Binding and Inhibition

Explore a ligand pocket, then change affinity and concentration in an equilibrium binding model.

Experimental trypsin cartoon cutaway exposing a gold benzamidine inhibitor and mint atomic spheres marking nearby pocket residues.

Structural referencePDB 3PTB

A defined equilibrium model

Binding depends on concentration as well as affinity.

Free ligand concentration10 nMLigand Kd10 nMSame-site competitor present

Solid curve: current model · Dashed curve: ligand alone · Point: selected concentration

Ligand-bound

50.0%

Competitor-bound

0.0%

Free sites

50.0%

With no competitor, free ligand equal to Kd gives 50% occupancy. Reducing Kd at the same free concentration increases occupancy.

Equation and assumptions

Ligand fraction = ([L]/Kd) / (1 + [L]/Kd + [I]/Ki)

One reversible binding site, mutually exclusive ligands, equilibrium, and prescribed free concentrations. There is no cooperativity, ligand depletion or kinetic simulation. All constants are illustrative; they are not measurements for the trypsin–benzamidine structure above. Occupancy alone does not establish efficacy or biological response.

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Quick reference

Affinity and occupancy

At equilibrium, a single-site binding model gives half occupancy when the free ligand concentration equals its dissociation constant, Kd. A lower Kd means higher affinity under the stated conditions. Occupancy also depends on how much free ligand is available.

Competition changes the balance

A second ligand competing for the same site reduces occupancy by the first. The concentration needed to halve that occupancy depends on the first ligand's concentration and affinity as well as the competitor's Ki. The sliders prescribe free concentrations and omit depletion.

A structure is a different kind of evidence

PDB 3PTB shows benzamidine bound to bovine trypsin in an X-ray structure at 1.70 Å. It provides a concrete pocket to inspect. The illustrative constants below do not come from those coordinates. Binding or displacement alone does not establish a biological effect.

Check your understanding Optional

A competitor halves ligand occupancy in the model. Has it necessarily halved the biological response?

Yes. Occupancy and response are always identical.Yes. Any ligand in a crystal is a complete inhibitor.No. Functional response needs its own evidence.

At the bench

Move from defined binding relationships to measured concentration–response data, controls, curve fitting and uncertainty in Assay. Protein provides structural context for the target.

Open Assay

Sources and model notes

Structure-based views of resolved experimental coordinates. Cutaways, category colours, camera choices and guided highlights are authored for teaching. Coordinates remain fixed; highlights are not experimental trajectories or simulation outputs.

RCSB PDB · Trypsin–benzamidine 3PTB

Experimental binding geometry; the numerical experiment is separate.

Assay Guidance Manual · Receptor binding

Equilibrium, free concentrations, saturation binding and competition assumptions.