17 August 1994 Detection of an intermediate late in the unfolding pathway of bacillus stearothermophilus lactate dehydrogenase
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In vivo proteins fold to form one active structure in minutes or seconds, ruling out the possibility that a polypeptide samples all possible conformational space during folding. We have used site directed mutagenesis to produce 15 single tryptophan containing mutants of Bacillus stearothermophilus lactate dehydrogenase (BS LDH) thus enabling the equilibria of unfolding to be seen from 15 defined positions. These mutant versions of BS LDH have the same X-ray structure as the wild type protein8. Previously Smith et al.11 had detected and assigned structures to 4 folding states. The first intermediate, a monomer with secondary and super secondary structure largely intact, is formed after the dimer dissociates at 0.55 M guanidinium hydrochloride (GuHCl). The second intermediate on the unfolding pathway is stable at 2.2 M GuHCl. It had been assumed previously that the transition from this molten-globule structure to the fully denatured form occurred as a single process. We have now identified a core folding motif. In this, helix (alpha) -1F forms a helix-sheet interaction with (beta) -K and (beta) -K has interactions with both (alpha) -2G and (alpha) -3G. This super secondary interaction forms the most stable folding motif in BS LDH and is lost at 2.8 M GuHCl leaving helix (alpha) -1F, (alpha) -2G, and (alpha) -3G which are stable until 3 M GuHCl.
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Roger N. Sleigh, Roger N. Sleigh, David J. Halsall, David J. Halsall, Anthony R. Clarke, Anthony R. Clarke, Moira Behan-Martin, Moira Behan-Martin, J. John Holbrook, J. John Holbrook, } "Detection of an intermediate late in the unfolding pathway of bacillus stearothermophilus lactate dehydrogenase", Proc. SPIE 2137, Time-Resolved Laser Spectroscopy in Biochemistry IV, (17 August 1994); doi: 10.1117/12.182740; https://doi.org/10.1117/12.182740

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