Polypeptides crosslinking

In another case in which a number of exemplary control experiments were done, Markwell and Fox (1980) crosslinked the outer membranes of enveloped viruses with methyl 3-(p-azidophenyl)dithio]propionimidate. Virus (4 mg protein/ml) was reacted with the imidate (0.1 to 0.5 mM) at 0°C for 30 min at pH 8.5. The reaction was quenched with 50 mM ammonium acetate, 50 mM NEM (30 min, 25 °C), and the vims recovered by centrifugation. After irradiation the crosslinked polypeptides were examined in a two-dimensional SDS-polyacrylamide gel. One complication was that the crosslinking pattern had to be compared with a native pattern of disulfide linkages, and a reagent with a different cleavable crosslink may have been a better choice. As mentioned above, the analysis was simplified by the use of surface labeling. 

Babcock, G.T., Widger, W.R., Cramer, W.A., Oertling, W.A. and Metz, J.G. 1985. Axial ligands of chloroplast cytochrome b-559 Identification and requirement for a heme-crosslinked polypeptide structure. Biochemistry, 24, 3638- 3645. 

A characteristic feature of a gel is its elastic behavior the gel deforms by an imposed force and it relaxes to its original state after releasing the force. Elastin, a crosslinked polypeptide network takes care of the elasticity of human and animal connective tissues, such as skin, ligaments, and arterial walls. For a more extensive discussion on the rheological properties of gels, the reader is referred to Chapter 17. 

Finally, a new water-soluble polyphosphazene was recently synthesized that has the structure shown in 36 (46). This polymer has two attributes as a biomedical macromolecule. First, the pendent carboxylic acid groups are potential sites for condensation reactions with amines, alcohols, phenols, or other carboxylic acid units to generate amide, ester, or anhydride links to polypeptides or bioactive small molecules. Second, polymer forms ionic crosslinks when brought into contact with di- or trivalent cations such as Ca or Ai3+. The crosslinking process converts the water-soluble polymer to a hydrogel, a process that can be reversed when the system... 

The first elastomeric protein is elastin, this structural protein is one of the main components of the extracellular matrix, which provides stmctural integrity to the tissues and organs of the body. This highly crosslinked and therefore insoluble protein is the essential element of elastic fibers, which induce elasticity to tissue of lung, skin, and arteries. In these fibers, elastin forms the internal core, which is interspersed with microfibrils [1,2]. Not only this biopolymer but also its precursor material, tropoelastin, have inspired materials scientists for many years. The most interesting characteristic of the precursor is its ability to self-assemble under physiological conditions, thereby demonstrating a lower critical solution temperature (LCST) behavior. This specific property has led to the development of a new class of synthetic polypeptides that mimic elastin in its composition and are therefore also known as elastin-like polypeptides (ELPs). 

This coacervation process forms the basis for the self-assembly, which takes place prior to the crosslinking. The assembly of tropoelastin is based on an ordering process, in which the polypeptides are converted from a state with little order to a more structured conformation [8]. The insoluble elastic fiber is formed via the enzymatic crosslinking of tropoelastin (described in Sect. 2.1). Various models have been proposed to explain the mechanism of elasticity of the elastin fibers. 

Fig. 19 Dityrosine and trityrosine crosslinks found in resilin. The R groups connect to the backbone of the polypeptide chain...

Fig. 20 Modular resilin-like polypeptide containing domains conferring elastomeric properties, heparin molecule interaction, cell adhesion, and matrix metalloproteinase (MMP) proteolysis. Lysine residues are encoded periodically to permit crosslinking...

In the synthesis of polypeptides with biological activity on a crosslinked polymer support as pioneered by Merrifield (1 2) a strict control of the amino acid sequence requires that each of the consecutive reactions should go virtually to completion. Thus, for the preparation of a polypeptide with 60 amino acid residues, even an average conversion of 99% would contaminate the product with an unacceptable amount of "defect chains". Yet, it has been observed (13) that with a large excess of an amino acid reagent —Tn the solution reacting with a polymer-bound polypeptide, the reaction kinetics deviate significantly from the expected exponential approach to quantitative conversion, indicating that the reactive sites on the polymer are not equally reactive. 

Reversible gelation is often encountered in bio-polymeric systems. Typical examples are solutions of polypeptide residues derived from animal collagen [82-84]. In these systems, ordered collagen-like triple helices form the physical crosslinks. 

Sonenberg, N., Morgan, M. A., Merrick, W. C., and Shatkin, A. J. (1978). A polypeptide in eukaryotic initiation factors that crosslinks specifically to the 5 -terminal cap in mRNA. Proc. Natl. Acad. Sci. USA 75, 4843-4847. 

Both of these observations are accounted for in the schematic representation in Fig. 3C and D of SBA crosslinked with Tn-PSM under saturation conditions. The schematic shows individual SBA molecules crosslinked to four different Tn-PSM molecules. Importantly, to form the crosslinked complex shown in Fig. 3D, lectin molecules bind to aGalNAc residues on all four sides of a Tn-PSM polypeptide chain. This allows staggering of individual SBA molecules along the Tn-PSM polypeptide chain, with concomitant decrease in steric interactions between lectin molecules. This is important, since calculations of the density of SBA molecules bound to Tn-PSM (knowing the diameter of SBA from X-ray crystal studies50 and the length of the Tn-PSM polypeptide chain) suggests that only 300 SBA tetramers can bind to the same side of a Tn-PSM polypeptide chain, which is less than the 540 bound monomers of SBA and 833 bound... 

Reported applications of BSOCOES include studying the polypeptide antigens on lymphocyte cell surfaces (Zarling et al., 1980), crosslinking labeled (3-endorphin to its opioid receptors (Howard et al., 1985), and isolation and characterization of calcitonin receptors in rat kidney (Bouizar et al., 1986). 

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