Charge Crowding Promotes Self-Assembly of Collagen Hetrotrimers

Abstract

The design of heterospecific collagen like peptides presents a large challenge in the field of protein design. Many rational and computational approaches have been used to achieve this goal. Our model suggests that crowding of charged amino acids could help in driving the desired heterospecificity. Stability and specificity of the desired state was targeted using positive and negative design. With this approach, we have targeted the folding of the heterotrimer state by destabilizing homotrimer states via electrostatic repulsion. In this study, we sought to investigate the specific impact of repulsive interactions at the molecular level. Three peptides have been designed by introducing charge crowded acidic amino acids either at N-terminal, central, or at C-terminal end of the Pro-Hyp-Gly sequence repeats. Our CD and NMR results demonstrate that charge crowding may destabilize the homotrimer states in our triple helical peptide system and can be utilized to promote the formation of the heterotrimer. Increase in salt concentration or decrease in pH result in an increase of homotrimer stability, which confirms the role of charge crowding on the destabilization of homotrimers via electrostatic repulsion. Further investigation is required to understand the molecular role of charge crowding and can be used in conjunction with other approaches to create specific collagen heterotrimers. We further extended our study to create higher order structure by designing basic amino acids sequences in place of acidic amino acids. Mixing of basic and acidic amino sequences resulted in higher order structures.