Structural characterisation of the hepatitis C envelope glycoprotein E1 ectodomain derived from a mammalian and a yeast expression system.

The structure of the ectodomain of the hepatitis C envelope glycoprotein E1 (E1s) was characterised by spectroscopic methods. Monomeric E1s was purified from a mammalian and from a Hansenula polymorpha cell lysate, and cysteine-blocked monomers were reconstituted into stable particles. Particles from yeast E1s and mammalian E1s showed a comparable reactivity in ELISA with sera from human chronic HCV carriers, similar antibody titers in the sera of immunised mice as well as a comparable structure as analyzed by spectroscopic methods (tryptophan fluorescence, circular dichroism, and Fourier transform infrared spectroscopy). The overall secondary structure of E1s was neither influenced by the degree of glycosylation nor by the nature of cysteine modification used during purification. The structural comparability of mammalian- and H. polymorpha-expressed E1s opens new perspectives for further development of E1s-based therapeutics as yeast systems generally allow a more easy scaling up.

Simulation of the activation of alpha-chymotrypsin: analysis of the pathway and role of the propeptide.

Alpha-chymotrypsin undergoes a reversible conformational change from an inactive chymotrypsinogen-like structure at high pH to an active conformation at neutral pH. In order to gain insight into this process on a structural level, we applied molecular dynamics and targeted molecular dynamics simulations in aqueous environment on the activation and inactivation processes of three different types of chymotrypsin. These are the wild-type bovine chymotrypsin containing the propeptide and the bovine and rat chymotrypsin lacking the propeptide. From these simulations, the importance of the propeptide and of the sequence differences between the rat and bovine variants from the viewpoint of activation could be evaluated and compared with previous fluorescence stopped flow results. The obtained results show the unambiguous influence of the propeptide on the explored conformational space, whereas the sequence differences between bovine and rat chymotrypsin play a minor role. The main features of activation are present in both the wild type and the variant lacking the propeptide, despite the fact that different parts of the conformational space were explored. The comparison of all trajectories shows that particular amino acid residues, such as 17, 18, 19, 187, 217, 218, and 223, undergo large dihedral transitions during the activation process, suggesting a role as hinge residues during the conformational change.

A fluorescence stopped-flow kinetic study of the conformational activation of alpha-chymotrypsin and several mutants.

The kinetic activation parameters (activation free energy, activation free enthalpy, and activation free entropy change) of the conformational change of alpha-chymotrypsin from an inactive to the active conformation were determined after a pH jump from pH 11.0 to pH 6.8 by the fluorescence stopped-flow method. The conformational change was followed by measuring changes in the protein fluorescence. For the bovine wild-type protein, the same kinetic parameters are obtained as in the study of proflavin binding. Several mutants were made with the goal to accelerate or decelerate this conformational transition. The inspiration for the choice of the mutants came from a previous modelling study done on the bovine wild-type chymotrypsin. The results of the fluorescence stopped flow experiments show that several mutants behaved as was expected based on the information provided by the modeling study on the wild-type variant. For some mutants our assumptions were not correct, and therefore additional modeling studies of the activation pathways of these mutant proteins are necessary to be able to explain the observed kinetic behavior.