Clark P L, Liu Z P, Zhang J, Gierasch L M
Molecular Biophysics Graduate Program, University of Texas Southwestern Medical Center at Dallas 75235, USA.
Protein Sci. 1996 Jun;5(6):1108-17. doi: 10.1002/pro.5560050613.
The native state fluorescence and CD spectra of the predominantly beta-sheet cellular retinoic acid-binding protein I (CRABPI) include contributions from its three tryptophan residues and are influenced by the positions of these residues in the three-dimensional structure. Using a combination of spectroscopic approaches and single Trp-mutants of CRABPI, we have deconvoluted these spectra and uncovered several features that have aided in our analysis of the development of structure in the folding pathway of CRABPI. The emission spectrum of native CRABPI is dominated by Trp 7. Trp 109 is fluorescence-silent due to its interaction with the guanidino group of Arg 111. Although the far-UV CD spectrum of CRABPI is largely determined by the protein's secondary structure, aromatic clustering around Trp 87 and the aromatic-charge interaction between Arg 111 and Trp 109 give rise to a characteristic feature in the CD spectrum at 228 nm. The near-UV CD bands of CRABPI arise largely from additive contributions of the three tryptophan residues. Trp 7 and Trp 87 give a negative CD band at 275 nm. The near-UV CD band from Trp 109 is positive and shifted to longer wavelengths (to 302 nm) due to the charge-aromatic interaction between Arg 111 and Trp 109. Our deconvolution of the equilibrium spectra have been used to interpret kinetic folding experiments monitored by stopped-flow fluorescence. These dynamic experiments suggest the early evolution of a well-populated, hydrophobically collapsed intermediate, which undergoes global rearrangement to form the fully folded structure. The results presented here suggest several additional strategies for dissecting the folding pathway of CRABPI.
主要为β-折叠的细胞视黄酸结合蛋白I（CRABPI）的天然态荧光和圆二色光谱包含其三个色氨酸残基的贡献，并受这些残基在三维结构中位置的影响。通过结合光谱学方法和CRABPI的单个色氨酸突变体，我们对这些光谱进行了反褶积，并发现了几个有助于我们分析CRABPI折叠途径中结构形成的特征。天然CRABPI的发射光谱以Trp 7为主。Trp 109由于与Arg 111的胍基相互作用而荧光沉默。尽管CRABPI的远紫外圆二色光谱在很大程度上由蛋白质的二级结构决定，但Trp 87周围的芳香族聚集以及Arg 111与Trp 109之间的芳香族-电荷相互作用在228 nm处产生了圆二色光谱的一个特征峰。CRABPI的近紫外圆二色带主要来自三个色氨酸残基的叠加贡献。Trp 7和Trp 87在275 nm处产生一个负的圆二色带。由于Arg 111与Trp 109之间的电荷-芳香族相互作用，Trp 109的近紫外圆二色带为正，并向更长波长（至302 nm）移动。我们对平衡光谱的反褶积已用于解释通过停流荧光监测的动力学折叠实验。这些动态实验表明，一个大量存在的、疏水塌缩中间体的早期演化，该中间体经历全局重排以形成完全折叠的结构。此处给出的结果为剖析CRABPI的折叠途径提出了几种额外的策略。
