Crosslinking, protein

Rat liver nuclei (chromato-graphically separated DNA fractions) DNA-protein crosslinks No data Keller and Heck 1988 =... 

Solomon MJ, Varshavsky A. Formaldehyde-mediated DNA-protein crosslinking a probe for in vivo chromatin structures. Proc. Natl. Acad. Sci. USA 1985 82 6470-6474. 

Figure 1.23 Protein crosslinking reactions done using homobifunctional reagents can result in large polymeric complexes of multiple sizes and indefinite structure.

Schiff base interactions between aldehydes and amines typically are not stable enough to form irreversible linkages. These bonds may be reduced with sodium cyanoborohydride or a number of other suitable reductants (Chapter 2, Section 5) to form permanent secondary amine bonds. However, proteins crosslinked by glutaraldehyde without reduction nevertheless show stabilities unexplainable by simple Schiff base formation. The stability of such unreduced glutaraldehyde conjugates has been postulated to be due to the vinyl addition mechanism, which doesn t depend on the creation of Schiff bases. 

The following list of homobifunctional imidoesters represent compounds that are commonly used for protein crosslinking and are currently available from commercial sources. 

The protocol for using GMBS or sulfo-GMBS in protein-protein crosslinking applications is similar to that of SMCC or sulfo-SMCC (see Section 1.3, this chapter). 

With bifunctional NHS ester reagents, one of three modification products can occur with proteins (1) a dead-end linkage wherein one end of the crosslinker has attached to an amine group within a protein and the other end has hydrolyzed and not formed an attachment, (2) an intra-protein crosslink wherein the PIR reagent has been coupled at both ends to amines within the same protein, or (3) an inter-protein crosslink wherein both ends of the PIR reagent have been coupled with amines on two different protein molecules (Figure 28.7). 

Figure 28.7 Reaction of a PIR compound with a protein sample can result in several products, all of which can be identified by mass spec analysis. A true inter-protein crosslink can occur that links two interacting proteins together, which is the desired product. However, the crosslinking process also may result in intra-protein crosslinks or dead ends wherein only one end of the PIR reagent has coupled to a protein.

The use of PIR compounds to study protein interactions is a significant advance over the use of standard homobifunctional crosslinkers. The unique design of the PIR reagent facilitates deconvolution of putative protein interaction complexes through a simplified mass spec analysis. The software can ignore all irrelevant peak data and just focus analysis on the two labeled peptide peaks, which accompany the reporter signal of appropriate mass. This greatly simplifies the bioinformatics of data analysis and provides definitive conformation of protein-protein crosslinks. 

Figure 28.21 The reactions of R u (11) pby 3 + are catalyzed by light at 452 nm that begins by forming an excited state intermediate. In the presence of persulfate, a sulfate radical is formed concomitant with the oxidative product Ru(III)bpy33+. This form of the chelate is able to catalyze the formation of a radical on a tyrosine phenolic ring that can react along with the sulfate radical either with a nucleophile, such as a cysteine thiol, or with another tyrosine side chain to form a covalent linkage. The result of this reaction cascade is to cause protein crosslinks to form when a sample containing these components is irradiated with light.

Fancy, D.A., and Kodadek, T. (1997) Site-directed oxidative protein crosslinking. Tetrahedron 53, 11953-11960. 

Fancy, D.A., Melcher, K., Johnston, S.A., and Kodadek, T. (1996) New chemistry for the study of multiprotein complexes The six-histidine tag as a receptor for a protein crosslinking reagent. Cbem. Biol. 3, 551-559. 

Fujii, N., Jacobsen, R.B., Wood, N.L., Schoenigeg J.S., and Guy, R.K. (2004) A novel protein crosslinking reagent for the determination of moderate resolution protein structures by mass spectrometry (MS3-D). Bioorg. Med. Chem. Lett. 14, 427-429. 

Salgo and Pryor [111] studied the effect of Trolox C (a water-soluble analog of vitamin E) on peroxynitrite-mediated DNA damage in rat thymocytes. They proposed that peroxynitrite mediated the formation of TBAR products, which caused the DNA-protein crosslinks. The latter were inhibited by the posttreatment of cells with Trolox. However, Trolox produced no effects on hydrogen peroxide- or bleomycin-induced DNA damage in human lymphocytes [108],... 

Graham DG, Anthony DC, Boekelheide K, et al. 1982. Studies of the molecular pathogenesis of hexane neuropathy. II. Evidence that pyrrole derivatization of lysyl residues leads to protein crosslinking. Toxicol Appl Pharmacol 64 415-422. 

F g- 13.13 Principle of protein crosslinking by enzymatic oxidation of lactose coupled with the Maillard reaction [35-37]. 

Fig. 14. The secondary structure of the E. coli ribosomal 23 S RNA, showing protein-binding sites, RNA-protein crosslink sites, and intra-RNA cross-link sites. The relationships among a—f are as in Figs. 2 and 3. Reproduced with permission from Brimacombe et al. (1983). 

TABLE 1. Characterization of the Coiled-Coil Protein Crosslinkers. 

Anthony DC, Boekelheide K, Anderson CW, et al. 1983. The effect of 3,4-dimethyl substitution on the neurotoxicity of 2,5-hexanedione. II. Dimethyl substitution acccelerates pyrrole formation and protein crosslinking. [Abstract] Toxicol AppI Pharmacol 71 372-382. 

Maltose binding protein crosslinked amylose Yes Bedouelle and Duplay, 1988... 

Branner-Jorgensen, S. (1978) On the mechanism of protein crosslinking with glutaraldehyde. 4 Enzyme Engineering Conference, M4. 

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