Chemistry of Crosslinking

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Protein functional groups

Despite the complexity of protein structure, including composition and sequence of 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 (called the alpha-amine) and in the side chain of lysine (Lys, K) residues (called the epsilon-amine). Because of its positive charge at physiologic conditions, primary amines are usually outward facing (i.e., on the outer surface) of proteins; thus, they are usually accessible for conjugation without denaturing protein structure.
• 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). Like primary amines, carboxyls are usually on the surface of protein structure.
• 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–). These must be reduced to sulfhydryls to make them available for crosslinking by most types of reactive groups.
• Carbonyls (–CHO): Ketone or aldehyde groups can be created in glycoproteins by oxidizing the polysaccharide post-translational modifications (glycosylation) with sodium meta-periodate.

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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Crosslinker reactive groups

A number of chemical reactive groups have been characterized and used to target the main kinds of protein functional groups. Many different crosslinking reagents can be synthesized when different combinations of two or more of these reactive groups are incorporated into one molecule. When combined with different sizes and types of chemical "backbones" (called spacer arms because they define the distance between respective reactive ends), the number of possible crosslinking compounds is enormous.

Popular crosslinker reactive groups for protein conjugation. Reactivity class titles link to specific sections of this article. Italicized chemical group names are not specifically discussed in this article.

Selecting crosslinkers

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

• Chemical specificity refers to the reactive target(s) of the crosslinker's reactive ends. A general consideration is whether the reagent has the same or different reactive groups at either end (termed homobifunctional and heterobifunctional, respectively; see below).
• Spacer arm length refers to the molecular span of a crosslinker (i.e., the distance between conjugated molecules). A related consideration is whether the arm is cleavable (i.e., whether the linkage can be reversed or broken when desired).
• Water-solubility and cell membrane permeability of a crosslinker affect whether it can permeate into cells and/or crosslink hydrophobic proteins within membranes. These properties are determined by the composition of the spacer arm and/or reactive group.
• Spontaneously reactive or photoreactive groups in a crosslinker affect whether it reacts as soon as it is added to a sample or can be activated at a specific time by exposure to UV light.

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Homobifunctional and heterobifunctional crosslinkers

Crosslinkers can be classified as homobifunctional or heterobifunctional.

Homobifunctional crosslinkers have identical reactive groups at either end of a spacer arm, and generally they must be used in one-step reaction procedures to randomly "fix" or polymerize molecules containing like functional groups. For example, adding an amine-to-amine crosslinker to a cell lysate will result in random conjugation of protein subunits, interacting proteins and any other polypeptides whose lysine side chains happen to be near each other in the solution. This is ideal for capturing a "snapshot" of all protein interactions but cannot provide the precision needed for other types of crosslinking applications. For example, when preparing an antibody-enzyme conjugate, the goal is to link one to several enzyme molecules to each molecule of antibody without causing any antibody-to-antibody linkages to form. This is not possible with homobifunctional crosslinkers.

Homobifunctional crosslinker example. DSS is a popular, simple crosslinker that has identical amine-reactive NHS-ester groups at either end of a short spacer arm. The spacer arm length (11.4 angstroms) is the final maximum molecular distance between conjugated molecules (i.e., nitrogens of the target amines).

Heterobifunctional crosslinkers possess different reactive groups at either end. These reagents not only allow for single-step conjugation of molecules that have the respective target functional groups, but they also allow for sequential (two-step) conjugations that minimize undesirable polymerization or self-conjugation. In sequential procedures, heterobifunctional reagents are reacted with one protein using the most labile group of the crosslinker first. After removing excess nonreacted crosslinker, the modified first protein is added to a solution containing the second protein where reaction through the second reactive group of the crosslinker occurs. The most widely-used heterobifunctional crosslinkers are those having an amine-reactive succinimidyl ester (i.e., NHS ester) at one end and a sulfhydryl-reactive group (e.g., maleimide) on the other end. Because the NHS-ester group is less stable in aqueous solution, it is usually reacted to one protein first. If the second protein does not have available native sulfhydryl groups, they can be added in a separate prior step using sulfhydryl-addition reagents.

Heterobifunctional crosslinker example.Sulfo-SMCC is a popular crosslinker that has an amine-reactive sulfo-NHS-ester group (left) at one end and a sulfhydryl reactive maleimide group (right) at the opposite end of a cyclohexane spacer arm. This allows for sequential, two-step conjugation procedures.

General reaction conditions

In many applications, it is necessary to maintain the native structure of the protein complex, so crosslinking is most often performed using near-physiologic conditions. Optimal crosslinker-to-protein molar ratios for reactions must be determined empirically, although product instructions for individual reagents generally contain guidelines and recommendations for common applications.

Depending on the application, the degree of conjugation is an important factor. For example, when preparing immunogen conjugates, a high degree of conjugation is desired to increase the immunogenicity of the antigen. However, when conjugating to an antibody or an enzyme, a low-to-moderate degree of conjugation may be optimal so that biological activity of the protein is retained.

The number of functional groups on the protein’s surface is also important to consider. If there are numerous target groups, a lower crosslinker-to-protein ratio can be used. For a limited number of potential targets, a higher crosslinker- to-protein ratio may be required. Furthermore, the number of components should be kept low or to a minimum because conjugates consisting of more than two components are difficult to analyze and provide less information on spatial arrangements of protein subunits.

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Carbodiimides (EDC and DCC)

EDC and other carbodiimides are zero-length crosslinkers; they cause direct conjugation of carboxylates (–COOH) to primary amines (–NH2) without becoming part of the final crosslink (amide bond) between target molecules.

