Maurice R. Eftink

Fluorescence quenching studies with proteins

Abstract A review is presented on the use of the technique of solute fluorescence quenching to study the structure and dynamics of proteins. A number of factors are discussed that must be considered in analyzing such data. Among these factors are the efficiency of the quenching process, the relative importance of static quenching, the heterogeneity of the emission, and the tendency of the quencher to interact with the protein.

The use of fluorescence methods to monitor unfolding transitions in proteins

This article discusses several strategies for the use steady-state and time-resolved fluorescence methods to monitor unfolding transitions in proteins. The assumptions and limitations of several methods are discussed. Simulations are presented to show that certain fluorescence observables directly track the population of states in an unfolding transition, whereas other observables skew the transition toward the dominant fluorescing species. Several examples are given, involving the unfolding of Staphylococcal aureus nuclease A, in which thermodynamic information is obtained for the temperature and denaturant induced transitions in this protein.

Fluorescence Quenching: Theory and Applications

Solute fluorescence quenching reactions were first applied to biochemical problems in the late 1960s and early 1970s, 7) and since that time they have been a very valuable research tool for studies with proteins, membranes, and other macromolecular assemblies. Quenching reactions are easy to perform, require only a small sample, usually are nondestructive, and can be applied to almost any system that has an intrinsic or extrinsic fluorescence probe. The most important characteristic, however, is the value of the information that these reactions can provide. Solute quenching reactions, using quenchers such as molecular oxygen, acrylamide, or iodide ion, provide information about the location of fluorescent groups in a macromolecular structure. A fluorophore that is located on the surface of a larger structure will be relatively accessible to a solute quencher that is dissolved in the aqueous phase. A fluorophore that is removed from the surface of a structure will be quenched to a lesser degree by the quencher. Thus, the quenching reaction can be used to probe topographical features of a macromolecular assembly and to sense any structural changes that may be caused by varying conditions or the addition of reagents. In addition, quenching reactions can, in some situations, provide information about conformational fluctuations. In Sections 2.3 and 2.4 I will discuss several examples of the use of solute quenchers in studies with proteins, membranes, and nucleic acids. Solute fluorescence quenching reactions can also be used to selectively alter the fluorescence properties of a sample in order to resolve contributions or aid in the measurement of data. To elaborate on this point, consider the different characteristics of fluorescence: the quantum yield, excitation and

[22] Use of multiple spectroscopic methods to monitor equilibrium unfolding of proteins

Publisher Summary This chapter comments on the methods used to monitor protein unfolding, with emphasis on spectroscopic methods. The chapter discusses the advantages of using multiple probing methods in order to gain more information about an unfolding process. To test various models, only equilibrium unfolding studies with proteins are considered, although many of the principles and methods can be extended to kinetics studies and to studies with other types of structured biomacromolecules, such as oligonucleotide duplexes. The unfolding of proteins can be induced in a number of ways. These include increasing (or decreasing) temperature, adding chaotropic chemical denaturants (for example, urea or guanidine), exposing to extreme pH, or increasing hydrostatic pressure. Differential scanning calorimetry (DSC) is the method of choice for studying the thermodynamics of the thermal unfolding of proteins. The chapter reviews the basic theory of pseudo-first-order conformational transitions in proteins. Examples are provided of how two or more different spectroscopic methods can be combined and/or how multiple types of spectroscopic data can be obtained with the same instrument.

Quenching-resolved emission anisotropy studies with single and multitryptophan-containing proteins.

Measurements of the anisotropy of protein fluorescence as a function of an added collisional quencher, such as acrylamide, are used to construct Perrin plots. For single tryptophan containing proteins, such plots yield an apparent rotational correlation time for the depolarization process, which, in most cases, is approximately the value expected for Brownian rotation of the entire protein. Apparent limiting fluorescence anisotropy values, which range from 0.20 to 0.32 for the proteins studied, are also obtained from the Perrin plots. The lower values for the limiting anisotropy found for some proteins are interpreted as indicating the existence of relatively rapid, limited (within a cone of angle 0 degrees--30 degrees) motion of the tryptophan side chains that is independent of the overall rotation of the protein. Examples of the use of this fluorescence technique to study protein conformational changes are presented, including the monomer in equilibrium dimer equilibrium of beta-lactoglobulin, the monomer in equilibrium tetramer equilibrium of melittin, the N in equilibrium F transition of human serum albumin, and the induced change in the conformation of cod parvalbumin caused by the removal of Ca+2. Because multitryptophan-containing proteins have certain tryptophans that are accessible to solute quencher and others that are inaccessible, this method can be used to determine the steady state anisotropy of each class of tryptophan residues.

