COMPACT DENATURED STATE OF A STAPHYLOCOCCAL NUCLEASE MUTANT BY GUANIDINIUM AS DETERMINED BY RESONANCE ENERGY-TRANSFER
COMPACT DENATURED STATE OF A STAPHYLOCOCCAL NUCLEASE MUTANT BY GUANIDINIUM AS DETERMINED BY RESONANCE ENERGY-TRANSFER
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DOI：
10.1021/bi00157a008
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发表时间：
1992-10-27
期刊：
影响因子：
2.9
通讯作者：
BRAND, L
中科院分区：
文献类型：
--
作者：
JAMES, E;WU, PG;BRAND, L
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 angstrom and an "apparent" full width at half-maximum (fwhm) of distance distribution of approximately 18 angstrom. 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.
