Probing local structural fluctuations in myoglobin by size-dependent thiol-disulfide exchange.
All proteins undergo local structural fluctuations (LSFs) or breathing motions. These motions are likely to be important for function but are poorly understood. LSFs were initially defined by amide hydrogen exchange (HX) experiments as opening events, which expose a small number of backbone amides to (1)H/(2)H exchange, but whose exchange rates are independent of denaturant concentration. Here, we use size-dependent thiol-disulfide exchange (SX) to characterize LSFs in single cysteine-containing variants of myoglobin (Mb). SX complements HX by providing information on motions that disrupt side chain packing interactions. Most importantly, probe reagents of different sizes and chemical properties can be used to characterize the size of structural opening events and the properties of the open state. We use thiosulfonate reagents (126-274 Da) to survey access to Cys residues, which are buried at specific helical packing interfaces in Mb. In each case, the free energy of opening increases linearly with the radius of gyration of the probe reagent. The slope and the intercept are interpreted to yield information on the size of the opening events that expose the buried thiol groups. The slope parameter varies by over 10-fold among Cys positions tested, suggesting that the sizes of breathing motions vary substantially throughout the protein. Our results provide insight to the longstanding question: how rigid or flexible are proteins in their native states?
Stratton
Margaret M
MM
Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210, USA.
Cutler
Thomas A
TA
Ha
Jeung-Hoi
JH
Loh
Stewart N
SN
D002850:Chromatography, Gel / Q000379:methods*; D004220:Disulfides / Q000737:chemistry*; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D009211:Myoglobin / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D017510:Protein Folding; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D004220:Disulfides; D009211:Myoglobin; D013438:Sulfhydryl Compounds