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The Use of a β-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions
Published on: February 1, 2018
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Conserved proline residues prevent dimerization and aggregation in the β-lactamase BlaC
A Chikunova1, M P Manley1, C N Heijjer1
1Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.
Protein Science : a Publication of the Protein Society
|March 27, 2024
Summary
Highly conserved prolines in beta-lactamase enzymes unexpectedly tolerate substitutions. Mutations can cause enzyme dimerization and instability, highlighting prolines
Area of Science:
- Biochemistry and Molecular Biology
- Enzymology
- Protein Structure and Function
Background:
- Amino acid conservation across protein families is driven by evolution.
- Conserved proline residues are typically crucial for protein folding and structural stability.
- Class A beta-lactamases exhibit conserved prolines that surprisingly tolerate substitutions.
Purpose of the Study:
- To investigate the functional roles of three conserved proline residues (107, 226, 258) in Mycobacterium tuberculosis beta-lactamase BlaC.
- To understand the impact of proline substitutions on BlaC enzyme activity, stability, and structural integrity.
Main Methods:
- Site-directed mutagenesis to introduce proline substitutions in BlaC.
- Enzyme activity assays to measure catalytic function.
- Crystallography to determine the three-dimensional structure of mutant variants.
- Analysis of protein aggregation and stability.
Main Results:
- Mutations in conserved prolines did not abolish enzyme activity but led to structural alterations.
- Proline substitutions induced enzyme dimerization, exemplified by the Pro107Thr variant showing domain swapping.
- Mutant proteins exhibited reduced stability and increased propensity for aggregation over time.
Conclusions:
- Conserved prolines in BlaC, while not essential for immediate activity, are critical for maintaining long-term structural integrity and preventing aberrant multimerization.
- Proline substitutions destabilize the enzyme structure, promoting dimerization and aggregation.
- The findings challenge the conventional view of proline's role, suggesting context-dependent functions in enzyme structure and stability.
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