# Proteomics/Protein - Protein Interactions/Cross-linking

This Section:

## Introduction

Cross-links are ionic or covalent bonds that form between two polymer chains. In the context of biological science, cross-linking generally refers to a molecular reaction that joins two molecular units in order to study their interactions.
In the process of studying protein-protein interactions, the technique of cross-linking has proven to be very important in obtaining information about the structure and function of proteins. Cross-linking also helps elucidate vital information about protein interactions in receptors, signaling cascades, and multiprotein complexes. The information obtained from cross-linking experiments is powerful because it provides a higher resolution of the structural data; to the point of mapping protein-protein interactions to specific amino acids or domains. The most important advantage of cross-linking is that it allows for noncovalent protein-protein interactions, those that are transient or dependent on specific physiological conditions, to be captured in long term covalent complexes that maintain the information even through further processing, including purification, enrichment, and analysis.

## Bifunctional cross-linking

Bifunctional cross-linking reagents contain two reactive groups, thus creating a way to covalently link two target groups. These target groups can be on the same protein, or on different proteins. The usual target for these reagents are nucleophilic side chains like tyrosine or cysteine.

#### Homobifunctional Cross-Linking

In homobifunctional cross-linking reagents both reactive groups are the same. The majority of homobifunctional cross-linking reagents create intramolecular crosslinks.

#### Heterobifunctional Cross-Linking

In heterobifunctional cross-linking reagents the reactive groups are different. This allows for intermolecular crosslinks. Possessing different reactive groups allows the crosslinker to make conjugations between specific functional groups on different molecules and help minimize self-conjugation.

## Crosslinker Reactivities

| Imidoesters | Imidoester crosslinkers react with primary amines to form amidine bonds. The resulting amidine is protonated and therefore has a positive charge at physiological pH. Imidoester homobifunctional crosslinkers can move through cell membranes, making them beneficial in crosslinking proteins within the membrane and with intrantramolecular crosslinking to studying protein subunit structure. |
| NHS-Esters | NHS-Esters react with α-amine groups present on the N-termini of proteins and ε-amines on lysine residues to form amide bonds. A covalent amide bond is formed during the reaction, releasing N-hydroxysuccinimide. NHS-ester crosslinking reactions are most commonly performed in phosphate, bicarbonate/ carbonate, or borate buffers. NHS-ester crosslinker that react with primary amines on the molecule’s surface could result in conformational change of the protein, which may result in loss of biological activity. |
| Maleimides | Maleimide groups react specifically with sulfhydryl groups when the pH of the reaction is between pH 6.5 and 7.5. The thioether linkage that forms is stable and is not reversible. |
| Haloacetyls | The most commonly used a-haloacetyl crosslinkers contain the iodoacetyl group that react with sulfhydryl groups at physiological pH. The reaction of the iodoacetyl group with a sulfhydryl proceeds by nucleophilic substitution of iodine with a thiol producing a stable thioether linkage. |

## Oxidative Cross-Linking

Oxidative cross-linking, introduced by Brown et al , is highly specific. The process is mediated by a Ni(II)-peptide reagent [N(II)-NH2-Gly-Gly-His-COOH] and the presence of a peracid such as magnesium monoperoxyphthalic acid or oxone. The initiation of the mechanism involves the oxidation of the Ni(II) to Ni(III) by the peracid, which creates a high valent oxo species. In turn, the species can then strip an electron from aromatic amino acid side chains, like a tyrosine residue. This radical cation can then, through electrophilic attack, covalently couple to a nearby tyrosine or cysteine. Finally, a hydrogen atom is abstracted by an unknown species. The final product is a cross-linked protein complex. To see the mechanism, as first described, in its entirety see Brown et al.
This cross-linking method is advantageous in that through an affinity cross-linking strategy the Ni(II)-peptide can be selectively delivered to a protein and activated at a desired time by the addition of the peracid. In turn, this reaction may be highly localized and avoids modifications to unrelated proteins making data interpretation less complicated.

