Proteins and Their Ligands: Their Importance and How to Crystallize Them

Abstract

usage of catalytically inactive mutants with bound ligand(s)• creation of an environment (i. g. buffer condition) which shifts the equilibrium constant so that the reaction cannot occurThe most important point concerning preparing co-crystallization trials is the knowledge of the corresponding kinetical parameters. Proteins bind their natural ligand(s) with high affinity, which means in the nM-up to low mM range. To successfully crystallize a protein with the ligand(s) bound, the affinity needs to be determined. There are numerous biophysical techniques to achieve this, for example Intrinsic Tryptophan Fluorescence, Isothermal Calorimetry, Surface Plasmon Resonance and many others. In principle the affinity is determined by the size of a ligand as well as the property of the binding site of the protein. As first approximation, one can state that affinity increases with a decrease in ligand size.

Crystal Structure of the Ectoine Hydroxylase, a Snapshot of the Active Site

“…The cocrystallization of a protein with its enzymatic reaction product has been successfully used to obtain informative crystal structures (54). When applied to the SaEctD protein, it yielded crystals with good diffraction properties, but none of them showed any electron density for 5-hydroxyectoine.…”

Efficient Screening Methodology for Protein Crystallization Based on the Counter-Diffusion Technique

“…One of the most important advantages of the CCD crystallization screening is its low protein consumption. With the 5 kits at 6 different pHs and capillaries of 0.1 mm inner diameter and 40 mm length, only 9 μL of protein solution is required for the full 30 protein solution for the 24 conditions. If compared with the amount of protein required to manually set up a typical HDVD (hanging-drop vapor diffusion) full screening (approximately 100 μL), the use of CCD represents approximately a 4-fold reduction.…”

Protein crystallization: Eluding the bottleneck of X-ray crystallography

“…These types of additives might include cofactors or ligands required for the biological activity of the protein. Such molecules not only facilitate successful crystallization but also provide functional insight into the protein by revealing the substrate or cofactor binding site [ 23 , 24 , 25 ].…”