Chemical Cross-Linking in Biology

Abstract

+9131The study of diverse biological phenomena by chemical cross-linking tech nology has greatly intensified during the past 5 years. A number of excel lent, timely, and chemically oriented reviews of the cross-linking literature have appeared (4, 68, 87). Therefore, we have attempted to present a critical evaluation of the principles and strategies that govern the applica tion of cross-linking technology to the solutions of biological problems.The use of chemical cross-linking reagents as molecular rulers in the determination of quaternary structure in oligomeric protein complexes in solution and in membranes has enjoyed wide application. Another as pect of cross-linking technology is the exploitation of heterobifunctional reagents to identify ligand-binding components in complex biological sys tems. It is often difficult to determine how proteins function in multicompo nent, complex systems. The classical biochemical approach of isolation and characterization of protein components does not always reveal tran sient interactions or migratory behavior of proteins, which occur in the natural environment of the cell. The covalent linkage of a ligand to a specifi c protein provides a strategy fo r visualizing this behavior. PHOTO REACTIVE CROSS-LINKING REAGENTSArylazide reagents that can be employed fo r photochemical attachment of ligands to recognition sites on macromolecules are described in Table

The Highly Conserved Layer-3 Component of the HIV-1 gp120 Inner Domain Is Critical for CD4-Required Conformational Transitions

“…In this procedure, glutaraldehyde (GA) cross-linking is used to fix the conformation of Env, and then the relative occupancy of gp120 conformations is assessed by quantifying the binding of ligands with different requirements for specific gp120 conformations. GA cross-links lysine residues and thereby fixes the conformation of the protein component of the envelope glycoproteins (55). Cells were left untreated or cross-linked (5 mM GA) and incubated with a polyclonal mixture of sera (PS) from HIV-1-infected individuals, CD4-Ig, or monoclonal Abs that recognize different conformations of the HIV-1 Env trimers.…”

Oligomer-Specific Conformations of the Human Immunodeficiency Virus (HIV-1) gp41 Envelope Glycoprotein Ectodomain Recognized by Human Monoclonal Antibodies

“…As GA targets primary amines on the protein component, [44][45][46] this may exert subtle effects on the envelope glycoprotein epitopes. To avoid any potential artifact generated by GA cross-linking, we performed MAb-depletion experiments to examine whether the oligomer-specific epitopes exist in native HIV-1 envelope glycoproteins.…”

A novel photoactivatable cross‐linker for the functionally‐directed region‐specific fluorescent labeling of proteins

“…The characteristics of nitrenes also provide a basis for the cross-linking specificity of SAED since the short-lived nitrenes (z 1 ms lifetime; Kiehm and Ji, 1977) react rapidly and minimize nonspecific coupling from random bimoleciilar collisions. The cross-linking selectivity can be further increased when high protein concentrations are required by using excess scavenger molecules during photolysis (Das and Fox, 1979). These small nucleophiles (such as Tris) eventually capture those nitrenes unable to reach a target without affecting the favored crosslinking process.…”

“…The short cross-linking span of SAED ( 5 1.8 nm) strictly limited the labeling to the vicinity of the contact region of trypsin with STI. Thus, this novel cross-linker permits the region-specific targeting of a fluorophore near a functionally important binding site.Chemical cross-linking reagents have been used for the determination of nearest-neighbor analysis, molecular interactions and orientations, as well as three-dimensional protein structures (Wang and Richards, 1974;Peters and Richards, 1977;Das and Fox, 1979). For these applications, the efficiency and the specificity of the coupling reaction has been increased by the development of photoaffinity heterobifunctional cross-linkers (Huang and Richards, 1977; Ji, 1979; Jaffe et al, 1980).…”