Single Molecule Protein Folding Studies

Proteins are one of the most diverse classes of biological macromolecule in both the structures that they form and the functions that they perform. Proteins are involved in every biological process, and their misfunction is associated with disease states including cancer in a very large number of cases. Having completed the human genome and many other mapping projects, there are now vast databases of information on the primary (amino acid) sequence of proteins in many organisms. In some cases, knowledge of the primary sequence itself may be sufficient to predict how a protein may fold and function. However, in many other cases, the primary sequence alone tells us little about the structure or activity of the protein. Being able to predict the structure of a protein from its amino acid sequence is a major goal in the 21st century and is part of what is termed the 'protein folding problem'. Knowledge of the structure that a protein adopts is crucial in the design of small molecules which will bind to it and affect its activity (the basis of many therapeutic drugs). Another important and related aspect of the protein folding problem is the determination of the pathways by which proteins fold. The energy landscapes for folding which determine the pathway(s) by which a protein will fold are complex. There are some fundamental questions about these energy landscapes which cannot be addressed by studying protein folding in bulk solution, looking at the average behaviour of many molecules. In order to address such questions as the heterogeneity of folding pathways and to get information on the individual behaviour of unfolded protein molecules in the denatured ensemble (the unfolded state of the protein and the starting point for any folding study) single-molecule experiments are required. Single-molecule spectroscopy has advanced significantly over the last ten years, however, there remain very few studies of single molecule protein folding. These type of experiments are increasingly important as the number of computational studies which simulate the folding of a single protein chain has increased dramatically in the last five years. Experimental single molecule data are desperately needed to compare with computational results to facilitate the benchmarking and improvement of the computational methods. The main difficulty in measuring single molecule protein folding is the requirement to study a reaction far from equilibrium. Technical developments and new methodologies have been recently developed in the Klenerman group which will allow us to make the single molecule measurements needed. The two proteins which will be studied are the small, stable protein ubiquitin, and the large beta-barrel protein GFP. These are two proteins whose folding pathways have been studied in detail from bulk solution results in the Jackson laboratory and which show quite different folding behaviours. They make ideal systems with which to test the single molecule experiments proposed and to develop the techniques further which can then be generally applied.

Despite the many technical advances in single-molecule measurements over the past ten years, there are still remarkably few single-molecule studies of protein folding. This is largely due to the inherent difficulties in making single-molecule measurements on systems far from equilibrium. The Klenerman group has recently developed novel single-molecule techniques to do just this based on new nanopipette technology. We have used this technique to successfully monitor the unfolding of a dye-labelled citrine variant of GFP at a single-molecule level. We now propose to extend these measurements on citrine to study i) the unfolding reaction under different conditions, ii) the refolding of GFP and iii) to characterise the ensemble of denatured states. Results will be compared to those obtained from bulk solution. In order to do this, a number of variants of GFP will be synthesised in which the protein will be doubly labelled with suitable dyes to allow a series of fluorescence resonance energy transfer. The fluorophores will be located on different structural elements in GFP allowing us to obtain specific information on the formation of different regions of the complex beta-barrel structure and identify any intermediates formed. In addition, the small monomeric protein, ubiquitin, will also be studied using similar single-molecule and labelling techniques. In collaboration with the Searle group, measurements on mutants of ubiquitin which are known to fold via stable intermediate states will also be undertaken. This set of experiments will not only establish the methodology for single-molecule folding measurements, which can then be applied to other proteins, but it will also provide new information on the pathways taken from the unfolded to folded state for these two proteins and hence the energy landscape. These results can also be compared with the single-molecule simulations of unfolding/folding.