 5 ms refolding period. Native levels of protection throughout the molecule accrue more slowly in tow kinetic phases (k approximately 2s-1, k less than 0.01s-1). Protection factors were determined by varying the high pH labeling pulse after refolding for 100 ms. Little or no native or unfolded protein is present; instead, most molecules are in one or more partially folded states. The intermediate state has modest, yet significant, protection for residues in the beta-sheets (protection factors 10-60), and almost no protection in the alpha- helices (protection factors less than 10). The pattern of labeling is consistent with a role for beta turns and beta-hairpins in the formation of the early intermediate. Recently a chemical cleavage method has been developed where an EDT-Fe based reagent (EPD-Fe) can be attached to a protein via a cysteine side chain. The addition of ascorbate generates hydroxyl radicals at the iron center which diffuse and cleave the polypeptide backbone in a region close to the cysteine attachment site at residues accessible to solvent. The observed cleavage sites can be mapped by amino acid sequencing. The cleavage is dependent on protein conformation. We propose characterize the molten globule state of apomyoglobin and staphylococcal nuclease fragment structures using this newly developed chemical cleavage technique. We will prepare a number of cysteine variants of these proteins and characterize the cleavage patterns observed in the native and molten globule states.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM051332-02
Application #
2634753
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1997-01-01
Project End
2000-12-31
Budget Start
1998-01-01
Budget End
1998-12-31
Support Year
2
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Texas Medical Br Galveston
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041367053
City
Galveston
State
TX
Country
United States
Zip Code
77555
Schultz, David A; Friedman, Alan M; White, Mark A et al. (2005) The crystal structure of the cis-proline to glycine variant (P114G) of ribonuclease A. Protein Sci 14:2862-70
Ferguson, Monique R; Fan, Xiuzhen; Mukherjee, Munia et al. (2004) Directed discovery of bivalent peptide ligands to an SH3 domain. Protein Sci 13:626-32
Zhao, Yonghong; Liu, Deqian; Kaluarachchi, Warna D et al. (2003) The crystal structure of Escherichia coli heat shock protein YedU reveals three potential catalytic active sites. Protein Sci 12:2303-11
Biswas, R; Ledman, D W; Fox, R O et al. (2000) Mapping RNA-protein interactions in ribonuclease P from Escherichia coli using disulfide-linked EDTA-Fe. J Mol Biol 296:19-31
Hall, K B; Fox, R O (1999) Directed cleavage of RNA with protein-tethered EDTA-Fe. Methods 18:78-84
Bhat, M G; Ganley, L M; Ledman, D W et al. (1997) Stability studies of amino acid substitutions at tyrosine 27 of the staphylococcal nuclease beta-barrel. Biochemistry 36:12167-74