Proteins, fluorescence folded state

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 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... 

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. 

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. 

Pyrene fluorophores are also used as probes. Derivatives of pyrene show /.max/ Xem 340/376 nm, e 4.3 x 104 M 1 cm-1, and environmental sensitivity, this fluorophore can be used to report on RNA folding [102]. Pyrene also displays a long-lived excited state (x > 100 ns), which allows for an excited pyrene molecule to associate with a pyrene in the ground state. The resulting eximer exhibits a red-shift in fluorescence intensity (A,em 490 nm). This characteristic can be used to study important biomolecular processes, such as protein conformation [103]. 

Intermediates are usually detected in reaction pathways by the appearance of additional phases. Most small proteins have only one non-proline-related folding phase in the accessible time range. But the simple kinetics can be deceptive, and there can be hidden intermediates, besides the high energy states discussed in the last section. In Chapter 17, section A4, procedures were described to test for equilibrium two-state transitions these procedures are also effective for detecting intermediates that may accumulate close to the transition region. Simitar tests, such as the simultaneous change of tryptophan fluorescence or near... 

CD complements fluorescence as a technique for characterizing the folded conformation in solution and for providing, in a single spectrum, a highly specific fingerprint for the native state. It requires only modest amounts of protein and measurements are relatively quick to make. Like fluorescence, its use as a comparative method requires a control spectrum from a carefully characterized sample of the natural or recombinant protein, recorded under defined and optimal conditions, using a spectrometer in good condition. 

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