Award details

Mapping out transition states in membrane protein folding: a phi value analysis

Reference	BB/D001676/1
Principal Investigator / Supervisor	Professor Paula Booth
Co-Investigators / Co-Supervisors	Professor Bonnie Wallace
Institution	University of Bristol
Department	Biochemistry
Funding type	Research
Value (£)	332,331
Status	Completed
Type	Research Grant
Start date	01/11/2005
End date	31/10/2009
Duration	48 months

Abstract

This application proposes the first investigation of the structures of transition states during the folding reaction of an integral membrane protein. We will apply a procedure that has been developed for water-soluble proteins; that of phi value analysis. Phi values are the only way to determine the structure of a folding transition state but have never been applied to integral membrane proteins. Here we propose a phi value study on a helical transmembrane protein. Transition states are the critical states in reactions when bonds are broken and made. However, these states cannot be directly observed for protein reactions. Phi value analysis was developed to gain such molecular level knowledge on transition states. Knowledge of protein structure formation during folding and in particular in these critical transition states, illuminates the vital interactions that are made during folding. In turn, this provides important information on the factors that determine the stability of the folded state. This information is unfortunately currently lacking for integral membrane proteins - a situation that is hampering structure and function work. Indeed one of the major barriers in membrane protein research, as acknowledged by the NIH and EC, is the difficulty in obtaining sufficient stable and correctly folded proteins for study. Over-expression of integral membrane proteins is difficult, but even if proteins can be over-expressed they frequently crash out of solution and aggregate during purification or crystallisation trials. A rational way to overcome this problem is to understand the molecular determinants of membrane protein folding and stability. Phi values are probably the most powerful method to achieve this. The phi value approach involves making a single point mutation in a protein and determining how this amino acid change affects the free energy of a folding transition state. A phi value is the comparison of the change in the transition state energy to that in the folded state and thus can indicate whether the structure at the site of the mutation is the same in the transition state as in the folded state. Our previous work (largely BBSRC funded) has laid the groundwork for a phi value analysis. We have already achieved the key requirements of a reversible folding system (to enable free energies of folding to be determined) together with kinetic studies and a detailed knowledge of the folding reaction scheme. A phi value analysis is impossible without the latter. Other requirements for the analysis are the ability to make mutant protein as well as a known high-resolution protein structure. Bacteriorhodopsin is the only helical transmembrane protein where a phi value analysis is anywhere near possible and thus is the focus of this first study. The proposed work on bacteriorhodopsin is vital if the powerful phi value approach is to be translated to the membrane domain. This will allow us to test many of the assumptions of phi value analysis and adapt the procedures for determining free energies to membrane systems - only then can we begin to design appropriate folding methodologies to apply phi value analyses to other helical transmembrane proteins. It is important to pave the way for such future studies. Thus, we aim to extend the method to another protein and choose the next most promising candidate, the potassium channel KcsA. Since a phi value analysis on this protein is not currently possible we wish to investigate whether a reversible folding system can be established for KcsA to determine the folding free energy. The work will involve preparation of bacteriorhodopsin mutants, determination of folding kinetics, together with free energies of intermediates and transition states during folding, allowing determination of phi values. This involves kinetic folding studies (pioneered by PJB for membrane protein) and circular dichroism studies (in which BAW is the leading UK expert on membrane proteins).

Summary

Proteins are the worker molecules in life forms. An understanding of how proteins work is essential in the fight against disease - if the proteins do not work correctly this can cause disease and most medicines are designed to target protein work. This project focuses on a particular type of protein, membrane proteins. Membranes surround cells and subdivide the cells into compartments, with membrane-embedded proteins allowing information and matter to pass across the membranes and between cells. The human genome sequence carries the code for the proteins in our bodies. We have yet to identify what each gene means, which protein it encodes and how the genetic information is translated into a working protein. The most glaring gaps in our knowledge come with membrane proteins. These proteins live in cell membranes and constitute about a third of the proteins in our bodies. Moreover, most of the commonly taken pills and medicines are targeted to affect membrane proteins. Unfortunately there is very little understanding of how membrane proteins work, because they are notoriously unstable and thus difficult to study outside the body. This grant aims to find out how genetic information is translated to make these membrane proteins, and to use this knowledge to stabilise the proteins. Genes are first decoded to give a string of amino acids, which then has to fold to the proteins correct, and unique, three-dimensional shape. We propose to investigate how the protein folds correctly - if it mis-folds then the protein is unstable and cannot work properly. We will look at the protein building blocks (the amino acids) in a membrane protein that is made from about 250 of these blocks. We need to work out the order in which the building blocks are assembled to make a stable protein structure.

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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