Bonds in proteins

One of the most common PTMs of proteins is the formation of disulfide bonds by oxidation of the cysteinyl thiols. This functionality is critical for biological activity of proteins becanse it plays an important role in the folding-unfolding processes and in stabilizing the correct three-dimensional structure of proteins [1]. The disulfide bonds are also essential for the expression of the activity of small 

---

The thioredoxin domain (see Figure 2.7) has a central (3 sheet surrounded by a helices. The active part of the molecule is a Pa(3 unit comprising p strands 2 and 3 joined by a helix 2. The redox-active disulfide bridge is at the amino end of this a helix and is formed by a Cys-X-X-Cys motif where X is any residue in DsbA, in thioredoxin, and in other members of this family of redox-active proteins. The a-helical domain of DsbA is positioned so that this disulfide bridge is at the center of a relatively extensive hydrophobic protein surface. Since disulfide bonds in proteins are usually buried in a hydrophobic environment, this hydrophobic surface in DsbA could provide an interaction area for exposed hydrophobic patches on partially folded protein substrates. 

Disulfide bonds in proteins are generally stable and nonreactive, acting like bolts in the structure. However, oxidized DsbA is less stable than the reduced form and its disulfide bond is very reactive. DsbA is thus a strong... 

In this chapter we shall illustrate some fundamental aspects of enzyme catalysis using as an example the serine proteinases, a group of enzymes that hydrolyze peptide bonds in proteins. We also examine how the transition state is stabilized in this particular case. 

FIGURE 5.18 Methods for cleavage of disulfide bonds in proteins, (a) Oxidative cleavage by reaction with performic acid, (b) Reductive cleavage with snlfliydryl compounds. Disulfide bridges can be broken by reduction of the S—S link with snlfliydryl agents such as 2-mercaptoethanol or dithiothreitol. Because reaction between the newly reduced —SH groups to re-establish disulfide bonds is a likelihood, S—S reduction must be followed by —SH modification (1) alkylation with iodoac-etate (ICH,COOH) or (2) modification with 3-bromopropylamine (Br— (CH,)3—NH,). 

Carboxypeptidase A catalyses the hydrolysis of the terminal peptide bond in proteins during the process of digestion ... 

Peptidases are enzymes that catalyse the hydrolysis of peptide bonds - the bonds between amino acids that are found in peptides and proteins. The terms protease , proteinase and proteolytic enzyme are synonymous, but strictly speaking can only be applied to peptidases that hydrolase bonds in proteins. Because there are many peptidases that act only on peptides, the term peptidase is recommended. Peptidases are included in subclass 3.4 of enzyme nomenclature [1,5]. 

This is a variation of the proton-detected shift-correlation experiment via long-range couplings proposed by Bax and Summers (Bax and Summers, 1986), with the difference that the first C pulse is substituted by a frequency selective pulse (Fig. 7.14) (Bermel et al., 1989 Kessler et al., 1989b,1990). This significantly increases resolution in the F dimension. For example, this can be used to remove the overlap between the cross-peaks of the carbonyl resonances of peptide bonds in proteins that all occur within a... 

Recently it has been found that couplings between and C across H-bonds, e.g. in systems containing N—H..-0= C units (in proteins), can be directly detected and provide evidence for H-bonds in proteins and nucleic acids [89]. Although this technology is now standard for larger biomolecules it is rarely used for smaller molecules [90]. 

Proteases are enzymes that break peptide bonds in proteins. As such they lend themselves to a variety of homogeneous assay techniques. Most employ labeling both ends of the substrate with a different tag, and looking for the appearance (disappearance) of the signal generated in the intact substrate (product). As an example, for a fluorescence quench assay, the N-terminal of a peptide is labeled with DNP and the C-terminal with MCA. As such, the peptide is fluorescently silent since the fluorescence from DNP is quenched by absorption by the MCA. Another very popular donor/acceptor pair is EDANS 5-[(2-aminoethyl)amino] naphthalene-1-sulfonic acid and DABCYL 4-(4-dimethylaminophenylazo)benzoic acid) (a sulfonyl derivative (DABSYL) [27], Upon peptide cleavage, the two products diffuse, and due to a lack of proximity, the fluorescence increases. 

Mezey, P.G. (1998) Chemical bonding in proteins and other macromolecules. In Pauling s Legacy Modern Modelling of Chemical Bonding, Maksic, Z. and Orville-Thomas, J. (Eds.), Elsevier Science Publ., Amsterdam, The Netherlands. 

Peters, D., and J. Peters. 1982. Quantum Theory of the Structure and Bonding in Proteins Part 13. The p branched hydrocarbon side chains valine and isoleucine. J. Mol. Struct. (Theochem) 88,157-170. 

Many extracellular proteins like immunoglobulins, protein hormones, serum albumin, pepsin, trypsin, ribonuclease, and others contain one or more indigenous disulfide bonds. For functional and structural studies of proteins, it is often necessary to cleave these disulfide bridges. Disulfide bonds in proteins are commonly reduced with small, soluble mercaptans, such as DTT, TCEP, 2-mercaptoethanol, thioglycolic acid, cysteine, etc. High concentrations of mercaptans (molar excess of 20- to 1,000-fold) are usually required to drive the reduction to completion. 

Cleland (1964) showed that DTT and DTE are superior reagents in reducing disulfide bonds in proteins (see previous discussion, this section). DTT and DTE have low oxidation-reduction potential and are capable of reducing protein disulfides at concentrations far below that required with 2-mercaptoethanol. However, even these reagents have to be used in approximately 20-fold molar excess in order to get close to 100 percent reduction of a protein. 

Konigsberg, W. (1972) Reduction of disulfide bonds in proteins with dithiothreitol. In Methods in Enzymology, (C.H.W. Hirs, and S.N. Timaseff, eds.), Vol. 25 p. 185. Academic Press, New York. 

Traut, R.R., Casiano, C., and Zecherle, N. (1989) Cross-linking of protein subunits and ligands by the introduction of disulfide bonds. In Protein Function—A Practical Approach (T.E. Creighton, ed.), pp. 101-133. IRL Press at Oxford University, Oxford. 

---