Protein crosslinking reagents or crosslinkers can be accurately defined as molecules containing two or more reactive ends that are capable of chemically attaching to specific functional groups on proteins or other molecules. These reagents are generally used in creating detectable scientific probes to facilitate a number of proteomics methods, including Western blotting, ELISA and other strategies for studying protein-protein interactions.
Crosslinkers: Understanding Their Structure and Chemistry
While the protein structure is relatively complex, there are only four functional groups that can be targeted for crosslinking and chemical modification techniques. This includes primary amines (-NH2), carboxyls (-COOH), sulfhydryls (-SH) and carbonyls (-CHO).
Primary amines are positively charged at physiologic conditions and are usually found on the outer surface of the proteins. Due to their location, primary amines are easily accessible for conjugation without denaturing the protein. Like primary amines, carboxyls can also be found on the outer surface of the protein structure while sulfhydryls usually exists in the side chain of cysteine. On the other hand, carbonyls such as aldehydes and ketones are formed by oxidative treatment of glycoproteins.
To facilitate the investigation of protein-protein interactions, a number of chemical reactive groups have been used to target these protein functional groups. This includes the following:
Primary amine reactive crosslinkers
Primary amines react with imidoesters to form amidine bonds. They also react with NHS-esters to form amide bonds and release N-hydroxysuccinimide. Imidoesters are best used for protein subunit studies, as well as in studying membrane composition and structure, and investigating protein-protein and molecular interactions.
On the other hand, NHS-esters are ideally used for studying cell surface proteins since they will only react with the protein molecules on the surface of the plasma membrane.
Note: When using amine reactive crosslinkers, avoid buffers containing amines such as Tris or glycine.
Sulfhydryl-reactive crosslinkers
Sulfhydryl reactive reagents react exclusively with free sulfhydryl residues so if there are no sulfhydryl residues available in the side chain of the amino acid cysteine, they should be generated through (1) the reduction of disulfide bonds or (2) the modification of the lysine ε-amine. If the former method is used, excess reducing agent must be removed and a metal chelating agent such as EDTA should be used to prevent the reoxidation of sulfhydryls to disulfides.
Cross-linking to sulfhydryl residues can be facilitated through the use of maleimides, haloacetyls or pyridylthiol groups. Maleimide-activated crosslinkers react exclusively with sulfhydryl groups at near neutral pH while haloacetyls react with sulfhydryls between pH 7.2 to 9.0 to form stable thioether linkages. Pyridyldithiols (pyridyl disulfides) react with free sulfhydryls over a wide range of pH (between pH 6.0 and 9.0) to form a disulfide linkage. The reaction releases pyridine-2-thione which absorbs light at 343nm.
Special precautions should be observed when using maleimides and haloacetyls to ensure accurate results. When using maleimide reagents, you must remove any excess thiols from the reaction buffer to prevent them from competing for coupling sites. On the other hand, if you are using haloacetyl reagents, you need to perform your experiment in the dark to limit the formation of free iodine which may react with tyrosine, tryptophan and histidine residues.
Carboxyl-reactive crosslinkers
Carbodiimides conjugate carboxyls to primary amines to form amide or hydrazone bonds without being a part of the final crosslink. Conjugation is usually performed between pH 4.5 to 7.5 (pH 4.5 to 5.0 is considered ideal) and only takes a few minutes to complete. This reaction chemistry is widely used in immobilization procedures and immunogen preparation.
Please take note that the presence of excess carboxyls and amines in your sample may cause random polymerization of polypeptides so you may want to make sure that there are no extraneous carboxyls and amines before proceeding with your experiment.
Carbonyl-reactive crosslinkers
Hydrazides react with carbonyl groups at pH 5.0 to 7.0 to form stable hydrazone bonds. As such, these reagents are ideally used for labeling, immobilizing and conjugating glycosylated proteins.
When using carbonyl-reactive crosslinkers, you need to determine the optimal temperature and pH condition for both oxidation and hydrazide reactions since the extent of glycosylation varies for each protein. You should also avoid buffers containing amines (e.g. Tris or glycine) since they react with aldehydes, quenching their reaction with hydrazides.
