Influence of Posttranslational Modifications on Peptide and Protein Activity

Abstract

Peptides and proteins represent a class of biomolecules that play a crucial role in almost all processes of life. The modification of proteins following translation on the ribosome – so called posttranslational modifications (PTMs) - serves as a key mechanism to expand the complexity, structure and function of the proteome. Importantly, PTMs have been shown to positively affect the structure, stability and/or activity of a number of proteins. Given the proposed importance of PTMs, access to homogeneously modified peptide and protein variants is crucial to enable the meaningful investigation of the specific effects of particular modifications. This thesis will describe the effect of specific PTMs on the activity of peptides and proteins. Chapter 2 describes the semi-synthesis of differentially sulfated ACA-01 proteins, a chemokine-binding evasin protein produced within the salivary glands of the Amblyomma cajennense tick. Tyr sulfation was shown to significantly enhance the chemokine-binding affinity and signalling-inhibitory potency of ACA-01 for six CC chemokines. Chapter 3 of this thesis continues to explore the unique family of evasin proteins produced by ticks. To allow for access to a large library of fully sulfated evasin proteins the use of an amber codon suppression system was proposed. Initial efforts toward the expression of four sulfated evasin proteins are described. Chapter 4 of this thesis describes the synthesis and evaluation of 16 cyclic peptides, bearing the acetyl Lys PTM, that were selected against bromodomain-containing and extra-terminal domain-containing proteins via reprogrammed mRNA display technology. Many peptides showed specificity for either bromodomain 1 or bromodomain 2 with affinities as low as 130 pM. Five crystal structures were obtained from the peptides in complex with the bromodomain proteins and showed diverse binding modes with the corresponding bromodomains.