How are proteins mechanically unfolded? A study spanning fundamental principles and biological complexity

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To be functional most proteins need to fold up into a well defined three dimensional structure. The probability that a protein remains in this state over a period of time is related to its thermodynamic stability: the greater the stability, the greater the proportion of folded active proteins. Proteins have many different functions in cells including energy generation, locomotion and structural roles. Mechanical force, which can denature proteins, is applied onto these proteins during many of these activities and, to maintain their function, proteins have to be mechanically resistant. Using techniques that allow the manipulation of single protein molecules, it is now possible to measure the mechanical strength of proteins in the laboratory. This technique uses an instrument called the atomic force microscope (AFM). However, the mechanical stability of proteins has been found to be unrelated to a protein's thermodynamic stability but correlates well with how the different types of a protein's sub-structure are arranged in each protein. The reason for this is unclear but it is thought that regions in proteins that are bound less tightly to the rest of the structure may be more likely to unfold when force is applied onto this part of the protein. As a consequence, the force at which a protein unfolds depends upon the points at which the force is exerted onto the protein. It has recently been discovered that cells possess large cylindrical protein complexes that are able to unfold and digest proteins which have been tagged for destruction in order to control cellular processes. It is thought that these 'unfoldases' (ClpXP for example) unfold even very stable proteins by applying force onto the proteins to be degraded. The rate at which proteins are unfolded by the Clp system appears to correlate with the stability of the protein local to the position of the degradation tag - an observation similar to that reported for the unfolding process measured by the AFM. The underlying mechanism for either process is, at present, unknown. This project aims to probe the fundamental origins of the mechanical properties of proteins by measuring how the presence of regions of local instability in proteins correlates with the mechanical strength of proteins when denatured using the AFM and when degraded by the cellular unfoldase ClpXP.