Proteins, fluorescence unfolding

Uversky, V.N., S. Winter, and G. Lober. 1996. Use of fluorescence decay times of 8-ANS-protein complexes to study the conformational transitions in proteins which unfold through the molten globule state. Biophys. Chem. 60(3) 79-88. 

Permyakov et al. (1985) studied the binding of Na(I) and K(I), as well as of Ca(ll) and Mg(II), to bovine a-lactalbumin by intrinsic protein fluorescence. Urea- and alkali-induced unfolding transitions involve stable partially unfolded intermediates for the ion-bound forms of this protein (see also Section IX,E). 

Acid Denaturation. LADH loses activity and zinc at pH 5 while still in the dimeric state 177,182). At lower pH dissociation occurs into subunits 182-184) and there are drastic changes in the protein fluorescence spectrum 185-187) and the fluorescence polarization spectrum 182). Different time dependences for the changes of the tyrosine and tryptophan difference fluorescence peaks are observed 187), which is consistent with a slower quenching of the buried Trp-314 (Section II,C,3,c) compared to the more exposed tyrosines. This interpretation implies that a partial unfolding of the tertiary structure occurs prior to the dissociation into subunits at acid pH. 

For eYFP, Fernandez and coworkers determined an unfolding force of approx. 60 pN. As the fluorescence is closely linked to the native state of the protein, mechanical unfolding switches its fluorescence off. ... 

Figure 11.15 Fluorescent protein as a mechanophore at the fibre-epoxy resin interface in self-reporting fibre-reinforced composites, (a) The formation of microdamages promotes interfacial debonding between resin and fibre, therefore causing the protein to unfold and to lose its fluorescence. (b) Confocal fluorescence microscopy image of a damaged glass fibre-eYFP/epo>y composite, (c) Z-stack projection of confocal fluorescence microscopy images of a damaged carbon fibre-eYFP/ epoxy composite. (F yellow fluorescence channel, O overlay of fluorescence and transmission images).

FIGURE 7 Fluorescence emission of a folded and unfolded protein. Fluorescence emission of tryptophan side chains can be to used monitor a protein s tertiary structure in solution. Tryptophan residues often have very different emission maxima depending on whether they are buried in a folded protein or exposed to solvent (unfolded protein). 

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. 

WHAT FLUORESCENCE CORRELATION SPECTROSCOPY CAN TELL US ABOUT UNFOLDED PROTEINS... 

Fluorescence correlation spectroscopy (FCS) measures rates of diffusion, chemical reaction, and other dynamic processes of fluorescent molecules. These rates are deduced from measurements of fluorescence fluctuations that arise as molecules with specific fluorescence properties enter or leave an open sample volume by diffusion, by undergoing a chemical reaction, or by other transport or reaction processes. Studies of unfolded proteins benefit from the fact that FCS can provide information about rates of protein conformational change both by a direct readout from conformation-dependent fluorescence changes and by changes in diffusion coefficient. 

Fig. 8. Dependence of (A) corrected diffusion coefficient (D), (B) steady-state fluorescence intensity, and (C) corrected number of particles in the observation volume (N) of Alexa488-coupled IFABP with urea concentration. The diffusion coefficient and number of particles data shown here are corrected for the effect of viscosity and refractive indices of the urea solutions as described in text. For steady-state fluorescence data the protein was excited at 488 nm using a PTI Alphascan fluorometer (Photon Technology International, South Brunswick, New Jersey). Emission spectra at different urea concentrations were recorded between 500 and 600 nm. A baseline control containing only buffer was subtracted from each spectrum. The area of the corrected spectrum was then plotted against denaturant concentrations to obtain the unfolding transition of the protein. Urea data monitored by steady-state fluorescence were fitted to a simple two-state model. Other experimental conditions are the same as in Figure 6.

Alexa488 bound to IFABP monitored by steady-state fluorescence was fitted to a two-state reversible unfolding model. This modified protein is slightly less stable (midpoint of 4.5 M compared to 4.7 M for wild-type IFABP). 

The material presented in this chapter demonstrates the utility of fluorescence correlation spectroscopy in the study of unfolded proteins. 

The fluorescence of purified histones has been studied by several different groups, 90 95) with the most detailed studies being on calf thymus histone HI. Histone HI, which binds to the outside of core particles, contains one tyrosine and no tryptophan. This protein exhibits a substantial increase in fluorescence intensity in going from a denatured to a folded state.<90) Collisional quenching studies indicate that the tyrosine of the folded HI is in a buried environ-ment.(91) Libertini and Small(94) have identified three emissions from this residue when in the unfolded state with peaks near 300, 340, and 400 nm. The 340-nm peak was ascribed to tyrosinate (vide infra), and several possibilities were considered for the 400-nm component, including room temperature phosphorescence, emission of a charge transfer complex, or dityrosine. Dityrosine has the appropriate spectral characteristics, but would require... 

Native fluorescence of a protein is due largely to the presence of the aromatic amino acids tryptophan and tyrosine. Tryptophan has an excitation maximum at 280 nm and emits at 340 to 350 nm. The amino acid composition of the target protein is one factor that determines if the direct measurement of a protein s native fluorescence is feasible. Another consideration is the protein s conformation, which directly affects its fluorescence spectrum. As the protein changes conformation, the emission maximum shifts to another wavelength. Thus, native fluorescence may be used to monitor protein unfolding or interactions. The conformation-dependent nature of native fluorescence results in measurements specific for the protein in a buffer system or pH. Consequently, protein denatur-ation may be used to generate more reproducible fluorescence measurements. 

The characteristics that discourage the use of RPLC for preparative isolation of bioactive proteins favor its use as an analytical tool for studying protein conformation. Chromatographic profiles can provide information on conformational stability of a protein and the kinetics of folding and unfolding processes. Information about solvent exposure of certain amino acid residues (e.g., tryptophan) as a function of the folding state can be obtained by on-line spectral analysis using diode array UV-vis detection or fluorescence detection. 

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