The disulfide structure of denatured epidermal growth factor: preparation of scrambled disulfide isomers

The conformational stability of human epidermal growth factor (EGF) and the structure of denatured EGF were investigated using the technique of disulfide scrambling. Under denaturing conditions and in the presence of a thiol catalyst, the native EGF denatures by shuffling its three native disulfide bonds and converts to a mixture of scrambled isomers. Analysis by HPLC reveals that the denatured EGF is composed of about 10 fractions of scrambled isomers. The heterogeneity varies under different denaturing conditions, with the heat-denatured samples exhibiting the highest degree of heterogeneity. The disulfide structures of eight major scrambled isomers of EGF were determined. The most predominant isomer adopts the bead-form structure with disulfide bonds bridged by three pairs of neighboring cysteines: Cys⁶-Cys¹⁴, Cys²⁰-Cys³¹, and Cys³³-Cys⁴². The denaturation curve of EGF is determined by the relative yield of the scrambled and native species of EGF. EGF is a highly stable molecule and can be effectively denatured only by guanidine chloride at a concentration of greater than 4–5 M. At 8 M urea, less than 16% of the native EGF was denatured. The unusual conformational stability of EGF was compared with that of eight different disulfide proteins that were similarly characterized by the method of disulfide scrambling.     

Unfolding of hirudin characterized by the composition of denatured scrambled isomers

The native core structure of hirudin, a thrombin specific inhibitor, contains 24 hydrogen bonds, two stretches of -sheet and three disulfide bonds. Hirudin unfolds in the presence of denaturant and thiol catalyst by shuffling its native disulfide bonds and converting to scrambled structures that consist of 11 identified isomers. The composition of scrambled isomers, which characterizes the structure of denatured hirudin, varies as a function of denaturing conditions. The unfolding pathway of hirudin has been constructed by quantitative analysis of scrambled isomers unfolded under increasing concentrations of various denaturants. The results demonstrate a progressive expansion of the polypeptide chain and the existence of a structurally defined stable intermediate along the pathway of unfolding.     

Testing the role of chain connectivity on the stability and structure of dihydrofolate reductase from E. coli: fragment complementation and circular permutation reveal stable, alternatively folded forms

The effects of chain cleavage and circular permutation on the structure, stability, and activity of dihydrofolate reductase (DHFR) from Escherichia coli were investigated by various spectroscopic and biochemical methods. Cleavage of the backbone after position 86 resulted in two fragments, (1--86) and (87--159) each of which are poorly structured and enzymatically inactive. When combined in a 1 : 1 molar ratio, however, the fragments formed a high-affinity (K(a) = 2.6 x 10(7) M(-1)) complex that displays a weakly cooperative urea-induced unfolding transition at micromolar concentrations. The retention of about 15% of the enzymatic activity of full-length DHFR is surprising, considering that the secondary structure in the complex is substantially reduced from its wild-type counterpart. In contrast, a circularly permuted form with its N-terminus at position 86 has similar overall stability to full-length DHFR, about 50% of its activity, substantial secondary structure, altered side-chain packing in the adenosine binding domain, and unfolds via an equilibrium intermediate not observed in the wild-type protein. After addition of ligand or the tight-binding inhibitor methotrexate, both the fragment complex and the circular permutant adopt more native-like secondary and tertiary structures. These results show that changes in the backbone connectivity can produce alternatively folded forms and highlight the importance of protein-ligand interactions in stabilizing the active site architecture of DHFR.     

Heteromeric assembly of the cytosolic glutamine synthetase polypeptides of Medicago truncatula: complementation of a glnA Escherichia coli mutant with a plant domain-swapped enzyme

We have cloned and sequenced the cDNAs corresponding to the two cytosolic glutamine synthetase (GS) polypeptides (a and b) of Medicago truncatula. Using these two cDNAs we have prepared a construct encoding the N-terminal domain of b and the C-terminal domain of a in order to produce a domain-swapped polypeptide which should assemble to give an enzyme containing chimeric active sites. Both the native and the domain-swapped enzymes were expressed in Escherichia coli where they were catalytically and physiologically active as they were able to rescue a glnA deletion mutant. The expressed polypeptides were of the correct size and the isoenzymes behaved similarly to their native homologues on ion-exchange chromatography. We have found slight differences in the kinetic properties of the purified enzymes and in the modulation of their activities by several putative cellular effectors. In vitro dissociation of the purified a and b homo-octamers, followed by reassociation, showed that the subunits are able to self-assemble, perhaps randomly, to form heteromeric isoenzymes. Moreover, heteromeric isoenzymes occur in the plant as revealed by studies on the GS isoenzymes of nodules, roots, stems and stipules.     

茶树茶氨酸合成酶基因的酶活性验证与蛋白三维结构分析

针对NCBI上已登录的茶氨酸合成酶与谷氨酰胺合成酶基因序列进行克隆、原核表达与酶活性验证,利用多种生物信息学数据库和软件,对Cs TS与Cs GS基因进行结构、性质和功能预测,采用同源建模法对蛋白三维结构进行预测,比较并预测催化作用位点的差异;用系统进化树分析从裸子植物到高等被子植物的谷氨酰胺合成酶基因序列,推测其进化的演变过程;通过对原核表达的基因工程菌提取粗酶液进行酶活性测定。结果表明:尽管茶树TS与GS序列高度同源,但是原核表达后的融合蛋白仍然显示了不同的催化能力,蛋白一、二级结构分析显示Cs TS与Cs GS差异不大,但是通过同源建模形成的蛋白三级结构分析显示,Cs TS与Cs GS存在3个催化位点上的差异,这可能是导致其酶活性差异的关键。系统进化分析结果首次确定茶氨酸合成酶应为谷氨酰胺合成酶基因家族成员,按照其细胞定位预测应为胞质型GS,其亲缘关系与同为双子叶植物的葡萄、陆地棉、巴西橡胶树、拟南芥较接近。