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Conserved Binding Sites

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...

Ligand Binding Sites

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...

The Equilibrium Binding Constant and Binding Strength

The Equilibrium Binding Constant and Binding Strength

The equilibrium binding constant (Kb) quantifies the strength of a protein-ligand interaction. Kb can be calculated as follows when the reaction is at equilibrium:

Ligand Binding and Linkage

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...

Protein Organization

Protein Organization

Proteins are polymers of amino acid residues. They are versatile and responsible for different cellular functions, including DNA replication, molecular transport, catalysis, and structural support. Proteins have a hierarchical structure comprising at least three levels of organization: primary, secondary, and tertiary structure. Some large proteins have a quaternary structure where individual protein subunits are linked together.
The primary structure of a protein is its amino acid sequence....

Noncovalent Attractions in Biomolecules

Noncovalent Attractions in Biomolecules

Noncovalent attractions are associations within and between molecules that influence the shape and structural stability of complexes. These interactions differ from covalent bonding in that they do not involve sharing of electrons.
Four types of noncovalent interactions are hydrogen bonds, van der Waals forces, ionic bonds, and hydrophobic interactions.
Hydrogen bonding results from the electrostatic attraction of a hydrogen atom covalently bonded to a strong-electronegative atom like oxygen,...

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Related Experiment Video

Updated: Oct 16, 2025

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

Published on: September 13, 2014

Temperature artifacts in protein structures bias ligand-binding predictions.

Shanshan Y C Bradford¹, Léa El Khoury², Yunhui Ge²

¹Department of Chemical Biology & Therapeutics, St. Jude Children's Research Hospital Memphis TN 38105 USA marcus.fischer@stjude.org.

Chemical Science

|October 20, 2021

Summary

Room temperature X-ray crystallography reveals protein conformations missed at cryogenic temperatures, impacting ligand discovery and computational predictions. Cryo-artifacts can conceal errors, misleading drug development.

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A Protocol for Computer-Based Protein Structure and Function Prediction

A Protocol for Computer-Based Protein Structure and Function Prediction

Published on: November 3, 2011

Area of Science:

Structural Biology
Computational Chemistry
Biophysics

Background:

X-ray crystallography is crucial for understanding protein function and drug discovery.
Cryogenic (cryo) temperatures are standard for data collection, but may miss functionally relevant protein conformations.
Room temperature (RT) data collection may reveal alternative states important for biological activity.

Purpose of the Study:

To investigate the impact of temperature on protein structure and ligand binding.
To assess how temperature-induced structural changes affect computational predictions.
To evaluate the reliability of cryo-structures for computational validation.

Main Methods:

Systematic investigation of T4 lysozyme L99A cavity under varying temperatures and ligand-bound states.
Structural analysis of apo and ligand-bound conformations at cryo and RT.
Evaluation of computational methods (docking, binding free energy calculations) using cryo and RT structural data.

Main Results:

Shifting from cryo to RT revealed new global and local structural changes in T4 lysozyme.
An apo helix conformation, hidden at cryo, was uncovered and found relevant for ligand binding.
Temperature significantly influenced side chain and ligand conformations, impacting computational predictions.
Cryo-artifacts were found to limit the utility of structures for computational validation across multiple protein classes.

Conclusions:

Cryogenic data collection can miss important protein conformations, potentially hindering ligand discovery.
Temperature artifacts in cryo-structures can mislead computational methods, affecting drug design and development.
Consulting only cryogenic structural data requires caution due to potential concealed errors and computational prediction failures.