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Structural characterisation of recombinant and synthetic proteins and peptides by biophysical techniques

Brazier, Stephen Paul; (1997) Structural characterisation of recombinant and synthetic proteins and peptides by biophysical techniques. Doctoral thesis (Ph.D.), University College London (United Kingdom). Green open access

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Abstract

Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopy were applied in the structural analysis of two proteins 1) plasminogen activator inhibitor-1 (PAI-1), whose active form is spontaneously converted to an inactive latent conformation, thus preventing a crystal structure of the active molecule to be elucidated and 2) potassium channels, which are large membrane bound proteins expressed in low amounts not ideal for x-ray crystallography. FTIR and CD spectroscopy are ideal techniques for studying the structure of these proteins; requiring relatively low amounts of protein, quick speed of data acquisition and the ability to determine the structure of a protein in a physiologically relevant state. PAI-1 is a member of the serpin superfamily, the major regulators of protease activity within the body. Recombinant proteins corresponding to active and latent PAI-1 were analysed by FTIR. The spectra of these proteins indicate that they are predominantly [beta] sheet with some [alpha]-helical structure. However upon conversion of active to latent conformation an increase in the [beta] sheet structure at the expense of [alpha]-helical structure is observed. This finding is probably due to insertion of the reactive centre loop (RCL) into [beta] sheet A of the protein, a well defined characteristic of other members of the serpin family. To determine which residues within the RCL are most important in the active/latent transition a number of mutations were inserted into the RCL of PAI-1. The secondary structure and thermal stabilities of these mutant PAI-1 proteins were characterised by FTIR, indicating that the residues closest to the N-terminal side of the RCL are most important in the active/latent transition. Potassium channel proteins regulate the transport of K+ ions across the membranes of both excitable and non-excitable cells. To date, three families have been identified; the inward rectifier channels, the voltage gated channels and a third group with only one member termed the minimal potassium channel (minK). Synthetic peptides corresponding to functionally important domains of ROMK1 (rat outer medulla inward rectifier potassium channel), the transmembrane domain of minK and the S4 'voltage sensor' of the Drosophila Shaker A channel were used to determine the secondary structure content in membrane mimetic and aqueous environments. Spectroscopic analysis of the two transmembrane spanning domains (M1 and M2) and the putative 'pore' region (P) of ROMK1 indicate that these domains are predominantly [alpha]-helical in a membrane mimetic environment. These results have been used to build a model of the ion conductance pathway of the channel. Structural analysis of the recombinantly expressed C-terminal was prevented by the inability to express sufficient quantities of protein for purification in various E.coli strains. FTIR spectra of the S4 segment of Shaker indicated that this domain has a high degree of conformational flexibility. The secondary structure adopted by the S4 peptide is influenced by a number of factors, the nature of the surrounding environment ([alpha]-helical in phospholipid, random in aqueous), temperature and pH. This property may have important implications for its proposed role as the 'voltage sensor' during channel activation in response to changes in the electric potential across the membrane. In addition to the structural results, a number of techniques have been employed in successfully reconstituting membrane proteins into phospholipids without exhibiting peptide aggregation. This is illustrated by the transmembrane region of minK and the M1 domain of ROMK1, which typically display spectra characteristic of protein aggregation, however when successfully reconstituted by the film method into phospholipids both these domains adopt an [alpha]-helix.

Type: Thesis (Doctoral)
Qualification: Ph.D.
Title: Structural characterisation of recombinant and synthetic proteins and peptides by biophysical techniques
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: (UMI)AAIU532720; Biological sciences
URI: https://discovery.ucl.ac.uk/id/eprint/10098059
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