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Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability

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Using experimental evolution to probe molecular mechanisms of protein function.

Marlies Fischer^1,2, Mandeep Kang^1,2, Nicholas Pj Brindle^1,2

¹Department of Molecular and Cell Biology, University of Leicester, Henry Wellcome Building, Lancaster Road, Leicester, LE1 9HN, United Kingdom.

Protein Science : a Publication of the Protein Society

|October 29, 2015

Summary

Directed evolution engineers protein function through iterative cycles of diversification and selection. High-throughput sequencing and computational analysis now offer deeper insights into protein sequence-function relationships and mechanisms.

Keywords:

directed evolution evolution high throughput sequencing mutation next generation sequencing protein function protein sequence

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Area of Science:

Biochemistry
Molecular Biology
Protein Engineering

Background:

Directed evolution is a key method for engineering protein function.
It involves cycles of sequence variation, activity assays, and selection.
This process generates valuable data on sequence-function relationships.

Purpose of the Study:

To highlight the utility of directed evolution for understanding protein function.
To emphasize the impact of recent technological advancements on the field.
To showcase the potential for deeper mechanistic insights.

Main Methods:

Iterative sequence diversification and variant screening.
High-throughput sequencing for comprehensive data acquisition.
Computational analysis for high-resolution residue contribution assessment.

Main Results:

Identification of key residues critical for protein function.
Elucidation of combinatorial effects on protein activity.
Revealing potential functional mechanisms through evolutionary trajectories.

Conclusions:

Directed evolution, enhanced by high-throughput sequencing and computation, provides unprecedented insight into protein function.
This approach enables detailed analysis of individual residue contributions to activity.
Future applications promise to significantly deepen our understanding of protein mechanisms.