Compact denatured state of a staphylococcal nuclease mutant by guanidinium as determined by resonance energy transfer.
The protein from a mutant clone of staphylococcal nuclease with a cysteine substituting for a lysine at position 78 was prepared and labeled with a cysteine-specific fluorescent probe 5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1-sulfonic acid (IAEDANS). Time-resolved nonradiative energy-transfer studies were done using the single tryptophan at position 140 as the energy donor and the IAEDANS as the receptor. Changes in distance and distance distributions were observed as a function of increasing guanidinium (GuHCl) concentration (0-2 M) and in the presence or absence of Ca2+ and inhibitor 2'-deoxythymidine 3',5'-diphosphate (pdTp). In the native state, both the ternary complex and the noncomplexed protein are best fit with one population having an average donor-acceptor distance of approximately 23 A and an "apparent" full width at half-maximum (fwhm) of distance distribution of approximately 18 A. Besides the contribution of linker arm of the acceptor, it appears that there are some conformational heterogeneties either due to the disordering of the tryptophan region or due to the whole protein in the native state. During GuHCl unfolding, the average distance remains relatively constant up to GuHCl concentrations where both the ternary complex and the ligand-free protein are denatured (1-1.3 M). The compact denatured states persist up to 2 M GuHCl. At 2 M GuHCl, the heterogeneity of the denatured state in the ternary complex is much larger than that of the ligand-free nuclease. The results show that the denatured states of staphylococcal nuclease mutant K78C by GuHCl are compact and these compact denatured states are likely due to residual structures or incompletely disrupted hydrophobic cores under these conditions.
James
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Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
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D000595:Amino Acid Sequence; D003545:Cysteine; D004735:Energy Transfer; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology*; D007700:Kinetics; D008239:Lysine; D008433:Mathematics; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009282:Naphthalenesulfonates; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry; D014364:Tryptophan
D005456:Fluorescent Dyes; D006146:Guanidines; D009282:Naphthalenesulfonates; D011994:Recombinant Proteins; C006421:1,5-I-AEDANS; D014364:Tryptophan; D008836:Micrococcal Nuclease; D019791:Guanidine; D008239:Lysine; D003545:Cysteine