Collagenolysis, the catabolism of triple helical collagen, is essential for the physiological remodeling of connective tissues during growth and development. Aberrant collagen degradation is a feature of both inflammatory diseases and cancer cell invasion. Matrix metalloproteinase-1 (MMP-1) is a known collagenase and previous studies have implicated its hemopexin (HPX) domain in binding and possibly destabilising collagen in preparation for hydrolysis by the catalytic (CAT) domain. More recently, conformational freedom and domain separation of the CAT and HPX domains has been proposed to play a role in collagen degradation. This study aims to explore HPX mediated collagen recognition and the postulated flexible state of MMP-1, in order to enhance our understanding of the collagenolytic mechanism. Here, biophysical methods have been used to study the complex formed between the MMP-1 HPX domain and a synthetic triple helical peptide (THP) that encompasses the MMP-1 cleavage site of the collagen α1(I) chain. A programme of site-directed mutagenesis was used to produce an extensive library of recombinant proteins. Surface plasmon resonance (SPR) has been used to characterise a previously unknown collagen binding site (exosite) located in blade 1 of the HPX domain and small angle x-ray scattering has been used to probe the conformational freedom and transient domain separation in mutant forms of both zymogen and mature MMP-1 enzymes. Significant reductions in THP affinity were observed on mutation of either Phe301, Val319 and Asp338, residues forming part of a “ball and socket” joint in the CAT-HPX interface. Disruption of the CAT-HPX interface by mutagenesis, of any of these three residues, severely impacts collagen recognition. Conversely, the reduced collagen binding activity of a "stapled" mutant (in which the CAT and HPX domains are constrained with a disulphide bridge) indicates that the ability of the domains to transiently dislocate is also important. Thus, a balanced equilibrium between these compact and dislocated states is an essential feature of MMP-1 collagenolytic activity.

Transcription factors are a family of DNA-binding proteins involved in the regulation of all cellular processes. One particular area of transcription factor function is the process of apoptosis, the deregulation of which is involved in cancer. The study of transcription factors may therefore provide a novel perspective on cancer aetiology, as well as insight into potential treatment opportunities. However, research into transcription factor activity has traditionally been hindered by technical limitations. Firstly, transcription factors being such potent molecules need only be present at a low concentration within a cell, thus requiring sensitive methods of detection, or a large cell sample for analysis. Secondly, their tertiary protein structures present problems for interactions with inhibitory pharmaceutical agents, limiting their clinical application. The aim of this project was to develop a method to overcome these obstacles; a method for the quantification of active transcription factors, with the sensitivity for analysing endogenous transcription factors, and the high throughput adaptability as a screen for testing molecules altering a transcription factor’s DNA-binding activity. This method is based on the isolation of a synthetic oligonucleotide probe complexed with the transcription factor of interest, using Protein-A conjugated magnetic bead immunopurification. The synthetic oligonucleotide is subsequently quantified using quantitative Polymerase Chain Reaction. The complex of synthetic oligonucleotide, transcription factor and antibody was shown by the Electromobility supershift assay to form under a defined set of experimental conditions, and the synthetic oligonucleotide reliably detected by quantitative Polymerase Chain Reaction. Following optimisation, this method was able to quantify purified p53, with a sensitivity of 5 ng, However, in the scope of this project, performance was not sufficient for detection of endogenous p53 from LS174T cells. Further optimisations are required to reach this goal in order to compete with commercially available Enzyme Linked Immunosorbent Assay based methods.