Protein unfolding fluorescence spectroscop

Because an unfolded or partly folded protein consists of a conformational ensemble containing a wide range of different structures, it is impossible to obtain meaningful results from crystal structures even if the molecule will form crystals, the resulting structure will not be representative of the ensemble in solution. It is necessary to obtain information on unfolded proteins in solution. Spectroscopic methods are therefore employed to give information on conformational preferences within the ensemble. These include circular dichro-ism, fluorescence, Raman and NMR spectroscopy. NMR gives a great deal of site-specific information, and is preferred when NMR spectra are possible. 

The apparent molecular mass of D-Ser toxin was dramatically increased by the addition of guanidine hydrochloride to the elution buffer, although that of the L-Ser toxin was not altered by the denaturing reagent. In the presence of 5.2 M guanidine hydrochloride, the D-form toxin was eluted at the same position as the L-form toxin and the apparent molecular masses of the two toxins were estimated as 6 kDa based on calibration with the standard proteins. CD and fluorescence spectroscopic analyses revealed that the two toxins were unfolded and lost their secondary and tertiary structure in 5.2 M guanidine hydrochloride at pH 7.4, as described below. It, therefore, appears that the D-Ser toxin forms a compact folded structure, whereas the L-Ser toxin has a relatively unfolded or extended structure. 

It has been observed for several proteins that the intermediate structures are formed as the protein unfolds from N state to D state [26]. As the protein unfolds, protein loses tertiary structure and, frequently, secondary structure. In some instances, the secondary structure remains intact while the tertiary structure is lost [12], which is clear from spectral studies that measure loss of secondary and tertiary structural changes. One spectroscopic technique that is sensitive to tertiary structure (e.g., fluorescence) would detect changes, whereas other techniques that are sensitive to secondary structures (e.g., far UV CD) do not show any spectral changes. This molecular property is defined as molten globule or structured intermediate [12]. These intermediates expose hydrophobic domains, and thus promote aggregation or surface adsorption. 

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. 

Folding Equilibrium Studies. E. coli RTEM p-lactamase is a monomeric protein. Its amino acid sequence has been determined (79). It has one disulfide bond between the residues Cys S and Cys. The presence of four tyrosines and four tryptophans allows the use of spectroscopic method for the conformational characterization of the enzyme. In this study, the effect of denaturants on the unfolding of p-lactamase was determined from activity measurements, difference spectroscopy and fluorescence intensity measurements. 

Utilizing time resolved internal reflection spectroscopic technique (Fig. 6), we were able to isolate the tryptophan intrinsic fluorescence and observe its = 20 ns fluorescence lifetime for albumin in bulk and in the surface microenvironment of a hydrophilic quartz material. The pH dependence of bulk albumin fluorescence lifetime served to "calibrate albumin in terms of native ( 7 ns time constant) protein at pH 7.2 and unfolded (c 4 ns) protein at the isoelectric pH 3.8. The fluorescence lifetime data (Tables I/II) supported the hypothesis that the adsorbed albumin exists in two forms on a hydrophilic quartz surface, each with a possibly different structure (] ). A loosely held "layer," consisting of microaggregates, native and partially unfolded albumin molecules with... 

An important problem in the study of protein folding is the control of the initial state (i.e., obtaining as starting material a completely unfolded protein). Therefore a very careful characterization of the denatured protein is the first step of the experimental work the second step is finding good conditions for reversibility. Different methods can be used to characterize the unfolded proteins hydrodynamic methods such as viscosity and sedimentation coefficient determination spectroscopic methods (absorption and fluorescence), fluorescence depolarization, CD, ORD and optical rotation, and NMR. Chemical methods provide a more direct tool for measuring the accessibility... 

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