Peptide bond, quenching

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. 

Cowgill pointed out that there are essentially two distinct quenching processes of tyrosine fluorescence resulting from association with the peptide bond.(3) Tyrosines affected by these mechanisms are classified in Table 1.3 as... 

Using a rapid quench-fiow kinetic assay for post-complex fragment formation, Nair and Cooperman showed that the ET encounter complex of serpin and enzyme forms both E I and the post-complex fragment with the same rate constant, indicating that both species arise from ET conversion to E I. These results support the conclusions (a) that the peptide bond remains intact within the ET complex, and (b) that E I is likely to be either the acyl-enzyme or the tetrahedral intermediate formed after water attack on acyl-enzyme. 

Other measures of protein flexibility have been found to correlate with thermal stability. One is resistance to proteolysis (Daniel et al., 1982 Fontana, 1988). Another is the quenching of buried tryptophan fluorescence by acrylamide, used in a study by Varley and Pain (1991). Both these processes are mediated by the same combination of local and global unfolding events that determine rates of hydrogen exchange. Their rates will depend on the ability of another molecule, acrylamide or a proteolytic enzyme, to penetrate into normally buried regions of the protein in order to either quench fluorescence or cleave peptide bonds. 

With site-directed mutation and femtosecond-resolved fluorescence methods, we have used tryptophan as an excellent local molecular reporter for studies of a series of ultrafast protein dynamics, which include intraprotein electron transfer [64-68] and energy transfer [61, 69], as well as protein hydration dynamics [70-74]. As an optical probe, all these ultrafast measurements require no potential quenching of excited-state tryptophan by neighboring protein residues or peptide bonds on the picosecond time scale. However, it is known that tryptophan fluorescence is readily quenched by various amino acid residues [75] and peptide bonds [76-78]. Intraprotein electron transfer from excited indole moiety to nearby electrophilic residue(s) was proposed to be the quenching... 

The 100 e.v. yield for the radiolytic degradation of the peptide bond, as measured in terms of G( NH3) after mild hydrolysis, has been determined for a variety of aliphatic, aromatic and sulfur-containing amino acids in the N-acetyl form. These data are summarized in Table I. In the case of the aliphatic series, we note that the length of the side-chain has relatively little effect on the yield of main-chain degradation. The effect of the aromatic groups of acetylphenylalanine and of acetyltyrosine is to quench in part the yields of those reactions that lead to formation of amide ammonia. The sulfur moiety of methionine on the other hand appears to be relatively ineffective in quenching such reactions. 

In this work, the solutions of human serum albumin (HSA) (>96%, Sigma) and of bovine serum albumin (BSA) (>98%, MP Biomedicals) in a phosphate buffer (0.01 M, pH 7.4) have been used. The proteins concentrations were lO- (absorption spectra measurement) and 10- M (fluorescence measurement at the nanosecond laser fluorimeter). All of the experiments were performed at a temperature of 25 1 °C. The structure and biological functions of HSA and BSA can be found in (Peters, 1996). Tryptophan, tyrosine, and phenylalanine (with relative contents of 1 18 31 in HSA and 2 20 27 in BSA) are the absorption groups in these proteins (as in many other natural proteins). The tyrosine fluorescence in HSA and BSA (as in many other natural proteins) is quenched due to the effect of adjacent peptide bonds, polar groups (such as CO, NH2), and other factors, and phenylalanine has a low fluorescence quantum yield (0.03) (Permyakov, 1992). Therefore, the fluorescence signal in these proteins is determined mainly by tryptophan groups. In that case the fluorescence, registered in nonlinear and kinetic laser fluorimetry measurements, correspond to tryptophan residues (this fact will be used in Section 6.1). 

A number of studies on the fluorescence decay of tyrosine, tyrosine derivatives, and small tyrosyl peptides have been carried out. 36-38 Whereas the tyrosine zwitterion and tyrosine derivatives with an ionized a-carboxy group exhibited monoexponential fluorescence decay (x = 3.26-3.76 ns), double- or triple-exponential decay was observed in most other cases. As in the case of the tryptophan model compounds, the complex decay kinetics were again interpreted in terms of rotamer populations resulting from rotation around the C —Cp bond. There is evidence to indicate that the shorter fluorescence lifetimes may arise from rotamers in which the phenol ring is in close contact with a hydrated carbonyl group 36 37 and that a charge-transfer mechanism may be implicated in this quenching process. 39 ... 

FIGURE 2.5 FRET quench readout principle. In the intact peptidic substrate (amino acids symbolized by X, Y and Z) labeled with a fluorophore (dye) and a quencher (Q) at the opposite sites of the scissile bond, the fluorescence emission is quenched through an energy transfer (ET) from the fluorophore to the quencher. After cleavage of the substrate between amino acids X and Y by a protease, the energy transfer is disrupted and an increase in fluorescence emission is observed (light gray arrow). An increase of fluorescence intensity over time dependent on the enzymatic velocity is recorded. 

Recombinant TOP and neurolysin activities were compared using seven series of peptides based on Abz-GFSPFRQ-EDDnp, an internally quenched fluorogenic substrate [377]. Most of the peptides were hydrolyzed at the bond corresponding to P(4)-F(5) in the reference substrate. Others were cleaved at this bond or at F(2)-S(3). The best substrates for TOP had at P(l), Phe, Ala or Arg and for neurolysin, Asn or Arg. 

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