EDC crosslinking reactions must be performed in conditions devoid of extraneous carboxyls and amines. Acidic (pH 4.5 to 5.5) MES buffer (4-morpholino-ethane-sulfonic acid) is most effective, but phosphate buffers at pH ≤ 7.2 are also compatible with the reaction chemistry. N-hydroxysuccinimide (NHS) or its water-soluble analog (Sulfo-NHS) is often included in EDC coupling protocols to improve efficiency or to create a more stable, amine-reactive intermediate (see next section).

Because peptides and proteins contain multiple carboxyls and amines, direct EDC-mediated crosslinking usually causes random polymerization of polypeptides. Nevertheless, this reaction chemistry is used widely in immobilization procedures (e.g., attaching proteins to a carboxylated surface) and in immunogen preparation (e.g., attaching a small peptide to a large carrier protein).

NHS-ester crosslinking reactions are usually performed in phosphate buffer at pH 7–9.0 for 0.5 to 4 hours at room temperature or 4°C. Primary amine buffers such as Tris (TBS) are not compatible because they compete for reaction; however, in some procedures, it is useful to add Tris or glycine buffer at the end of a conjugation procedure to quench (stop) the reaction.

Sulfo-NHS esters are identical to NHS esters except that they contain a sulfonate (–SO₃) group on the N-hydroxysuccinimide ring. This charged group has no effect on the reaction chemistry, but it does tend to increase the Spelling suggestion water-solubility of crosslinkers containing them. In addition, the charged group prevents Sulfo-NHS crosslinkers from permeating cell membranes, enabling them to be used for cell surface crosslinking methods.

Because the resulting amidine bond is protonated, the crosslink has a positive charge at physiological pH, much like the primary amine which it replaced. For this reason, imidoester crosslinkers have been used to study protein structure and molecular associations in membranes and to immobilize proteins onto solid-phase supports while preserving the isoelectric point (pI) of the native protein. Although imidoesters are still used in certain procedures, the amidine bonds formed are reversible at high pH. Therefore, the more stable and efficient NHS-ester crosslinkers have steadily replaced them in most applications.

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Short homobifunctional maleimide crosslinkers enable disulfide bridges in protein structures to be converted to permanent, irreducible linkages between cysteines. More commonly, the maleimide chemistry is used in combination with amine-reactive NHS-ester chemistry in the form of heterobifunctional crosslinkers that enable controlled, two-step conjugation of purified peptides and/or proteins.

During the reaction, a disulfide exchange occurs between the –SH group of the target molecule and the 2-pyridyldithiol group of the crosslinker. Pyridine-2-thione (MW = 111 kDa; λ_max = 343 nm) is released as a byproduct that can be monitored spectrophotometrically and removed from protein conjugates by dialysis or desalting.

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Hydrazide chemistry is useful for labeling, immobilizing or conjugating glycoproteins through glycosylation sites, which are often (as with most polyclonal antibodies) located at domains away from the key binding sites whose function one wishes to preserve.

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When an aryl azide compound is exposed to UV light, it forms a nitrene group that can initiate addition reactions with double bonds or insertion into C-H and N-H sites or can undergo ring expansion to react with a nucleophile (e.g., primary amine). Reactions can be performed in a variety of amine-free buffer conditions to conjugate proteins or even molecules devoid of the usual functional group "handles".

Photoreactive reagents are most often used as heterobifunctional crosslinkers to capture binding partner interactions. A purified bait protein is labeled with the crosslinker using the amine- or sulfhydryl-reactive end. Then this labeled protein is added to a lysate sample and allowed to bind its interactor. Finally, photo-activation with UV light initiates conjugation via the phenyl azide group.

Photo-activation of diazirine creates reactive carbene intermediates. Such intermediates can form covalent bonds through addition reactions with any amino acid side chain or peptide backbone at distances corresponding to the spacer arm lengths of the particular reagent. Diazirine-analogs of amino acids can be incorporated into protein structures by translation, enabling specific recombinant proteins to be activated as the crosslinker.

The following representative example demonstrates photo-reactive crosslinking of an intracellular protein complex with SDA reagents. Protein interactions involving the early endosome antigen 1 (EEA1) protein were examined in the immortalized human cervical cancer cell line, HeLa. EEA1 forms a homodimer through a coiled coil domain, binds to phospholipid vesicles and is involved in endosomal trafficking.

Capture and detection of intracellular protein homodimerization with NHS-Diazirine Crosslinkers. HeLa cells were incubated with various NHS-diazirine derivatives and treated with UV light. Cells were then lysed and analyzed by SDS-PAGE and western blotting using an anti-EEA1 antibody. After UV exposure, reduced mobility forms of EEA1 were observed only with samples treated with the SDA and SDAD and not a mock-treated control sample. Because EEA1 is an intracellular protein complex, it was not crosslinked by Sulfo-SDA, which does not permeate cell membranes. In addition, slower mobility, SDAD-crosslinked EEA1 could be cleaved within the spacer using the reducing agent dithiothreitol (DTT). GAPDH detection was used to confirm equal protein loading.

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Chemoselective ligation refers to the use of mutually specific pairs of conjugation reagents. Unlike typical crosslinking methods used in biological research, this reaction chemistry depends upon a pair of unique reactive groups that are specific to one another and foreign to biological systems. Because these reactions (azide-alkyne or azide-phosphine) do not occur in cells, these functional groups react only with each other in biological samples, thus resulting in minimal background and few artifacts, hence the term “chemoselective”. This specialized form of crosslinking can be applied for both in vivo metabolic labeling and for bioconjugation using bioorthogonal coupling partners.

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1. Hermanson, Greg T. (2013) Bioconjugate Techniques, Third edition.