Protein Folding And Enzyme Catalysis Driven By Physical Fields

Current protein physics and chemistry focus mainly on two problems:protein folding problem and relationship between protein structures and functions. Enzyme catalyses are one of the most important functions of the proteins.Protein folding mechanism is explored by studying the unfolding pathway of a protein using urea or GdnHCl as a denaturant. In contrast to denaturant-induced unfolding, we use electric field to lengthen protein molecules and trigger unfolding reaction of the proteins. We observe that electrostatic field at power of1.5V/cm can efficiently induced unfolding process of bovine serum albumin (BSA). The electric field-induced unfolding behavior is very different to the denaturant-induced one, but it is similar to the creep of polymer gels. After removing electric field, protein conformation can recover to native state in a limited degree. But this refolding is a slow process, and it does not obey the refolding rule after the denaturant-induced unfolding. The electro-driven protein unfolding and refolding have emerged to be a simple and easily controlled technique to measure folding pathway and kinetics of the proteins. It may provide a new tool for the study of protein folding problem.Proteins fold by either two-or multi-state kinetics. Amino acids play very different roles in different kinetics. Many residues that are easy to form α helices, β sheets and turns regular secondary structures can speed up folding rate of small, two-state proteins. The hydrophilic, flexible residues can promote the rate of large, multi-state folding proteins. For an amino acid, polarity, volume, accessible surface area, isoelectric point, phase transfer energy and residue exposure have little contributed to protein folding kinetics. Cys is a special amino acid, this residue strongly accelerates two-state folding and however decelerate multi-state folding. This result not only provides an insight into protein structure prediction, but also is employed to direct the point mutations that change folding kinetics. Electrochemical biosensors are frequently used to detect trace levels of many substances. Here we develop an alternative methodology of biosensors for application of enzymatic assays, in which the electrocatalytically kinetic behavior of enzymes is monitored by measuring the Faradaic current for a variety of substrate and inhibitor concentrations. A steady-state and pre-steady-state reduction of H2O2on the horseradish peroxidase electrode is examined. The result shows that only the substrate-concentration dependence of the steady-state electric current follows a strict rule Michaelis-Menten equation, whereas the dependence of the steady-state electric current on inhibitor concentration has a obvious discontinuity. During the pre-steady-state phase, catalysis and inhibition process all display an abrupt change of the output current. The phenomena are universal and there would be underlying biochemical rationale.We use the electrochemical biosensor designed to monitor the response of enzymatic activity to ultrasound stimulation. H2O2reduction with horseradish peroxidase (HRP) is accompanied by a increasing biosensor current in proportion to the enzyme activity. The fast response of the biosensor allowed continuous measurement of dynamic changes of enzyme activity. Thus, we present an enzymatic assay based on the biosensor for the real-time assessment of the effect of ultrasound to enzyme activity. The finding shows that the enzymatic activity tended towards a maximum value when the ultrasonic frequency was closed to a given value. As ultrasonic power is increased the rate of enzymatic reaction is more rapid. However, the changes in the enzymatic activity are insensitive to ultrasonic power at high frequencies. Ultrasonic oscillation not only is responsible largely for the mass transfer improvement but by stereochemistry also brings the enzyme conformational refinement.We also examine the effect of ultrasonic irradiation and acoustic wave on enzymatic hydrolysis reaction. Ultrasonic and acoustic waves are mechanical vibration in different frequency range; they can affect the catalytic power of different enzyme proteins. This observation can provide a starting point for understanding enzyme catalysis mechanism.