Fluorescence Quenching Reactions

The quenching of the fluorescence of biomacromolecules by solute quenchers has become a widely used and powerful technique (Lehrer, 1976; Lehrer and Leavis, 1989; Lakowicz, 1983; Eftink and Ghiron, 1981; Eftink, 1991). Such quenching reactions have been used primarily to obtain topographical information about proteins, nucleic acids, and membrane systems. The accessibility of intrinsic or extrinsic fluorescence probes (e.g., the amino acid, tryptophan), which are attached to a biomacromolecule, to small quenchers (e.g., iodide, acrylamide, oxygen) is directly determined by such reactions. Conformational changes in the biomacromolecule can then be monitored in terms of changes in the accessibility to the quencher of the fluorophore. In addition to such topographical information, in some cases information about the conformational dynamics of globular proteins has been obtained with solute quenching reactions.

Frequency domain measurements of the fluorescence lifetime of ribonuclease T1

Using multifrequency phase/modulation fluorometry, we have studied the fluorescence decay of the single tryptophan residue of ribonuclease T1 (RNase T1). At neutral pH (7.4) we find that the decay is a double exponential (tau 1 = 3.74 ns, tau 2 = 1.06 ns, f1 = 0.945), in agreement with results from pulsed fluorometry. At pH 5.5 the decay is well described by a single decay time (tau = 3.8 ns). Alternatively, we have fitted the frequency domain data by a distribution of lifetimes. Temperature dependence studies were performed. If analyzed via a double exponential model, the activation energy for the inverse of the short lifetime component (at pH 7.4) is found to be 3.6 kcal/mol, as compared with a value of 1.0 kcal/mol for the activation energy of the inverse of the long lifetime component. If analyzed via the distribution model, the width of the distribution is found to increase at higher temperature. We have also repeated, using lifetime measurements, the temperature dependence of the acrylamide quenching of the fluorescence of RNase T1 at pH 5.5. We find an activation energy of 8 kcal/mol for acrylamide quenching, in agreement with our earlier report.

ANALYSIS OF MULTIDIMENSIONAL SPECTROSCOPIC DATA TO MONITOR UNFOLDING OF PROTEINS

Publisher Summary This chapter illustrates the use of global analysis to analyze data from a modified Aviv 62DS circular dichroism (CD) spectrophotometer, which is capable of making multidimensional spectroscopic measurements on a single sample during a thermal melt. This instrument was modified by adding to it a photomultiplier (PM) tube at right angles to the beam path, allowing fluorescence and light scattering measurements to be made along with the CD and absorbance measurements that the instrument is normally capable of making. Profiles from the different measurement types were analyzed globally to take advantage of the greater accuracy that is gained from an analysis of a larger number of data points in the transition region, and to uncouple the thermodynamic parameters from the baseline parameters. When comparing data from multiple experiments, a decision must be made as to how much emphasis, or significance, should be given to each data set. Measurements made at different times frequently will have different noise levels, and therefore they should make proportionally different contributions to the fitting process.

Spectral displacement techniques for studying the binding of spectroscopically transparent ligands to cyclodextrins

A spectroscopic displacement method is used to determine association constants of beta-cyclodextrin with compounds that are spectroscopically transparent. These compounds are adamantanecarboxylate and structurally related compounds. Association constants obtained are compared to values obtained by other methods. It is shown that for all types of displacement techniques a distinction must be made between free and total concentrations of ligand in cases of strong binding.

Intrinsic Fluorescence of Proteins

These are some thoughts to introduce this volume on protein fluorescence. The following articles will describe several specific protein systems and fluorescence techniques. There will be examples that focus on understanding the fluorescence properties of a protein, articles that exploit fluorescence to gain information about protein dynamics, and articles that apply the fluorescence of tryptophan or other fluorophores to gain kinetic or thermodynamic information. The applications of fluorescence are vast.