Ultraviolet resonance Raman studies reveal the environment of tryptophan and tyrosine residues in the native and partially folded states of the E colicin-binding immunity protein Im7

被引:27
作者
Rodriguez-Mendieta, IR
Spence, GR
Gell, C
Radford, SE
Smith, DA [1 ]
机构
[1] Univ Leeds, Sch Phys & Astron, Inst Mol Biophys, Leeds LS2 9JT, W Yorkshire, England
[2] Univ Leeds, Sch Biochem & Microbiol, Leeds LS2 9JT, W Yorkshire, England
[3] Univ Leeds, Sch Phys & Astron, Astbury Ctr Struct Mol Biol, Leeds LS2 9JT, W Yorkshire, England
基金
英国生物技术与生命科学研究理事会;
关键词
D O I
10.1021/bi047746k
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Understanding the nature of partially folded proteins is a challenging task that is best accomplished when several techniques are applied in combination. Here we present ultraviolet resonance Raman (UVRR) spectroscopy studies of the E colicin-binding immunity proteins, Im7* and Im9*, together with a series of variants of Im7* that are designed to trap a partially folded state at equilibrium. We show that the environments of the tryptophan and tyrosine residues in native wild-type Im7* and Im9* are indistinguishable, in contrast with models for their structures based on X-ray and NMR methods. In addition, we show that there is a general increase in the hydrophobicity in the environment of Trp75 in all of the variants compared with wild-type Im7*. These data suggest that a significant rearrangement of the tryptophan pocket occurs in the variants, which, together with an overall decrease in solvent accessibility of Trp75 as judged by time-resolved fluorescence lifetime measurements and fluorescence quenching experiments, rationalize the unusual fluorescence properties of the variants reported previously. The data highlight the power of UVRR in analyzing the structural properties of different conformational states of the same protein and reveal new information about the structural rearrangements occurring during Im7* folding, not possible using other spectroscopic methods alone. Finally, we describe a previously unreported dependence of the tryptophan Fermi doublet on excitation wavelength in the ultraviolet region revealed by these protein spectra. We corroborated this observation using tryptophan-containing model compounds and conclude that the conventional interpretation of this UVRR feature at these wavelengths is unreliable.
引用
收藏
页码:3306 / 3315
页数:10
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