Tailoring protein nanomechanics through chemical reactivity with single bond resolution

• Main Author: Beedle, Amy Elizabeth McIver
• Other Authors: Garcia-Manes, Sergi ; Richards, David Robert
• Published: King's College London (University of London) 2018
• Subjects: 530
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.745409

It is now well established that the overall mechanical properties of a wide variety of tissues can be described by the cumulative nanomechanical properties of each of the individual constituent proteins. However, on a deeper, atomistic level, it is far less understood how the chemical reactivity of the individual chemical bonds forming the protein’s architecture can tune its nanomechanics. The amino acid cysteine is unique among all the protein amino acids due to its reactive sulfhydryl group. The particular chemical properties of the sulfur atom enable cysteine to exhibit a wide range of oxidation states, resulting in a rich and varied chemical reactivity that spans from the formation of organo-metallic bonds when coordinating a plethora of metal ions to the formation of the highly covalent and mechanically rigid disulfide bonds. It is therefore tempting to speculate that the incorporation of an individual cysteine residue into a protein sequence is a natural strategy for modulating chemical reactivity, mostly through post-translational modifications, with knock-on effects for protein mechanics. In this thesis work I employ single molecule force spectroscopy AFM to investigate how the different chemical reactivity of cryptic cysteines affects the folding and the nanomechanical properties of individual proteins. Firstly, I characterize the mechanical stability of the copper-thiolate bond in the metalloproteins azurin and plastocyanin. Furthermore, I use a constant force approach to probe two chemically distinct strategies to achieve non-enzymatic disulfide bond formation within the context of oxidative folding; through a transient sulfenic acid species or through the creation of a mixed disulfide species between a protein cysteine and a low molecular weight thiol. Altogether this work highlights that subtle adjustments to the chemical reactivity of a single thiol-bond within a protein, has unanticipated yet profound effects on the overall protein nanomechanics.