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

Lead Research Organisation: University of Leeds
Department Name: Institute of Molecular BioPhysics


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.

Technical Summary

Understanding the determinants of a protein's mechanical resistance and the factors that modulate it is a fascinating topic of fundamental importance because not only do cells use biological machines to mechanically unfold proteins in diverse processes such as protein degradation, translocation, signalling, adhesion and movement, but also because proteins have great potential as biocompatible tissue engineering scaffolds and nanoelectronics components. Over the last six years the atomic force microscope has been used to characterise the mechanical properties of proteins. Although there are a wealth of data on the mechanical properties of I27 and proteins of related fold, the determinants of a protein's mechanical resistance are still largely unknown. Furthermore, over the last five years, a large body of research has suggested that proteins are actively unfolded by application of force in many cellular processes such translocation, protein degradation and protein-complex disassembly. Interestingly, the rate at which a protein is unfolded seems to correlate with the stability of the regions of the protein which are local to the application of the force for both processes. By characterising the local dynamic behaviour of three model proteins by hydrogen exchange experiments using NMR, this project aims to probe the fundamental origins of the mechanical properties of proteins, to derive predictive rules that allow mechanical resistance to be designed and tailored ab initio and, ultimately, to relate these to the control of biological activity and homeostasis in vivo. This will be achieved by the combination of single-molecule manipulation, molecular dynamics simulations, nuclear magnetic resonance experiments and assays of protein degradation using the bacterial degradation machine ClpXP.


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Description Two variants of protein L that differ two-fold in mechanical strength display the same dynamic behaviour. These variants are conservative mutations at a single site (I60V and I60F) that decrease or increase hydrophobic contacts across the mechanical interface. This suggests that protein dynamics do not play a major role in determining mechanical strength but stabilising the mechanical interface does increase mechanical strength. This knowledge adds to the growing appreciation of how Nature has utilised force and evolved protein structure to respond to it in diverse ways.

We demosntrated this diversity by showing that stabilisation of a protein (E9) by complexation with its ligand (Im9 which binds distal to the site of force application), does not affect the rate of degradation of E9 by ClpXP (an enzyme evolved to unfold proteins that have been targeted for degradation).
Exploitation Route This knwoledge may help in the design of force sensors or inhibitors that target force activated proteins/complexes. This research is important for both fundamental and applied science. The lack of mechanical stabilisation observed upon binding of a ligand is surprising and suggests that very stable complexes can be broken apart relatively easily by application of low levels of force. This highlights the importance when interpreting measurements obtained by non-force methods on systems where force may be applied in vivo.
Sectors Manufacturing/ including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology