Assessing the functional roles of coevolving PHD finger residues

Area of Science:

• Biochemistry
• Structural Biology
• Computational Biology

Background:

• In silico methods using coevolving residue constraints have advanced protein structure prediction.
• The functional and physical roles of these coevolving residues require experimental validation.
• The PHD finger module, a histone reader, exhibits subtype-specific coevolving residues.

Purpose of the Study:

• To experimentally assess the contributions of coevolving residues to protein folding and stability.
• To clarify the specific roles of coevolving residues within different PHD finger subtypes.
• To develop and validate a combined computational and experimental strategy for analyzing residue contributions.

Main Methods:

• Experimental analysis using proteolysis and thermal unfolding assays on wildtype and mutant PHD finger proteins.
• Computational sequence design to identify residues critical for folding.
• Generative model-based energy estimates of individual protein structures.

Main Results:

• Coevolving residues demonstrate varied contributions to protein stability and folding, despite strong in silico constraints.
• Residue positions with significant coevolutionary constraints impacted stability in certain PHD finger subtypes but not others.
• Computational sequence design and energy estimations successfully distinguished between coevolving residues that contribute to folding and those that do not.
• Experimental validation confirmed the findings from computational analyses regarding folding-critical residues.

Conclusions:

• Coevolving residues play context-dependent roles in protein folding and stability.
• A combined approach of experimental assays, sequence design, and energy estimation provides a robust strategy for dissecting residue contributions.
• This methodology can be applied to understand residue importance in other large protein families.