Fluorescence tryptophan

Measuring Protein Sta.bihty, Protein stabihty is usually measured quantitatively as the difference in free energy between the folded and unfolded states of the protein. These states are most commonly measured using spectroscopic techniques, such as circular dichroic spectroscopy, fluorescence (generally tryptophan fluorescence) spectroscopy, nmr spectroscopy, and absorbance spectroscopy (10). For most monomeric proteins, the two-state model of protein folding can be invoked. This model states that under equihbrium conditions, the vast majority of the protein molecules in a solution exist in either the folded (native) or unfolded (denatured) state. Any kinetic intermediates that might exist on the pathway between folded and unfolded states do not accumulate to any significant extent under equihbrium conditions (39). In other words, under any set of solution conditions, at equihbrium the entire population of protein molecules can be accounted for by the mole fraction of denatured protein, and the mole fraction of native protein,, ie. 

The intramolecular distances measured at room temperature with the AEDANS FITC pair were similar in the Ca2Ei and E2V states [297]. Ca and lanthanides are expected to stabilize the Ej conformation of the Ca -ATPase, since they induce a similar crystal form of Ca -ATPase [119,157] and have similar effects on the tryptophan fluorescence [151] and on the trypsin sensitivity of Ca -ATPase [119,120]. It is also likely that the vanadate-stabilized E2V state is similar to the p2 P state stabilized by Pi [418]. Therefore the absence of significant difference in the resonance energy transfer distances between the two states implies that the structural differences between the two conformations at sites recorded by currently available probes, fall within the considerable error of resonance energy transfer measurements. Even if these distances would vary by as much as 5 A the difference between the two conformations could not be established reliably. 

Since tryptophan is also a fluorescence molecule, we monitored the disassembly through spectroscopic measurements. It was found that within the AB6 dendron (32), the tryptophan fluorescence was significantly quenched due to the confined proximity, which is forced by the dendritic skeleton. Upon the disassembly, we observed a gradual increase of two bands on 400 and 760 nm that correspond to free tryptophan molecules (Fig. 5.28). 

Fluorescence detectors, discussed in Chapter 1, are extremely sensitive picogram quantities of sample can sometimes be detected. However, most polymers (with the exception of certain proteins) are not fluorescent and thus these detectors are rarely used in GPC. Proteins, particularly those containing tryptophan, fluoresce intensely and are readily detected. Because both the IR and the fluorimetric detector are selective for certain functional groups, rather than being sensitive to analyte mass, there are many pitfalls in quantitation. These and other detectors have been reviewed.177178... 

Merola, R, Rigler, R., Holmgren, A., and Brochon, J-C., Picosecond tryptophan fluorescence of thioredoxin evidence for discrete species in slow exchange, Biochemistry, 28, 3383, 1989. 

Callis PR, Burgess BK (1997) Tryptophan fluorescence shifts in proteins from hybrid simulations an electrostatic approach. J Phys Chem 101 9429-9432... 

Vivian JT, Callis PR (2001) Mechanisms of tryptophan fluorescence shifts in proteins. Biophys J 80 2093-2109... 

Chen J, Callis PR, King J (2009) Mechanism of the very efficient quenching of tryptophan fluorescence in human gamma D- and gamma S-crystallins the gamma-crystallin fold may have evolved to protect tryptophan residues from ultraviolet photodamage. Biochemistry 48(17) 3708-3716... 

Callis PR, Petrenko A, Muino PL, Tusell JR (2007) Ab initio prediction of tryptophan fluorescence quenching by protein electric field enabled electron transfer. J Phys Chem B 111(35) 10335-10339... 

Liu T, Callis PR, Hesp BH, de Groot M, Buma WJ, Broos J (2005) Ionization potentials of fluoroindoles and the origin of nonexponential tryptophan fluorescence decay in proteins. J Am Chem Soc 127(11) 4104-4113... 

Kurz LC, Fite B, Jean J, Park J, Erpelding T, Callis P (2005) Photophysics of tryptophan fluorescence link with the catalytic strategy of the citrate synthase from Thermoplasma acidophilum. Biochemistry 44(5) 1394-1413... 

