Residues in the hinge region were the most flexible (exhibited the highest phi/psi entropy), along with residues at the N- and C-termini. different motions. Keywords: fluorescence anisotropy, molecular dynamics simulations, monoclonal antibodies, answer dynamics, therapeutic antibodies Introduction We are interested in understanding the solution dynamics of antibodies. The solution dynamics of antibodies is critical to antibody function. The main body of information comes from a small number of full-length antibody structures, but static structures do not give insight into the dynamics. The internal dynamics of multidomain ICAM4 proteins has been a FR901464 topic of interest in a number of different studies. 1-7 The focus of these studies has been on fluctuations in the solvent round the protein, the secondary structure, the domainCdomain interactions and the conformations of the amino acid side chains. Here, we study the internal dynamics of the IgG1 class of antibodies. The IgG1 class of antibodies is the most abundant human IgG subclass and the template for the majority of antibody drugs. IgG1 is composed of four polypeptide chains, two heavy chains (HC) and two light chains (LC). These four chains fold into three domains: two Fab domains that contain determinants for antigen binding and an Fc domain name responsible for the effector function and binding of the Fc receptor proteins.8 From the early studies of antibody structure, it was apparent that Fab domains were connected to the Fc through the unstructured linker (hinge region), rendering them capable of binding to antigens separated by a range of distances.9,10 In addition, electron microscopy reports on immobilized complexes provide evidence for the significant axial rotation of the Fab domain name.11,12 In subsequent years, a wide range of biophysical techniques have been used to investigate the dynamics of these versatile molecules and to look into the role of the hinge region in the structure and function of the antibodies.13-16 Substantial efforts have been made by many investigators to obtain high-resolution X-ray crystal structures of the intact antibodies, but the lack of a stable defined solution structure of the antibodies has prevented their crystallization and only few structures of full-length antibodies are currently available.17,18 Low-resolution cryo-electron tomography studies have been very insightful in defining the conformational space explored by the antibodies.19,20 Previous studies suggest that the dynamics of the antibodies could play a role in their function (antigen binding, complement activation) and solution stability; however, the molecular details of these observations remain unclear.21-25 The purpose of the present work is to look at the inter- and intra-domain motions, their timescales of motion, and to understand which amino acid residues contribute to the local and global FR901464 flexibility. We do this by combining the insights obtained by time-resolved spectroscopy experiments and molecular dynamics simulations. Our MD simulations lengthen an order of magnitude longer than prior simulations on full-length antibodies, and we use this data to construct a Markov model characterizing the major conformational species in antibody solutions. Our results indicate that Fab and Fc fragments form multiple meta-stable proteinCprotein interactions, with heterogeneous proteinCprotein conversation surfaces based on multiple polar interactions. Results and Conversation Comparison of experimental and computational results In this work, we used two independent approaches to look at the dynamics of a large multidomain biological molecule: time-resolved fluorescence anisotropy that allows determination of the rotational correlation times of the fluorescence probes FR901464 site-specifically conjugated to the molecule of interest and all-atom molecular.