## Photoreactive crosslinking

Cross-linking is a useful tool in the study of protein-protein reactions, but the nature of most cross-linking methods prevents their use in live cells. Recently, the use of photo-reactive amino acid analogs to create cross-links between interacting proteins has allowed scientists to study protein complexes in-vivo. Analogs of leucine and methionine, both featuring photosensitive diazirine rings, are fed to growing cells. These analogs are incorporated into proteins and create cross-links between interacting proteins when exposed to ultraviolet light.
Photo-sensitizers, like naturally occurring riboflavin, undergo photo-excitation with exposure to light, transferring energy to reactions. The photo-excited molecules are generally very reactive as well, frequently binding readily to nearby compounds. This property has been used in new clinical treatments of keratonconus, a degenerative disorder of the cornea. A solution of riboflavin is added to the eye and activated with UV light. The reactive riboflavin creates new cross-linking bonds across the cornea, restoring some of its mechanical strength.
The amino acid analogs used to analyze protein-protein interactions take advantage of the reactivity of photo-excited molecules. The analogs are identical to the natural amino acids, except for a photosensitive diazirine ring. When exposed to UV light, the nitrogen is released and a reactive carbene is formed. The activated carbene has a very short half-life, so reactions must occur with groups in a very close proximity. In a protein complex, the carbenes will react with nearby groups to form stable cross-links that “freeze” the complex in its current position, allowing scientists to analyze the protein-protein interactions within the complex.
Cells are grown in a medium featuring these analogs, with the cells including them into proteins during synthesis where the corresponding natural amino acids would normally occur. Three amino acid analogs were created. Photo-leucine and photo-methionine were produced in large amounts, while photo-isoleucine was produced in almost 20-fold less amounts.
The exciting thing about these amino acids analogs is their lack of toxicity. Studies have shown that the photo-amino acids are incorporated well into the cells, and growing cells in a medium lacking natural leucine and methionine, but with plentiful amounts of their photo-analogs, slows the growth only slightly but the viability of the cells is unchanged. Also, photo-activation of the cells does not affect viability. This allows these amino acid analogs to be used in growing cells to study protein-protein interactions in-vivo, with minimal effect on the cell.
Photo-excitable diazirine has been incorporated into sugars to analyze membrane reactions. Monosaccharides with diazirine are incorporated in cell surfaces, excited with ultraviolet light, then analyzed as part of glycoprotein complexes within the membrane. These photo-sugars are metabolically incorporated into the cell’s glycoproteins and can trap interations between glycoproteins. Because of diazirine’s small size, the functionality of the glycoproteins is relatively unaffected.

## References

* ↑ Lynch and DE Koshland Jr. Disulfide cross-linking studies of the transmembrane regions of the aspartate sensory receptor of Escherichia coli. Proceedings of the National Academy of Sciences, Vol 88, 10402-10406.
* ↑ A. Fancy, Karsten Melcher, Stephen Albert Johnston, and Thomas Kodadek. Chemistry & Biology. July 1996, 3:551-559. New chemistry for the study of multiprotein complexes: the six-histidine tag as a receptor for a protein crosslinking reagent.
* ↑ A. Trakselis, Stephen C. Alley, and Faoud T. Ishmael. Identification and Mapping of Protein-Protein Interactions by a Combination of Cross-Linking, Cleavage, and Proteomics. Bioconjugate Chemistry. Vol 16, Number 4, pgs 741-750.
* 1 2 C. Brown, Sang-Hwa Yang, and Thomas Kodadek. Highly Specific Oxidative Cross-Linking of Proteins Mediated by a Nickel-Peptide Complex. Biochemistry 1995, 34, 4733-4739.
* ↑ A. Fancy. Elucidation of protein–protein interactions using chemical cross-linking or label transfer techniques. Current Opinion in Chemical Biology. Vol 4, Issue 1, 1 February 2000, Pages 28-33.
* ↑ (2008, March 20). In Wikipedia, The Free Encyclopedia. Retrieved 14:59, March 30, 2008.
* ↑ (2008, March 19). In Wikipedia, The Free Encyclopedia. Retrieved 15:20, March 30, 2008.
* ↑ M., Radzikowska, A., and Thiele, C. (2005) Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells. Nature Methods. 2, 261 – 268
* ↑ Tanaka and Jennifer J. Kohler. Photoactivatable Crosslinking Sugars for Capturing Glycoprotein Interactions. J. Am. Chem. Soc.; 2008; 130(11) pp 3278 – 3279
Authors: Anthony Corbett, Joshua Horn