Qiu W, Li T, Zhang L, Yang Y, Kao Y-T, Wang L, Zhong D (2008) Ultrafast quenching of tryptophan fluorescence in proteins interresidue and intrahelical electron transfer. Chem Phys 350 154-164... 

S. S. Lehrer, Solute perturbation of protein fluorescence. The quenching of other tryptophan fluorescence of model compounds and of lysosome by iodide ion, Biochemistry 10, 3254-3263 (1971). 

Tyrosine fluorescence emission in proteins and polypeptides usually has a maximum between 303 and 305 nm, the same as that for tyrosine in solution. Compared to the Stokes shift for tryptophan fluorescence, that for tyrosine appears to be relatively insensitive to the local environment, although neighboring residues do have a strong effect on the emission intensity. While it is possible for a tyrosine residue in a protein to have a higher quantum yield than that of model compounds in water, for example, if the phenol side chain is shielded from solvent and the local environment contains no proton acceptors, many intra- and intermolecular interactions result in a reduction of the quantum yield. As discussed below, this is evident from metal- and ionbinding data, from pH titration data, and from comparisons of the spectral characteristics of tyrosine in native and denatured proteins. 

A protein induced after coliphage N4 infection has been studied. Although it has one or two tryptophans, its intrinsic fluorescence is dominated by the ten tyrosines/1111 Tryptophan fluorescence is seen after denaturing the protein. Upon binding to single-stranded DNA, the tyrosine fluorescence is quenched. This signal has been used to demonstrate that the binding affinity is very dependent on salt concentration and is also very sensitive to the nucleotide sequence. 

The indole chromophore of tryptophan is the most important tool in studies of intrinsic protein fluorescence. The position of the maximum in the tryptophan fluorescence spectra recorded for proteins varies widely, from 308 nm for azurin to 350-353 nm for peptides lacking an ordered structure and for denatured proteins. (1) This is because of an important property of the fluorescence spectra of tryptophan residues, namely, their high sensitivity to interactions with the environment. Among extrinsic fluorescence probes, aminonaphthalene sulfonates are the most similar to tryptophan in this respect, which accounts for their wide application in protein research.(5)... 

Proteins having one chromophore per molecule are the simplest and most convenient in studies of fluorescence decay kinetics as well as in other spectroscopic studies of proteins. These were historically the first proteins for which the tryptophan fluorescence decay was analyzed. It was natural to expect that, for these proteins at least, the decay curves would be singleexponential. However, a more complex time dependence of the emission was observed. To describe the experimental data for almost all of the proteins studied, it was necessary to use a set of two or more exponents.(2) The decay is single-exponential only in the case of apoazurin.(41) Several authors(41,42) explained the biexponentiality of the decay by the existence of two protein conformers in equilibrium. Such an explanation is difficult to accept without additional analysis, since there are many other mechanisms leading to nonexponential decay and in view of the fact that deconvolution into exponential components is no more than a formal procedure for treatment of nonexponential curves. 

A. Grinvald and J. Z. Steinberg, Fast relaxation processes in a protein revealed by the decay kinetics of tryptophan fluorescence, Biochemistry 13, 5170-5177 (1974). 

J. R. Lakowicz and A. Baiter, Resolution of initially excited and relaxed states of tryptophan fluorescence by differential-wavelength deconvolution of time-resolved fluorescence decays, Biophys. Chem. 15, 353-360 (1982). 

Room temperature phosphorescence can be observed from dried proteins. Sheep wool keratin(47) has a phosphorescence lifetime of 1.4 s. Six lyophilized proteins were shown to exhibit phosphorescence at room temperature.(48) The spectra were diffuse, and the lifetime was non-single-exponential, which the authors interpreted as due to inhomogeneous distribution of tryptophans. As the protein was hydrated, the phosphorescence lifetime decreased. This decrease occurred over the same range of hydration where the tryptophan fluorescence becomes depolarized. Hence, these results are consistent with the idea that rigidity of the site contributes to the lifetimes. 

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