Tryptophan location

The long lifetime of phosphorescence allows it to be used for processes which are slow—on the millisecond to microsecond time scale. Among these processes are the turnover time of enzymes and diffusion of large aggregates or smaller proteins in a restricted environment, such as, for example, proteins in membranes. Phosphorescence anisotropy is one method to study these processes, giving information on rotational diffusion. Quenching by external molecules is another potentially powerful method in this case it can lead to information on tryptophan location and the structural dynamics of the protein. 

Fluorescence emission lifetime of one tryptophan residue located in five different positions within an 18-residue amphiphatic peptide (Table 7.5) was also analyzed with the rotamers model. These peptides are unstructured in aqueous solution and become stmctured when associated to a lipid. A blue shift in the emission maximum of the Trp residue is observed, indicating that the fluorophore is in contact with a hydrophobic environment when the lipid is bound to the peptides. Only peptide 18D-12 shows an emission maximum located at 354 revealing that in this peptide tryptophan is in contact with aqueous environment. These data are consistent witli the fact that association of lipid with peptide yield an a-helix where the tryptophan residues located on the hydrophobic surface of the a-helix are in the hydrophobic environment provided by the lipid surface, whereas tryptophans located on the hydrophilic face of the helix are directed toward the aqueous phase. Also, the helix axis of the peptides is parallel to the lipid surface (Fig. 7.9). 

Hydrophobic interactions among the amino acid side chains also determine tertiary structure. Most globular proteins have their hydrophobic side chains, for example, those of phenylalanine, valine, or tryptophan, located on the inside of the protein structure. Conversely, the hydrophilic amino acids, such as glutamic acid, serine, or asparagine, are generally found on the outside surface of the protein, where they are available for interaction with water. Alternatively, when these groups are found on the inside of soluble... 

Water-soluble globular proteins usually have an interior composed almost entirely of non polar, hydrophobic amino acids such as phenylalanine, tryptophan, valine and leucine witl polar and charged amino acids such as lysine and arginine located on the surface of thi molecule. This packing of hydrophobic residues is a consequence of the hydrophobic effeci which is the most important factor that contributes to protein stability. The molecula basis for the hydrophobic effect continues to be the subject of some debate but is general considered to be entropic in origin. Moreover, it is the entropy change of the solvent that i... 

Fig. 15. The two Fe-S clusters are some 12-13 A apart and within possible electron transfer range. A tyrosine residue, Y493, is situated roughly halfway between the two clusters, but whether it plays a role in any electron transfer is unclear. Two adjacent tryptophan residues are also located close to cluster 2 again, their possible roles in any enzymatic reaction remain to be defined.

Both enzymes belong to the family of a,p-hydrolases." The active site of MeHNL is located inside the protein and connected to the outside through a small channel, which is covered by the bulky amino acid tryptophane 128." It was possible to obtain the crystal structure of the complex with the natural substrate acetone cyanohydrin with the mutant SerSOAla of MeHNL. This complex allowed the determination of the mode of substrate binding in the active site." A summary of 3D structures of known HNLs was published recently." " ... 

Fluorescent probes are divided in two categories, i.e., intrinsic and extrinsic probes. Tryptophan is the most widely used intrinsic probe. The absorption spectrum, centered at 280 nm, displays two overlapping absorbance transitions. In contrast, the fluorescence emission spectrum is broad and is characterized by a large Stokes shift, which varies with the polarity of the environment. The fluorescence emission peak is at about 350 nm in water but the peak shifts to about 315 nm in nonpolar media, such as within the hydrophobic core of folded proteins. Vitamin A, located in milk fat globules, may be used as an intrinsic probe to follow, for example, the changes of triglyceride physical state as a function of temperature [20]. Extrinsic probes are used to characterize molecular events when intrinsic fluorophores are absent or are so numerous that the interpretation of the data becomes ambiguous. Extrinsic probes may also be used to obtain additional or complementary information from a specific macromolecular domain or from an oil water interface. 

However, diffusion of the reactive QM out of the enzyme active site is a major concern. For instance, a 2-acyloxy-5-nitrobenzylchloride does not modify any nucleophilic residue located within the enzyme active site but becomes attached to a tryptophan residue proximal to the active site of chymotrypsin or papain.23,24 The lack of inactivation could also be due to other factors the unmasked QM being poorly electrophilic, active site residues not being nucleophilic enough, or the covalent adduct being unstable. Cyclized acyloxybenzyl molecules of type a could well overcome the diffusion problem. They will retain both the electrophilic hydroxybenzyl species b, and then the tethered QM, in the active site throughout the lifetime of the acyl-enzyme (Scheme 11.1). This reasoning led us to synthesize functionalized... 

A typical fluorescence EEM results for leachate samples from R-landfill demonstrate five distinctive and intense fluorescence peaks in Figure 2, such as at Ex/Em=230-250/400-440 nm (labeled as A ), which was relative to UV humic fraction identified in location to the diagnostic fluorescence centre observed previously at Ex/Em=220-230/340-370 nm (labeled as D ), a poorly understood fluorescent centre widely attributed to a component of the UV fulvic-like (Coble 1996) at Ex/Em=320-350/400-440 nm (labeled as C ), which can be attributed to aromatic and aliphatic groups in the DOM fraction and commonly labeled as fulvic-like (Coble 1996) at Ex/Em=350-400/420-460 nm (labeled as E ), which is attributed to humic-like and a final fluorescence centre at Ex/Em= 275-280/350-360 nm (labeled as B ), which is attributed to the protein tryptophan, and widely observed in polluted river waters (Baker 2001 2002) and clean estuaries (Mayer et al. 1999). 

Fluorescent probes can be divided into three classes (i) intrinsic probes-, (ii) extrinsic covalently bound probes and (iii) extrinsic associating probes. Intrinsic probes are ideal but there are only a few examples (e.g. tryptophan in proteins). The advantage of covalently bound probes over the extrinsic associating probes is that the location of the former is known. There are various examples of probes covalently... 

Fluorescence quenching has proven to be a powerful means to determine location of tryptophans. Small organic molecules, such as acetone, acrylamide, and amino acids, have been used to quench fluorescence of tryptophans which are exposed to the solvent.(50 51) These molecules apparently quench by close interaction and so provide a tool to determine the surface accessibility7 of tryptophan in a protein. 

If a collisional quencher of the fluorophore is also incorporated into the membrane, the lifetime will be shortened. The time resolution of the fluorescence anisotropy decay is then increased,(63) providing the collisional quenching itself does not alter the anisotropy decay. If the latter condition does not hold, this will be indicated by an inability to simultaneously fit the data measured at several different quencher concentrations to a single anisotropy decay process. This method has so far been applied to the case of tryptophans in proteins(63) but could potentially be extended to lipid-bound fluorophores in membranes. If the quencher distribution in the membrane differed from that of the fluorophore, it would also be possible to extract information on selected populations of fluorophores possibly locating in different membrane environments. 

Fluorescence is not widely used as a general detection technique for polypeptides because only tyrosine and tryptophan residues possess native fluorescence. However, fluorescence can be used to detect the presence of these residues in peptides and to obtain information on their location in proteins. Fluorescence detectors are occasionally used in combination with postcolumn reaction systems to increase detection sensitivity for polypeptides. Fluorescamine, o-phthalaldehyde, and napthalenedialdehyde all react with primary amine groups to produce highly fluorescent derivatives.33,34 These reagents can be delivered by a secondary HPLC pump and mixed with the column effluent using a low-volume tee. The derivatization reaction is carried out in a packed bed or open-tube reactor. 

---