Studies On The Determinants Of A Protein’s Mechanical Stability At The Single Molecule Level

Mechanical properties of proteins are essential in a wide range of biological processes, ranging from cell-cell adhesion, muscle contraction to protein degradation and translocation. Understanding how proteins are designed in nature to achieve desired mechanical stability not only is important for understanding biological processes but also holds the promise to design novel protein-based biomaterials for biomedical and material science applications. Over the past decade, single molecule atomic force microscopy (AFM), in combination with protein engineering and molecular dynamics simulations, has enabled the characterization of mechanical properties of proteins at the single molecule level in great detail, and some general molecular determinants of protein mechanical stability have emerged. Although there have been great advances in the engineering of mechanical stability of proteins, the molecular determinants of mechanical stability are still largely unknown. It is still impossible to directly predict the mechanical stability of a protein from its three dimensional structure or even its sequence. It is also difficult to tune the mechanical stability of a protein in a rational fashion.This dissertation includes4chapters. In chapter1, the techniques that are used to study the mechanical properties of elastomeric proteins, with the focus of the use of single molecule AFM, are introduced. We present the general experimental procedures, the interpretation/analysis of data and the models for unfolding of elastomeric proteins. Also, the proteins that have been studied by AFM so far are summarized and the important advances are highlighted. In the following chapters, we combine single molecule AFM and protein engineering techniques to investigate the molecular determinants of mechanical stability of protein GB1from three aspects. In chapter2, we engineered seven point mutants and carried out mechanical φ-value analysis of the mechanical unfolding of GB1. We found that three mutations, which are across the surfaces of two subdomains that are to be sheared by the applied stretching force, in the hydrophobic core (F30L, Y45L, and F52L) result in significant decrease in mechanical unfolding force of GB1. The mechanical unfolding force of these mutants drop by50-90pN compared with wild-type GB1, which unfolds at around180pN at a pulling speed of400nm/s. These results indicate that hydrophobic core packing plays an important role in determining the mechanical stability of GB1. Also, the φ-value analysis for the mechanical unfolding of GB1reveals the structure of the mechanical unfolding transition state of GB1, which is consistent with the predicted one by molecular dynamics simulations.In chapter3, we build a host system based on protein GB1, denoted AASS. We chose four amino acid(Val, Leu, Gln, Ala) with quite different beta-sheet forming propensity to substitute guest site (T53) which is in AASS system to check the effect of beta-sheet forming propensity on mechanical stability of protein GB1. Also, we chose amino acid(His, Ala, Asp, Gly) with dramatic low propensity to be substituted at residue T53in wtGB1to investigate the contribution of sidechain interaction on beta-sheet to the mechanical stability of protein GB1. Surprisingly, we found that only sidechain interaction is crucial in determining mechanical stability of GB1, whereas beta-sheet forming propensity which can be seen in thermodynamic scale does not have effect on the mechanical stability of GB1.These results suggest that optimizing sidechain interactions across the surfaces that are to be sheared will likely be an efficient method to enhance the mechanical stability of GB1and GB1homologues, these information may open a new way toward the design of elastomeric-protein based biomaterials.In chapter4, based on the results of last chapter, we engineered a pairwise mutation which located at a solvent-exposed site between the two central parallel beta strands to optimize the sidechain interactions across the surfaces that are to be sheared. The results show that pairwise mutations can enhance the mechanical stability of protein GB1by achieving preferential stabilization of the native state over the mechanical unfolding transition state. This general approach in protein mechanics will enable the rational tuning of the mechanical stability of proteins and will open a new avenue toward engineering proteins of tailored nanomechanical properties.