Olofsson Maria, Kalinin Stanislav, Zdunek Janusz, Oliveberg Mikael, Johansson Lennart B-A
Biophysical Chemistry and Biochemistry, Department of Chemistry, Umeå University, S-901 87 Umeå, Sweden.
Phys Chem Chem Phys. 2006 Jul 14;8(26):3130-40. doi: 10.1039/b601313a. Epub 2006 May 31.
We demonstrate that Tryptophan (Trp) and N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl iodoacetamide (BODIPY) is a suitable donor-acceptor (D-A) pair for intraprotein distance measurements, applicable to the study of protein folding. The suitability of the Trp-BODIPY electronic energy transfer is exemplified on the extensively-characterised two-state protein, S6, from Thermus thermophilus. This protein has proved to be useful for the elucidation of folding cooperativity and nucleation, as well as the changes upon induction of structural transitions. For a comprehensive structural coverage, BODIPY molecules were anchored by Cys insertions at four different positions on the S6 surface. Trp residues at position 33 or 62 acted as donors of electronic energy to the BODIPY groups. None of the D-A pairs show any detectable difference in the folding kinetics (or protein stability), which supports the notion that the two-state transition of S6 is a highly concerted process. Similar results are obtained for mutants affecting the N- and C-terminus. The kinetic analyses indicate that changes of the transition state occur through local unfolding of the native state, rather than by a decrease of the folding cooperativity. The distances obtained from the analysis of the time-resolved fluorescence experiments in the native state were compared to those calculated from X-ray structure. As an additional measure, molecular dynamics simulations of the different protein constructs were performed to account for variability in the BODIPY location on the protein surface. The agreement between fluorescence and X-ray data is quite convincing, and shows that energy transfer measurements between Trp and BODIPY can probe distances between ca. 17 to 34 A, with an error better than 10%.
我们证明，色氨酸（Trp）和N-（4,4-二氟-5,7-二甲基-4-硼-3a,4a-二氮杂-s-茚并[1,2-b]噻吩-3-基）甲基碘乙酰胺（BODIPY）是用于蛋白质内距离测量的合适供体-受体（D-A）对，适用于蛋白质折叠研究。Trp-BODIPY电子能量转移的适用性在来自嗜热栖热菌的广泛表征的两态蛋白质S6上得到了例证。这种蛋白质已被证明有助于阐明折叠协同性和成核作用以及结构转变诱导后的变化。为了进行全面的结构覆盖，通过在S6表面的四个不同位置插入半胱氨酸来锚定BODIPY分子。33位或62位的Trp残基作为向BODIPY基团的电子能量供体。没有一个D-A对在折叠动力学（或蛋白质稳定性）上显示出任何可检测到的差异，这支持了S6的两态转变是一个高度协同过程的观点。对于影响N端和C端的突变体也获得了类似的结果。动力学分析表明，过渡态的变化是通过天然态的局部解折叠发生的，而不是通过折叠协同性的降低。将在天然态下通过时间分辨荧光实验分析得到的距离与从X射线结构计算得到的距离进行了比较。作为一项额外的措施，对不同蛋白质构建体进行了分子动力学模拟，以考虑BODIPY在蛋白质表面位置的变异性。荧光数据和X射线数据之间的一致性非常令人信服，表明Trp和BODIPY之间的能量转移测量可以探测约17至34埃之间的距离，误差优于10%。
