Analytical Methods For Assessing The Effects Of Site-Directed Mutagenesis On Protein–Cofactor And Protein–Protein Functional Relationships

Keywords

Binding constant; Circular dichroism; Coenzymes; Metalloprotein; Polymerase chain reaction; Protein expression; Redox potential; Steady-state kinetics; Tryptophan fluorescence

Abstract

To completely understand the role of an amino acid residue that is targeted for site-directed mutagenesis a thorough analysis of the impact that the mutation has on the function of the protein is required. General methods for performing site-directed mutagenesis and expressing the recombinant protein variant are described. Protein–cofactor interactions are important because cofactors are often directly involved in facilitating catalysis by enzymes and in electron transfer by redox proteins. Many cofactors also have characteristic spectroscopic properties. As such, general methods are described to analyze the spectroscopic, redox and catalytic properties of protein-bound cofactors. Methods for assessing the effects of a mutation on protein–protein interactions are also described. Lastly, methods for assessing the overall structural integrity of the protein are described, as this is important to ensure that the mutation has not caused a global disruption of protein structure, rather than a specific effect on function.

Publication Date

1-1-2017

Publication Title

Methods in Molecular Biology

Volume

1498

Number of Pages

421-437

Document Type

Article; Proceedings Paper

Personal Identifier

scopus

DOI Link

https://doi.org/10.1007/978-1-4939-6472-7_29

Socpus ID

84990950529 (Scopus)

Source API URL

https://api.elsevier.com/content/abstract/scopus_id/84990950529

This document is currently not available here.

Share

COinS