Spectroscopic tools for improved protein secondary structure analysis in protein-heparan sulfate complexes

Lead Research Organisation: University of Liverpool
Department Name: Sch of Biological Sciences

Abstract

Many biological processes such as cell growth, division and responses to conditions in the body depend on interactions between proteins and cell-surface ligands. It is technically challenging to detect these interactions and understand what changes are occurring in the proteins when they bind their target ligand. However, such information is vital, not only for a better understanding of the process, but also if we are to be able to control them or to create new treatments. This problem is even more stark for one set of interactions, those between proteins and the cell-surface receptor heparan sulfate (HS). This is because the current techniques, based on circular dichroism (CD) and infra-red (FTIR) spectroscopy do not cope well with HS, which exhibits signals that interfere with the analysis of the protein component of the complex. We propose to explore two approaches which may enable us to selectively probe the structural changes in proteins in the presence of HS. The first is based on selection of an active but structurally modifed HS fragment from our existing library of structures. This will be selected so that it lacks particular groups and will present no interferring signal in infra-red (FTIR) but will retain biological activity. This we term a 'transparent' HS ligand. Using this, the signal for HS should disappear while that of the protein is detected. Structural changes should then only be reported from the protein. The second method utilises a technique called vibrational circular dichroism (VCD), which should detect the regular structures present in the protein but not those of the less-regularly structured HS ligand (a normal, not a modified HS ligand). This may enable the protein to be selectively observed even in the presence of HS. The proposed research is to test these two possibilities with two proteins that are known to change shape on binding HS. One is the normal (non-infectious) form of the prion protein (PrPC) and the other is an enzyme involved in plaque formation in Alzheimers' disease; BACE-1. If successful, the approach will be widely applicable to studying the great number of protein-HS interactions that occur between protein and cell-surface HS ligands, which currently cannot be accurately followed.

Technical Summary

An ability to measure confomational change in proteins when complexed with their ligand is vital to help understand fundamental molecular and biological processes. However, we recently observed that protein-heparan sulfate (HS) complexes are difficult to analyse by conventional CD or FTIR techniques because the HS interferes with the protein spectra. There are two related approaches which could be applied to resolve this problem. The first is the selection of active HS ligands free of amide groups from our existing libraries of HS analogue structures. These will be essentially 'transparent' in the FTIR regions used for protein structural analysis. This will provide a tool permitting the technique to be tested with active ligands with transmission FTIR as well as providing an independent external verification of the second method, which uses vibrational circular dichroism (VCD). This technique relies on the fact that right and left circularly polarised infra-red light are differentially absorbed by protein secondary structural elements. The opportunity here is to exploit a fundamental difference between the amide bonds of the protein backbone and those present in HS. The difference is that in HS, the amide groups are not connected to each other and so lack the regular geometric relationship of those of the protein backbone which gives rise to the VCD spectrum of protein secondary structure. It is therefore, highly likely that the VCD spectrum of HS will lack amide signals. Two questions will be asked: 1.Can FTIR 'transparent' HS ligands be selected and used for protein-HS complex analysis, free from interference of amide signals associated with normal HS ligands? 2.Can V-CD selectively detect protein signals in the presence of normal HS ligand? The answers will allow us to determine whether either, or both, approaches can be applied to the study of the many biologically important protein-HS complexes in solution.

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