Intrinsic amino acid tryptophan probes

II. Optical Probe of Intrinsic Amino Acid Tryptophan... 

II. OPTICAL PROBE OF INTRINSIC AMINO ACID TRYPTOPHAN... 

The aim of this Chapter is to review a method by which fluorescence properties of organic dyes can, in general, be predicted and understood at a microscopic (nm scale) by interfacing quantum methods with classical molecular dynamics (MD) methods. Some review of our extensive applications [1] of this method to the widely exploited intrinsic fluorescence probe in proteins, the amino acid tryptophan (Trp) will be followed by a discussion of electrochromic membrane voltagesensing dyes. 

Photoluminescence can be used to detect an analyte in three ways (1) the analyte itself is intrinsically fluorescent (direct sensing) (2) the analyte can be tagged with a fluorophore label or (3) the analyte interacts with a luminescent probe. Direct sensing and fluorophore-tags are widely used in biomedical applications to probe cell environments. Many proteins are intrinsic fluorophores due to the presence of the aromatic amino acids tryptophan, phenylalanine and tyrosine. Analytes such as pH, CO2, NH3, O2 and various cations and anions can be measured indirectly using luminescence probes. 

Fluorescence applications are ranging from in vivo and in vitro diagnostics to experimental biology. In the first case mainly intrinsic fluorescence is used, which arises from proteins (in particular the amino acid tryptophan [4,5]), extracellular fibres [6,7] or specific coenzymes, e.g. nicotinamide adenine dinucleotide (NADH). So far, fluorescence intensity of NADH has been correlated with metabolic functions [8,9] and was proposed to be an appropriate parameter for detection of ischemic [10,11] or neoplastic [12-14] tissues. The application of fluorescent probes for diagnostic purposes in humans has so far been limited to fluorescein or indocyanine green used... 

Melittin, which is an amphipathic peptide from honeybee venom, consists of 26 amino acid residues and adopts different conformations from a random coil, to an a-helix, and to a self-assembled tetramer under certain aqueous environments see Fig. 9. We have carried out our systematic studies of the hydration dynamics in these three conformations using a single intrinsic tryptophan ( W19) as a molecular probe. The folded a-helix melittin was formed with lipid interactions to mimic physiological membrane-bound conditions. The self-assembled tetramer was prepared under high-salt concentration (NaCl = 2 M). The tryptophan emission of three structures under three different aqueous environments is 348.5 nm, 341 nm, and 333.5 nm, which represents different exposures of aqueous solution from complete in random-coil, to locating at the lipid surface of a nanochannel (50 A in diameter) in a-helix and to partially buried in tetramer. Figure 10 shows... 

The selective resolution enhancement in derivative spectroscopy is pushed even further in the fourth derivative mode. As in the case of second derivative spectroscopy, the amplitude and the position of the derivative spectral bands of the aromatic amino acids are related to the polarity of the medium. We have undertaken a systematic investigation of these spectral features of the N-acetyl O-ethyl esters of tyrosine and tryptophan in various solvents of different polarity (from cyclohexane to water). Astonishingly, a simple relationship between the spectral parameters of the fourth derivatives and the dielectric constant was found [11]. As shown in Figure 5, for tyrosine it is the position of >.max, and for tryptophan it is the derivative amplitude which depends linearly on the dielectric constant er. Since in addition the fourth derivative spectra of these model compounds do not depend significantly on pressure (at least up to 500 MPa), these spectral features may be used as an intrinsic probe to sense the dielectric constant in the vicinity of tyrosine and tryptophan. 

One extreme view of chemical introduction of an extrinsic fluorescent probe is found in the case ofthe alanine derivative of the fluorophore 6-dimethylamino-2-acylnaphthalene (DAN) (Figure 4.23). This derivative fluorophore, given the trivial name Aladan, is incorporated into a polypeptide by solid-phase synthetic chemistry (although a molecular biology technique known as nonsense suppression is now available for the introduction of unnatural amino-acid residues into recombinant proteins). The fluorescent emission maximum (Tnax) of Aladan shifts dramatically on different solvent exposures, from 409 nm in heptane to 542 nm in water, yet at the same time remains only mildly changed by variations in pH or salt concentration. This compares to a maximum environment-mediated shift of around 40 nm for intrinsic tryptophan fluorescence. In addition, there is little spectral overlap between extrinsic Aladan fluorescence and intrinsic fluorescence from tryptophan or tyrosine. 

The majority of naturally occurring compounds are nonluminescent, including nucleic acids (DNA/RNA), mono- and polysaccharides, hpids, and most of the small biomolecules. Proteins contain in their structure three amino acids—phenylalanine, tyrosine, and tryptophan— that fluoresce in the UV range. Due to its long-wave emission and relatively low abundance, tryptophan is commonly used as an intrinsic luminescent probe to study proteins. Tryptophan also exhibits phosphorescence at room temperature. 

A fluorophore is a component of a protein or small molecule that exhibits fluorescence. It can also be called a fluorescent label, chromophore, or fluorescent probe. Each fluorophore has characteristic excitation and emission wavelengths. In other words, a fluorophore will fluoresce when the light of a particular energy, corresponding to the excitation wavelength, is used. Examples of fluorophores include GFP and related emissive proteins, and small molecules like fluorescein and coumarin. Many biomolecules have intrinsic fluorescence. For instance, tryptophan, an amino acid, fluoresces in the ultraviolet region of the electromagnetic spectrum. 

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