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Thermostabilizing mutations preferentially occur at structural weak spots with a high mutation ratio

Paper ID Volume ID Publish Year Pages File Format Full-Text
23774 43472 2012 10 PDF Available
Title
Thermostabilizing mutations preferentially occur at structural weak spots with a high mutation ratio
Abstract

We apply Constraint Network Analysis (CNA) to investigate the relationship between structural rigidity and thermostability of five citrate synthase (CS) structures over a temperature range from 37 °C to 100 °C. For the first time, we introduce an ensemble-based variant of CNA and model the temperature-dependence of hydrophobic interactions in the constraint network. A very good correlation between the predicted thermostabilities of CS and optimal growth temperatures of their source organisms (R2 = 0.88, p = 0.017) is obtained, which validates that CNA is able to quantitatively discriminate between less and more thermostable proteins even within a series of orthologs. Structural weak spots on a less thermostable CS, predicted by CNA to be in the top 5% with respect to the frequency of occurrence over an ensemble, have a higher mutation ratio in a more thermostable CS than other sequence positions. Furthermore, highly ranked weak spots that are also highly conserved with respect to the amino acid type found at that sequence position are nevertheless found to be mutated in the more stable CS. As for mechanisms at an atomic level that lead to a reinforcement of weak spots in more stable CS, we observe that the thermophilic CS achieve a higher thermostability by better hydrogen bonding networks whereas hyperthermophilic CS incorporate more hydrophobic contacts to reach the same goal. Overall, these findings suggest that CNA can be applied as a pre-filter in data-driven protein engineering to focus on residues that are highly likely to improve thermostability upon mutation.

Graphical abstractFigure optionsDownload full-size imageDownload as PowerPoint slideHighlights► Constraint Network Analysis for predicting protein thermostability and weak spots. ► Improved analysis by considering structure ensembles and new network representation. ► Very good correlation between predicted and experimental thermostabilities for 5 CS. ► Weak spots predicted on less stable CS are preferentially mutated in more stable CS. ► Method works as pre-filter in data-driven engineering for thermostabilization.

Keywords
CNA, Constraint Network Analysis; CS, citrate synthase; DCM, distance constraint model; TUS, thermal unfolding simulation; RCD, rigid cluster decomposition; MD, molecular dynamics; PDB, protein data bank; WSMR, weak spot mutation ratioCitrate synthase; Th
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Thermostabilizing mutations preferentially occur at structural weak spots with a high mutation ratio
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 159, Issue 3, 15 June 2012, Pages 135–144
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us