Changes in side chain packing during apomyoglobin folding characterized by pulsed thiol-disulfide exchange.
It is clear that close-packed side chain interactions play a dominant role in stabilizing native proteins, but the extent to which they stabilize kinetic intermediates and shape the energetic landscape of folding is not known. A method for characterizing structural changes at the level of individual side chains is presented and applied to study the refolding of apomyoglobin mutants containing engineered cysteine residues at key helical packing interfaces. The formation of buried side chain structure at the probe sites is followed by the extent of thiol-disulfide exchange during a pulse of thiol labeling reagent (either methyl methanethiosulfonate or 5,5'-dithiobis (2-nitrobenzoic acid)) applied at various stages of folding. The results suggest that the eight helices pack in at least three distinct stages, involving formation of two intermediates with time constants of <2 ms and 50 ms. In some parts of the refolding protein, stable side chain structure can be attained very rapidly, possibly in advance of backbone hydrogen bond formation as detected by previous pulsed amide hydrogen exchange experiments.
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Department of Biochemistry and Molecular Biology, SUNY Health Science Center at Syracuse, New York 13210, USA.
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D000603:Amino Acids, Sulfur; D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D003553:Cystine / Q000737:chemistry* / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D017022:Flow Injection Analysis; D007700:Kinetics; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009211:Myoglobin / Q000737:chemistry* / Q000235:genetics; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013439:Sulfhydryl Reagents
D000603:Amino Acids, Sulfur; D001059:Apoproteins; D004220:Disulfides; D009211:Myoglobin; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; C010857:apomyoglobin; C014674:methyl methanethiosulfonate; D003553:Cystine; D004228:Dithionitrobenzoic Acid; D008741:Methyl Methanesulfonate; D003545:Cysteine