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Related Concept Videos

Conserved Binding Sites01:49

Conserved Binding Sites

Many proteins’ biological role depends on their interactions with their ligands, small molecules that bind to specific locations on the protein known as ligand-binding sites. Ligand-binding sites are often conserved among homologous proteins as these sites are critical for protein function.
Binding sites are often located in large pockets, and if their location on a protein’s surface is unknown, it can be predicted using various approaches. The energetic method computationally analyses the...
Covalently Linked Protein Regulators02:04

Covalently Linked Protein Regulators

Proteins can undergo many types of post-translational modifications, often in response to changes in their environment. These modifications play an important role in the function and stability of these proteins. Covalently linked molecules include functional groups, such as methyl, acetyl, and phosphate groups, and also small proteins, such as ubiquitin. There are around 200 different types of covalent regulators that have been identified.
These groups modify specific amino acids in a protein.
Protein-protein Interfaces02:04

Protein-protein Interfaces

Many proteins form complexes to carry out their functions, making protein-protein interactions (PPIs) essential for an organism's survival. Most PPIs are stabilized by numerous weak noncovalent chemical forces. The physical shape of the interfaces determines the way two proteins interact. Many globular proteins have closely-matching shapes on their surfaces, which form a large number of weak bonds. Additionally, many PPIs occur between two helices or between a surface cleft and a polypeptide...
Ligand Binding and Linkage00:49

Ligand Binding and Linkage

Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked.  In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence the...
Ligand Binding Sites02:40

Ligand Binding Sites

Proteins are dynamic macromolecules that carry out a wide variety of essential processes; however, the activities of most proteins depend on their interactions with other molecules or ions, known as ligands.
Protein-ligand interactions are quite specific; even though numerous potential ligands surround a cellular protein at any given time, only a particular ligand can bind to that protein. Moreover, a ligand binds only to a dedicated area on the surface of the protein, known as the...

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Updated: Jul 14, 2026

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing
11:36

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Published on: July 3, 2016

Structure-based protocol for identifying mutations that enhance protein-protein binding affinities.

Deanne W Sammond1, Ziad M Eletr, Carrie Purbeck

  • 1Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7260, USA.

Journal of Molecular Biology
|July 3, 2007
PubMed
Summary

Scientists developed a computational method to enhance protein binding affinity by predicting beneficial mutations. This approach successfully increased binding in experimental tests, offering a valuable tool for protein engineering applications.

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Computational Prediction of Amino Acid Preferences of Potentially Multispecific Peptide-Binding Domains Involved in Protein-Protein Interactions
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Computational Prediction of Amino Acid Preferences of Potentially Multispecific Peptide-Binding Domains Involved in Protein-Protein Interactions
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Computational Prediction of Amino Acid Preferences of Potentially Multispecific Peptide-Binding Domains Involved in Protein-Protein Interactions

Published on: January 26, 2024

Area of Science:

  • Protein Engineering
  • Computational Biology
  • Biochemistry

Background:

  • Protein binding affinity is crucial for developing biosensors, therapeutics, and industrial reagents.
  • Rational design of protein interactions requires methods to predictably enhance binding affinities.

Purpose of the Study:

  • To develop and validate a structure-based computational method for predicting single amino acid mutations that enhance protein-protein binding affinities.
  • To experimentally verify the predictions of the computational protocol on two distinct protein complexes.

Main Methods:

  • A structure-based protocol was designed to identify mutations that increase buried hydrophobic surface area and/or decrease buried hydrophilic surface area at protein interfaces.
  • Mutations were selected based on criteria including amino acid property changes, predicted changes in binding free energy using Rosetta, and monomer stability.
  • The computational predictions were tested by synthesizing and characterizing 12 single-site mutations in the Galpha(i1)-RGS14 GoLoco motif and E2 (UbcH7)-E3 (E6AP) complexes.

Main Results:

  • Nine out of 12 experimentally tested mutations successfully increased protein-protein binding affinity.
  • Five of these mutations enhanced binding affinity by more than 1.0 kcal/mol.
  • Validation against literature data showed that the method accurately predicted affinity increases for 5 out of 8 identified mutations.

Conclusions:

  • The developed structure-based computational method is effective in rationally predicting single point mutations that enhance protein-protein binding affinities.
  • This approach provides a reliable tool for protein engineering, enabling the optimization of binding interactions for various applications.
  • The successful experimental validation demonstrates the practical utility of the computational protocol in protein design.