Probing the Effects of Molecular Crowding on Protein Structure Using Atomic Force Microscopy

Abstract

Enzymes exist in crowded cellular environments. However, conventionalin vitroandin silicoenzymatic studies are conducted in dilute conditions that do not consider the molecular crowding effects on an enzyme's structure and dynamics and therefore its function. Currently, we are investigating crowding effects on the structure and stability of a multidomain enzyme, prolyl-transfer RNA synthetase (ProRS) ofEscherichia coli. ProRS catalyzes the ligation of proline to tRNAProduring protein biosynthesis. Atomic Force Microscopy (AFM), a type of scanning probe microscopy, can develop high-resolution topographic images on the nanometer scale. Moreover, AFM allows the analysis of detailed topographic features and surface roughness of samples. Therefore, AFM is used to analyze the effects of synthetic crowder molecules like ficoll 70 and dextran 40 on Ec ProRS structure. Results and images of ProRS in diluted and crowded environments will be presented.