Conformational flexibility in a staphylococcal nuclease mutant K45C from time-resolved resonance energy transfer measurements.
Thermal fluctuations exist in native proteins and other macromolecules in solution. Some may play a role in ligand or receptor binding, control rates of enzymatic catalysis, or define a range of conformations a segment can adopt in solution. We apply the method of time-resolved resonance energy transfer to study the conformational flexibility of a staphylococcal nuclease mutant, K45C, where lysine 45 located at a flexible loop is replaced by a cysteine. We labeled the thiol group with DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) and used the TNB group covalently attached to the protein as an energy acceptor from a single tryptophan at residue 140 as the donor. Conformational flexibility occurring on the time scale of nanoseconds or longer is dispersed as an apparent distance distribution in time-resolved resonance energy transfer measurements. Below room temperature the apparent distance distribution was fitted with a symmetric Lorentzian model with a full width at half maximum height of about 6 A, indicating substantial degrees of heterogeneity between residues 45 and 140. At room or higher temperature where the protein is in its native state, the apparent distance distribution is asymmetric, indicating the presence of static disorders. Segments in the protein that contribute to the static disorder can be converted to mobile ones with the addition of denaturing guanidinium chloride.
Wu
P
P
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
Brand
L
L
D004735:Energy Transfer; D004795:Enzyme Stability; D019791:Guanidine; D006146:Guanidines; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000235:genetics; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics
D006146:Guanidines; D012996:Solutions; D008836:Micrococcal Nuclease; D019791:Guanidine