Corrêa Fernando, Farah Chuck S
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil.
Biophys J. 2007 Apr 1;92(7):2463-75. doi: 10.1529/biophysj.106.098541. Epub 2007 Jan 11.
Tropomyosin (Tm) is a dimeric coiled-coil protein, composed of 284 amino acids (410 A), that forms linear homopolymers through head-to-tail interactions at low ionic strength. The head-to-tail complex involves the overlap of approximately nine N-terminal residues of one molecule with nine C-terminal residues of another Tm molecule. In this study, we investigate the influence of 2,2,2-trifluoroethanol (TFE) and glycerol on the stability of recombinant Tm fragments (ASTm1-142, Tm143-284(5OHW269)) and of the dimeric head-to-tail complex formed by the association of these two fragments. The C-terminal fragment (Tm143-284(5OHW269)) contains a 5-hydroxytryptophan (5OHW) probe at position 269 whose fluorescence is sensitive to the head-to-tail interaction and allows us to accompany titrations of Tm143-284(5OHW269) with ASTm1-142 to calculate the dissociation constant (Kd) and the interaction energy at TFE and glycerol concentrations between 0% and 15%. We observe that TFE, but not glycerol, reduces the stability of the head-to-tail complex. Thermal denaturation experiments also showed that the head-to-tail complex increases the overall conformational stability of the Tm fragments. Urea and thermal denaturation assays demonstrated that both TFE and glycerol increase the stability of the isolated N- and C-terminal fragments; however, only TFE caused a significant reduction in the cooperativity of unfolding these fragments. Our results show that these two cosolvents stabilize the structures of individual Tm fragments in different manners and that these differences may be related to their opposing effects on head-to-tail complex formation.
原肌球蛋白（Tm）是一种二聚体卷曲螺旋蛋白，由284个氨基酸（410埃）组成，在低离子强度下通过头对头相互作用形成线性同聚物。头对头复合物涉及一个分子的大约九个N端残基与另一个Tm分子的九个C端残基的重叠。在本研究中，我们研究了2,2,2-三氟乙醇（TFE）和甘油对重组Tm片段（ASTm1-142、Tm143-284(5OHW269)）以及由这两个片段缔合形成的二聚体头对头复合物稳定性的影响。C端片段（Tm143-284(5OHW269)）在第269位含有一个5-羟基色氨酸（5OHW）探针，其荧光对头部到尾部的相互作用敏感，使我们能够在TFE和甘油浓度在0%至15%之间时，伴随ASTm1-142对Tm143-284(5OHW269)的滴定来计算解离常数（Kd）和相互作用能。我们观察到TFE而非甘油降低了头对头复合物的稳定性。热变性实验还表明，头对头复合物增加了Tm片段的整体构象稳定性。尿素和热变性分析表明，TFE和甘油都增加了分离的N端和C端片段的稳定性；然而，只有TFE导致这些片段展开的协同性显著降低。我们的结果表明，这两种共溶剂以不同方式稳定单个Tm片段的结构，并且这些差异可能与它们对头部到尾部复合物形成的相反作用有关。
