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Chicken egg white lysozyme variant I55T : Raman spectroscopic analysis and expression of random mutants Kanagawa, Saeko

Abstract

Chicken egg white lysozyme is often used in protein studies because of its well-known properties. I55T variant chicken egg white lysozyme is an analogue of I56T amyloidogenic human lysozyme which causes hereditary non-neuropathic amyloidosis. The mechanism of alteration of physical properties by a single substitution is not well-understood. Wild type and I55T lysozymes expressed in Pichia pastoris were compared using Raman spectroscopy to elucidate the conformational alteration caused by the single amino acid substitution. Wild type showed a similar spectrum to commercial lysozyme from chicken egg white; however, some structural changes were indicated. The I55T variant did not exhibit typical tryptophan signals, suggesting the exposure of tryptophan residue(s) to an aqueous environment. This Raman analysis suggests a structural change in I55T variant, which may be related to the aggregation property of I55T variant. It was suggested that P. pastoris expression system may affect protein folding. Mutations were introduced to the I55T variant using Random Centroid Optimization for Genetics (RCG) to attempt to decrease the amyloidogenic nature of 155T variant. Nine sets of two site mutations were introduced to the 155T template and these nine variants were expressed in P. pastoris along with wild type and I55T lysozymes. Two variants, K33V-C115F and P79I-G126K, were not secreted. The lack of secretion may indicate the protein structure was abnormal, which resulted in degradation of the protein by the yeast. Seven mutants exhibited comparable lysozyme expression levels to wild type but six displayed no measurable enzymatic activity. This may suggest the aggregation of the secreted protein and/or a conformation change around active sites which resulted in the loss of functionality. Effects of mutations were investigated using the Homology Similarity Analysis program; moderate changes in all propensities were found for T40E-A42K, the only mutant which exhibited enzymatic activity, and higher charge introduction in P79I-G126K. Aggregation property may have been enhanced in K33V-C115F and P79I-G126K due to the increased β-strand propensity. Results indicate that the local conformational characteristics relative to the whole protein structure may be more important than the local sequence propensity. Data indicate the difficulty in altering the amyloidogenic property of I55T lysozyme.

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