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Updated: Mar 5, 2026

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

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Published on: February 7, 2019

Creating a Homeodomain with High Stability and DNA Binding Affinity by Sequence Averaging.

Katherine W Tripp¹, Matt Sternke¹, Ananya Majumdar¹

¹The T. C. Jenkins Department of Biophysics and ‡Biomolecular NMR Center, Johns Hopkins University , 3400 North Charles Street, Baltimore, Maryland 21218, United States.

Journal of the American Chemical Society

|March 23, 2017

Summary

Consensus design created a hyperstable globular protein (homeodomain) that retains function. This stabilized protein binds DNA with higher affinity, demonstrating stability is compatible with protein function.

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Published on: July 14, 2015

Area of Science:

Protein engineering
Structural biology
Biophysics

Background:

Consensus design successfully enhances stability in linear repeat proteins.
The applicability of consensus design to globular proteins and its effect on function remain unclear.

Purpose of the Study:

To investigate the potential of consensus design for creating stable, functional globular proteins.
To characterize the structural and functional properties of a consensus-designed homeodomain.

Main Methods:

Consensus design strategy applied to a homeodomain sequence.
Circular dichroism and differential scanning calorimetry for stability assessment.
DNA binding assays and 15N relaxation studies for functional and dynamic characterization.

Main Results:

A consensus-designed homeodomain (HD) adopts a stable, folded structure.
The consensus-HD exhibits a 5 kcal·mol-1 higher unfolding free energy than the natural engrailed-HD.
Consensus-HD demonstrates ~100-fold higher DNA binding affinity and reduced backbone dynamics.

Conclusions:

Consensus design is effective for creating hyperstable globular proteins.
Greatly stabilized proteins can exhibit enhanced functional properties, such as increased substrate affinity.
High protein stability is compatible with and can improve protein function.