An interdisciplinary study of the mechanical and dynamic properties of α-solenoid repeat proteins

Tandem-repeat proteins differ from globular proteins, both in their biophysical characteristics and in how they interact with their respective partners, yet they comprise nearly one third of the human proteome and are central to many cellular processes and disease phenotypes. Repeat proteins have been shown to behave like nano-sized biological springs: they are flexible, dynamic and elastic. Using coarse-grained models, I discuss how intrinsic flexibility may arise in repeat proteins and how it could be crucial for the biological function of two systems: PR65, the scaffold protein of the protein phosphatase 2A, and Rap proteins, which are involved in quorum sensing. To interrogate α-solenoids at physiologically relevant forces, I performed force spectroscopy experiments using a dumbbell optical tweezers set up for which it is necessary to attach the relevant protein to DNA. As PR65 is not amenable to current DNA-protein attachment methods, I developed a protocol that allows the cross-linking of DNA oligos to proteins using bio-orthogonal chemistry. I then explored the mechanics of the natural repeat protein, PR65, and a series of designed TPR proteins. I find that these proteins respond to forces in a novel manner which is significantly different to what has been previously reported. TPRs unfold and refold in quasi-equilibrium at constant force without energy loss. In contrast, PR65 unfolds in separate domains and refolds along an entirely different pathway. In conclusion, my doctoral studies explore the physical characteristics of repeat proteins in more detail. Using both experimental and computational techniques, I provide unique perspectives on different aspects of their mechanical and dynamic capabilities. This work provides the basis for future investigations of how such interesting mechanical behaviour relates to biological function. Are repeat proteins simply a molecular recognition platforms for their multitude of binding partners, or do their mechanics matter in a biological context

Bioorthogonal protein-DNA conjugation methods for force spectroscopy.

Accurate and stable site-specific attachment of DNA molecules to proteins is a requirement for many single-molecule force spectroscopy techniques. The most commonly used method still relies on maleimide chemistry involving cysteine residues in the protein of interest. Studies have consequently often focused on model proteins that either have no cysteines or with a small number of cysteines that can be deleted so that cysteines can then be introduced at specific sites. However, many proteins, especially in eukaryotes, contain too many cysteine residues to be amenable to this strategy, and therefore there is tremendous need for new and broadly applicable approaches to site-specific conjugation. Here we present bioorthogonal approaches for making DNA-protein conjugates required in force spectroscopy experiments. Unnatural amino acids are introduced site-specifically and conjugated to DNA oligos bearing the respective modifications to undergo either strain-promoted azidealkyne cycloaddition (SPAAC) or inverse-electron-demand Diels-Alder (IE-DA) reactions. We furthermore show that SPAAC is compatible with a previously published peptide-based attachment approach. By expanding the available toolkit to tag-free methods based on bioorthogonal reactions, we hope to enable researchers to interrogate the mechanics of a much broader range of proteins than is currently possible

A quantitative description for optical mass measurements of single biomolecules

Label-free detection of single biomolecules in solution has been achieved using a variety of experimental approaches over the past decade. Yet, our understanding of the magnitude of the optical contrast and its relationship with the underlying atomic structure as well as the achievable measurement sensitivity and precision remain poorly defined. Here, we use a Fourier optics approach combined with an atomic structure-based molecular polarizability model to simulate mass photometry experiments from first principles. We find excellent agreement between several key experimentally determined parameters such as optical contrast-to-mass conversion, achievable mass accuracy, and molecular shape and orientation dependence. This allows us to determine detection sensitivity and measurement precision mostly independent of the optical detection approach chosen, resulting in a general framework for light-based single-molecule detection and quantification

A Quantitative Description for Optical Mass Measurement of Single Biomolecules

Label-free detection of single biomolecules in solution has been achieved using a variety of experimental approaches over the past decade. Yet, our understanding of the magnitude of the optical contrast and its relationship with the underlying atomic structure as well as the achievable measurement sensitivity and precision remain poorly defined. Here, we use a Fourier optics approach combined with an atomic structure-based molecular polarizability model to simulate mass photometry experiments from first principles. We find excellent agreement between several key experimentally determined parameters such as optical contrast-to-mass conversion, achievable mass accuracy, and molecular shape and orientation dependence. This allows us to determine detection sensitivity and measurement precision mostly independent of the optical detection approach chosen, resulting in a general framework for light-based single-molecule detection and quantification

Folding cooperativity and allosteric function in the tandem-repeat protein class.

The term allostery was originally developed to describe structural changes in one binding site induced by the interaction of a partner molecule with a distant binding site, and it has been studied in depth in the field of enzymology. Here, we discuss the concept of action at a distance in relation to the folding and function of the solenoid class of tandem-repeat proteins such as tetratricopeptide repeats (TPRs) and ankyrin repeats. Distantly located repeats fold cooperatively, even though only nearest-neighbour interactions exist in these proteins. A number of repeat-protein scaffolds have been reported to display allosteric effects, transferred through the repeat array, that enable them to direct the activity of the multi-subunit enzymes within which they reside. We also highlight a recently identified group of tandem-repeat proteins, the RRPNN subclass of TPRs, recent crystal structures of which indicate that they function as allosteric switches to modulate multiple bacterial quorum-sensing mechanisms. We believe that the folding cooperativity of tandem-repeat proteins and the biophysical mechanisms that transform them into allosteric switches are intimately intertwined. This opinion piece aims to combine our understanding of the two areas and develop ideas on their common underlying principles.This article is part of a discussion meeting issue 'Allostery and molecular machines'.LSI acknowledges the support of a Senior Fellowship from the UK Medical Research Foundation. AP was supported by a BBSRC Doctoral Training Programme scholarship and an Oliver Gatty Studentship. MS was supported by a BBSRC Doctoral Training Programme scholarship

RapI_mode3.mpg from Folding cooperativity and allosteric function in the tandem-repeat protein class

This clip shows the motion of RapI along the third lowest vibrational mode. This motion involves largely the N-terminal three-helix bundle which twists in a screw-like manner orthogonal to the superhelical axis, while the C-terminal repeats simply open and close with respect to the superhelix

RapI_mode2.mpg from Folding cooperativity and allosteric function in the tandem-repeat protein class

This clip shows the motion of RapI along the second lowest vibrational mode. The whole molecule moves in a screw-like motion that loosens and tightens the superhelical twist

Supplementary Information: Folding cooperativity and allosteric function in the tandem-repeat protein class

Supplementary Information providing the mathematical basis and structures used to generate Elastic Network Models, in addition to some figures that further insight to the results presented in the paper

RapI_mode2.mpg from Folding cooperativity and allosteric function in the tandem-repeat protein class

This clip shows the motion of RapI along the second lowest vibrational mode. The whole molecule moves in a screw-like motion that loosens and tightens the superhelical twist

Supplementary Information: Folding cooperativity and allosteric function in the tandem-repeat protein class

Supplementary Information providing the mathematical basis and structures used to generate Elastic Network Models, in addition to some figures that further insight to the results presented in the paper