Overview of Crosslinking and Protein Modification

A number of techniques for studying the structure and interaction of proteins, as well as for manipulating proteins for use in affinity purification or detection procedures, depend on methods for chemically crosslinking, modifying or labeling proteins. 

Crosslinking is the process of chemically joining two or more molecules by a covalent bond. Modification involves attaching or cleaving chemical groups to alter the solubility or other properties of the original molecule. "Labeling" generally refers to any form of crosslinking or modification whose purpose is to attach a chemical group (e.g., a fluorescent molecule) to aid in detection of a molecule and is described in other articles.

The entire set of crosslinking and modification methods for use with proteins and other biomolecules in biological research is often called "bioconjugation" or "bioconjugate" technology. (Conjugation is a synonym for crosslinking.)

Explore protein labeling products  Crosslinking Technical Handbook

Covalent modification and crosslinking of proteins depends on the availability of particular chemicals that are capable of reacting with the specific kinds of functional groups that exist in proteins. In addition, protein function and structure are either the direct focus of study or they must be preserved if a modified protein is to be useful in a technique. Therefore, the composition and structure of proteins, and the potential effects of modification reagents on protein structure and function, must be considered.

Proteins have four levels of structure. The sequence of its amino acids is the primary structure. This sequence is always written from the amino end (N-terminus) to the carboxyl end (C-terminus). Protein secondary structure refers to common repeating elements present in proteins. There are two basic components of secondary structure: the alpha helix and the beta-pleated sheet. Alpha helices are tight, corkscrew-shaped structures formed by single polypeptide chains. Beta-pleated sheets are either parallel or anti-parallel arrangements of polypeptide strands stabilized by hydrogen bonds between adjacent –NH and –CO groups. Parallel beta-sheets have adjacent strands that run in the same direction (i.e., N-termini next to each other), while anti-parallel beta sheets have adjacent strands that run in opposite directions (i.e., N-terminus of one strand arranged toward the C-terminus of adjacent strand). A beta-pleated sheet may contain two to five parallel or antiparallel strands.

Tertiary structure is the full three-dimensional, folded structure of the polypeptide chain and is dependent on the suite of spontaneous and thermodynamically stable interactions between the amino acid side chains. Disulfide bond patterns, as well as ionic and hydrophobic interactions greatly impact tertiary structure. Quaternary structure refers to the spatial arrangement of two or more polypeptide chains. This structure may be a monomer, dimer, trimer, etc. The polypeptide chains composing the quaternary structure of a protein may be identical (e.g., homodimer) or different (e.g., heterodimer).

The four levels of protein structure. The sequence of amino acids, represented by blue dots, joined by peptide bonds, comprise the primary structure. The properties of the constituent amino acids, in the context of the cellular environment, largely determine spontaneous formation of the higher-level structure that is essential for protein function.

The complete structure of a functioning protein involves more than polypeptide chains at the four levels of structure. Various covalent modifications often occur, either during or after assembly of the polypeptide chain. Most proteins undergo co- and/or post-translational modifications. Examples include phosphorylation (of serine, threonine or tyrosine residues), glycosylation, and ubiquitination.

Knowledge of these native modifications is extremely important because they may alter physical and chemical properties, folding, conformation distribution, stability, activity, and consequently, function of the proteins. The study of post-translational modifications (a different meaning from the protein modification being discussed in the present article) is an important area of research; see related articles for a discussion of that topic.

Because the structure of a protein dictates its biological activity, characterization of protein structure continues to be an important area of research. Proteins are relatively easy molecules to manipulate, and protein crosslinking and chemical modification methods are commonly used to determine the roles of individual amino acid side chains in the physical, chemical, and biological properties of proteins. Furthermore, once their biological properties are understood, proteins can often be used in various applications such as preparing antibody-enzyme conjugates for immunoassays.

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Despite the complexity of protein structure, including composition with 20 different amino acids, only a small number of protein functional groups comprise selectable targets for practical bioconjugation methods. In fact, just four protein chemical targets account for the vast majority of crosslinking and chemical modification techniques:

• Primary amines (–NH2): This group exists at the N-terminus of each polypeptide chain and in the side chain of lysine (Lys, K) residues.
• Carboxyls (–COOH): This group exists at the C-terminus of each polypeptide chain and in the side chains of aspartic acid (Asp, D) and glutamic acid (Glu, E).
• Sulfhydryls (–SH): This group exists in the side chain of cysteine (Cys, C). Often, as part of a protein's secondary or tertiary structure, cysteines are joined together between their side chains via disulfide bonds (–S–S–).
• Carbonyls (–CHO): These aldehyde groups can be created by oxidizing carbohydrate groups in glycoproteins.

