The exchange kinetics of over 70% of the 143 backbone amide hydrogens in staphylococcal nuclease H124L (nuclease H124L), both in its unligated state and in its ternary complex with Ca2+ and thymidine 3',5'-bisphosphate, have been quantified by nitrogen-15 resolved proton nuclear magnetic resonance spectroscopy. Protection factors for the slowly exchanging hydrogens in unligated nuclease H124L at 37 degrees C and pH 5.5 were found to vary by over one order of magnitude. This range of protection factors has been interpreted in the framework of global and local structural fluctuations. The three most highly protected hydrogens (K24, L25, M26) map to strand 2 of the central five-stranded beta-barrel. The free energy change for the opening reaction which exposes these hydrogens to the solvent (delta G(degree)op) was calculated from the exchange rates in the native and denatured states, the latter values being estimated from model peptide exchange studies [Molday, R. S., Englander, S. W., & Kallen, R. G. (1972) Biochemistry 11, 150-158]. Close agreement was found between delta G(degree)op and delta G(degree)u, the free energy change of unfolding as measured by urea denaturation experiments. Exchange of these hydrogens thus appears to occur via global unfolding of the protein. One region exhibited somewhat lower protection factors: it mapped to the C-terminal portions of helix 2 and helix 3 and to part of the intervening segment. This region has been identified as a minor hydrophobic domain of nuclease [Shortle, D., Stites, W. E., & Meeker, A. K. (1990) Biochemistry 29, 8033-8041].(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 4623, 4624 Extra Details: structural fluctuations; five-stranded beta-barre;,cis<->trans isomerization; hydrogen-bonded; hydrogen exchange
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:26 p.m.
|Number of data points||2|
|Proteins||Thermonuclease ; Thermonuclease|
|Assays/Quantities/Protocols||Experimental Assay: dG_H2O ; Experimental Assay: dG_H2O|
|Libraries||Mutations for sequence ATSTKKLHKEPATLIKAIDGDTVKLMYKGQPMTFRLLLVDTPETKHPKKGVEKYGPEASAFTKKMVENAKKIEVEFDKGQRTDKYGRGLAYIYADGKMVNEALVRQGLAKVAYVYKPNNTHEQHLRKSEAQAKKEKLNIWSEDNADSGQ|