Folding studies on proteases by the conventional hydrogen exchange experiments are severely hampered because of interference from the autolytic reaction in the interpretation of the exchange data. We report here NMR identification of the hierarchy of early conformational transitions (folding propensities) in HIV-1 protease by systematic monitoring of the changes in the state of the protein as i...

BACKGROUND Proteins with low sequence identity but almost identical tertiary structure and function have been valuable to uncover the relationship between sequence, tertiary structure, folding mechanism and functions. Two homologous chemokines, CCL11 and CCL24, with low sequence identity but similar tertiary structure and function, provide an excellent model system for respective studies. RES...

The structure and stability of the fluorescent protein monomeric Kusabira Orange (mKO), a GFP-like protein, was studied under different pressure levels and in different chemical environments. At different pH values (between pH 7.4 and pH 4.0) and under a pressure up to 600 MPa (at 25 °C), mKO did not show significant fluorescence spectral changes, indicating a structural stability of the protei...

The refolding of guanidine hydrochloride-denatured horse heart ferricytochrome c at pH 7.0 and 23 degrees C occurs in three kinetic phases as observed by stopped flow measurements using changes in Soret absorbance or in tryptophan fluorescence. The three kinetic phases have time constants of 10 +/- 5 ms, 240 +/- 30 ms, and 13 +/- 3 s accounting for 15 +/- 5%, 70 +/- 5%, and 15 +/- 5% of the tot...

The kinetics of electron transfer from the triplet-excited Zn-porphyrin to a Ru(NH(3))(5)(His-33)(3+) complex have been measured in Zn-substituted ruthenium-modified cytochrome c under denaturing conditions. In the folded protein, the electron-tunneling rate constant is 7.5 x 10(5) s(-1). As the protein is denatured with guanidine hydrochloride, a faster adiabatic electron-transfer reaction app...

A large proportion of adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermannii is isolated in an inactive form which contains a tightly bound cobalamin. Even when the enzyme was denatured in 5.0 M guanidine hydrochloride the cobalamin remained associated with the protein. However, when dithiothreitol was added to the denatured protein, the pink inhibitor was rapidl...

In 4 M guanidine hydrochloride (GdnHCl), the dimeric enzyme glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides (G6PD) dissociated to subunits and was extensively unfolded. Rapid dilution of this high GdnHCl concentration allowed G6PD to partially renature, as measured by enzyme reactivation, to a level which depended on the conditions employed. The fraction of the enzyme which did...

Baupain belongs to the α+β class of proteins with a secondary structure-content of 44% α-helix, 16% β-sheet and 12% β-turn. The structural transition induced by pH was found to be noncooperative, with no important differences observed in the pH range from 3.0 to 10.5. At pH 2.0 the protein presented substantial non-native structure with strong ANS binding. Guanidine hydrochloride (GdnHCl)-induc...

Nerve growth factor was insolubilized by covalent attachment to Sepharose beads. Nerve growth factor-Sepharose was biologically active in both the neurite outgrowth assay for nerve growth factor and in preserving responsive neurons in vitro. Modification of the bioassay to detect solubilized activity of nerve growth factor and histological examination of ganglia treated with nerve growth factor...

The glucagon immunoreactivity present in canine pancreatic extracts can be separated by gel filtration into two fractions; one fraction, comprising over 90% of the immunoreactivity, is similar in molecular size to the glucagonla11 marker, while the other appears to be at least twice as large. Incubation of this large glucagon immunoreactivity (LGI) in solutions such as 1 M acetic acid, 8 M urea...