Compact thermally-denatured state of a staphylococcal nuclease mutant from resonance energy transfer measurements.
Thermal denaturation of a staphylococcal nuclease mutant K78C, where lysine 78 is replaced by cysteine, was studied by circular dichroism (CD) and resonance energy transfer. CD spectra suggest that residual structures remain in the denatured state. Steady-state energy transfer from intrinsic tyrosines to a single and intrinsic tryptophan was measured at different temperatures. In the thermally-denatured state of K78C, there is still a substantial degree of energy transfer from tyrosine(s) to tryptophan, indicating residual structures in the denatured state. The cysteine residue in mutant K78C was labeled with a cysteine specific probe IAEDANS. Fluorescence decays of the tryptophan were measured to estimate distance distributions between Trp 140 and IAEDANS at position 78. Measurements were done as a function of temperature from 4 degrees C (native) to 65 degrees C (denatured) both with and without Ca2+ and inhibitor pdTp. Below 30 degrees C, the apparent distance distribution of both the ligand-free nuclease and the enzyme with bound pdTp can be adequately described by a Gaussian model. Above 40 degrees C, where the ligand-free nuclease but not the ternary complex begins to denature, two different populations are required to fit the data both with and without pdTp. One population has a compact structure and the other has an expanded structure. As temperature rises, the population of the expanded structure increases. At the highest temperature, the non-native compact structure is still the major form (60 to 70%). The overall thermally-denatured states of staphylococcal nuclease mutant K78C in the absence and presence of ligands are thus compact and heterogeneous.
Wu
P G
PG
Department of Biology, Johns Hopkins University, Baltimore, MD 21218.
James
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E
Brand
L
L
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002942:Circular Dichroism; D005456:Fluorescent Dyes / Q000737:chemistry; D006361:Heating; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D009282:Naphthalenesulfonates / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence / Q000379:methods; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D014443:Tyrosine; C006421:1,5-I-AEDANS; D014364:Tryptophan; D008836:Micrococcal Nuclease