Structural and functional studies on SCR domains from human complement receptor 1

Clark, Nicola Suzanne (1996) Structural and functional studies on SCR domains from human complement receptor 1. University of Southampton, Doctoral Thesis.

Abstract

SCR1-3, SCR3 and SCR3₂ are soluble recombinant proteins, consisting of the first three, third and two consecutive third N-terminal domains of CR1 respectively and provide the basis for the work described in this thesis.

SCR1-3 has four tryptophan residues, one conserved in each domain at positions 51, 113 and 184, and an additional non conserved residue located at position 7 in SCR1. Individual site-directed replacement of these residues by phenylalanine residues determined that the fluorescence properties of SCR1-3, observed at 338nm, are due to the contribution of the non-conserved tryptophan located in SCR1, whilst the fluorescence of the remaining three conserved tryptophan residues is quenched by the N-terminal disulphide bond of each SCR unit. Fluorescence and circular dichroism techniques were utilised to determine the conformational stabilities and folding properties of SCR1-3, SCR3 and SCR3₂. SCR1-3 is significantly more stable than SCR3 and SCR3₂. Kinetic analysis of the folding and unfolding processes of the triple domain SCR1-3 indicates the presence of folding intermediates, not detectable at equilibrium, which are possibly attributable to the formation of interdomain contacts. The single domain, SCR3, demonstrates a simple two state transition between the folded and unfolded forms, with no detectable folding intermediates. Kinetic analysis of the refolding process of the W7F mutant protein confirmed that the rate limiting intermediate step was not due to spectral perturbations caused by the refolding of the non-conserved tryptophan and was therefore most probably due to interdomain interactions.

Chemical modification and site directed mutagenesis studies have identified groups of residues and regions within SCR1-3 involved in the interaction with complement proteins.

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