Protein functional group targets located on a representative protein. This illustration depicts the generalized structure of an immunoglobulin (IgG) protein.  Heavy and light chains are held together by a combination of non-covalent interactions and covalent interchain disulfide bonds, forming a bilaterally symmetric structure. The V regions of H and L chains comprise the antigen-binding sites of the immunoglobulin (Ig) molecules. Each Ig monomer contains two antigen-binding sites and is said to be bivalent. The hinge region is the area of the H chains between the first and second C region domains and is held together by disulfide bonds. This flexible hinge (found in IgG, IgA and IgD, but not IgM or IgE) region allows the distance between the two antigen-binding sites to vary. Also shown are several functional groups that are selectable targets for practical bioconjugation.

For each of these protein functional-group targets, there exist one to several types of reactive groups that are capable of targeting them, and these have been used as the basis for synthesizing crosslinking and modification reagents. 

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Crosslinking is the process of chemically joining two or more molecules by a covalent bond. Crosslinking reagents (or crosslinkers) are molecules that contain two or more reactive ends capable of chemically attaching to specific functional groups (primary amines, sulfhydryls, etc.) on proteins or other molecules.

Attachment between two groups on a single protein results in intramolecular crosslinks that stabilize the protein tertiary or quaternary structure. Attachment between groups on two different proteins results in intermolecular crosslinks that stabilize a protein-protein interaction. Alternatively, if the sample were a mixture of two purified proteins (e.g., an antibody and an enzyme), the intermolecular crosslink creates a specific conjugate for use in detection procedures. Finally, attachment between a protein and a chemical group on a solid material, such as a glass slide or beaded resin, results in immobilization of the protein to the surface; protein immobilization is the basis for many kinds of assay and affinity purification systems.

Thus, crosslinking is used for many purposes, including to:

• Stabilize protein tertiary and quaternary structure for analysis.
• Capture and identify unknown protein interactors or interaction domains.
• Conjugate an enzyme or tag to an antibody or other purified protein.
• Immobilize antibodies or other proteins for assays or affinity-purification.
• Attach peptides to larger "carrier" proteins to facilitate handling/storage.

Crosslinkers are selected on the basis of their chemical reactivities (i.e., specificity for particular functional groups) and other chemical properties that facilitate their use in different specific applications:

• Chemical specificity, including whether the reagent has the same or different reactive groups at either end (i.e., does it have a homobifunctional or heterobifunctional structure?)
• Spacer arm length, including whether the arm is cleavable (i.e., can the linkage be reversed or broken when desired?)
• Water-solubility and cell membrane permeability (i.e., can the reagent be expected to permeate into cells and/or crosslink hydrophobic proteins within membranes?)
• Spontaneously reactive or photo-reactive groups (i.e., will the reagent react as soon as it is added to a sample or can its reaction be activated at a specific time?)

An example of a crosslinker: BS3.

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Protein analysis and detection techniques often require more than direct conjugation with a bifunctional crosslinker or activated labeling reagent. For example, in many situations, specialized protein modifications are needed to add molecular mass, increase solubility for storage, or create a new functional group that can be targeted in a subsequent reaction step.

Simply stated, protein modification reagents are chemicals that block, add, change or extend the molecular reach of functional groups. (In a more general sense, protein modification also includes proteases and reducing agents for cleaving polypeptides, but those are distinct topics that are better discussed in other articles.) Three examples are sufficient to describe the types and purposes of modification reagents:

• Pegylation: Chemically attaching single- or branched-chain polyethylene glycol (PEG) groups to proteins is a form of labeling or modification that is primarily used to confer water-solubility and/or inert molecular mass to proteins. Forms of PEG that have been synthesized to contain reactive chemical groups comprise ready-to-use, activated reagents for pegylation. 

Examples of single chain, amine reactive PEGylation reagents.

• Block sulfhydryls: Protein sulfhydryls (side chain of cysteine) are important regulators of protein structure and function. Certain reagents are capable of reacting permanently or reversibly with sulfhydryl groups (e.g., NEM or MMTS, respectively). These reagents add a very small "cap" on the native sulfhydryl, enabling the activity of certain enzymes to be controlled for specific assay purposes.

Sulfhydryls can be blocked using NEM and MMTS.

• Convert amines to sulfhydryls: SATA and related reagents contain an amine-reactive group and a protected sulfhydryl group. By reacting the compound to a purified protein, the side chain of lysine residues can be modified to contain a sulfhydryl group for targeting with sulfhydryl-specific crosslinkers or immobilization chemistries. The method does not actually convert the amine into a sulfhydryl; rather it attaches a sulfhydryl-containing group to the primary amine. The effect is also to extend the length of the side chain by several angstroms.

Sulfhydryls can be converted to amines using SATA or Traut’s Reagent.

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1. A practical approach to crosslinking. Mattson, G., et al. Molecular Biology Reports (1993) 17:167-183
2. Redox-regulated dynamic interplay between Cox19 and the copper-binding protein Cox11 in the intermembrane space of mitochondria facilitates biogenesis of cytochrome c oxidase. Bodea M, Woellhafa MW, Bohnertb M, Laanb MVD, Sommerd F, Junge M, Zimmermanne R, Schrodad M, and Herrmanna JM. Molecular Biology of the Cell, Volume 26, July 1, 2015.