William R. Laws

Calcium Binding to Calmodulin Mutants Monitored by Domain-Specific Intrinsic Phenylalanine and Tyrosine Fluorescence

Cooperative calcium binding to the two homologous domains of calmodulin (CaM) induces conformational changes that regulate its association with and activation of numerous cellular target proteins. Calcium binding to the pair of high-affinity sites (III and IV in the C-domain) can be monitored by observing calcium-dependent changes in intrinsic tyrosine fluorescence intensity (lambda(ex)/lambda(em) of 277/320 nm). However, calcium binding to the low-affinity sites (I and II in the N-domain) is more difficult to measure with optical spectroscopy because that domain of CaM does not contain tryptophan or tyrosine. We recently demonstrated that calcium-dependent changes in intrinsic phenylalanine fluorescence (lambda(ex)/lambda(em) of 250/280 nm) of an N-domain fragment of CaM reflect occupancy of sites I and II (VanScyoc, W. S., and M. A. Shea, 2001, Protein Sci. 10:1758-1768). Using steady-state and time-resolved fluorescence methods, we now show that these excitation and emission wavelength pairs for phenylalanine and tyrosine fluorescence can be used to monitor equilibrium calcium titrations of the individual domains in full-length CaM. Calcium-dependent changes in phenylalanine fluorescence specifically indicate ion occupancy of sites I and II in the N-domain because phenylalanine residues in the C-domain are nonemissive. Tyrosine emission from the C-domain does not interfere with phenylalanine fluorescence signals from the N-domain. This is the first demonstration that intrinsic fluorescence may be used to monitor calcium binding to each domain of CaM. In this way, we also evaluated how mutations of two residues (Arg74 and Arg90) located between sites II and III can alter the calcium-binding properties of each of the domains. The mutation R74A caused an increase in the calcium affinity of sites I and II in the N-domain. The mutation R90A caused an increase in calcium affinity of sites III and IV in the C-domain whereas R90G caused an increase in calcium affinity of sites in both domains. This approach holds promise for exploring the linked energetics of calcium binding and target recognition.

Fluorescence quenching studies : analysis of nonlinear Stern-Volmer data

Publisher Summary This chapter illustrates the way two of the situations, static quenching and multiple species, cause the nonlinear deviations. The chapter evaluates the ability of the commonly used Marquardt nonlinear least-squares algorithm to recover known parameters from synthetic data. The results of the study point out the experimental and analysis criteria that must be met to obtain optimal information from fluorescence quenching studies. In particular, the chapter demonstrates the need to perform time-resolved fluorescence quenching studies. In the same way, the dynamic quenching parameters are either confirmed or evaluated, the distinction between single and multiple species is verified, and the existence of static quenching, or other process, is established. Multiple chromophores can arise in several ways even in a chemically pure system. Unless steady-state quenching experiments are performed to sufficiently high quencher concentrations, an incorrect analysis can occur because data that appear linear are actually nonlinear. It is difficult to distinguish between the two static quenching models for both single species and multiple species systems. It is necessary to obtain the dynamic quenching parameters as well as the fractional intensities to enable an analysis of multiple species quenching data.

LINKED-FUNCTION ANALYSIS OF FLUORESCENCE DECAY KINETICS: RESOLUTION OF SIDE-CHAIN ROTAMER POPULATIONS OF A SINGLE AROMATIC AMINO ACID IN SMALL POLYPEPTIDES

A linked‐function approach to fluorescence decay data analysis is presented that permits complex systems to be resolved from a single decay curve. The method involves linking fluorescence decay parameters based on a relationship established by independent physical measurements. As an example, by correlating the fluorescence data with 1H‐NMR results, the complex fluorescence decay kinetics of tyrosine analogs and single tyrosyl residues in simple polypeptides can be explained by ground‐state rotameric populations of the phenol ring about the Cα‐Cβ bond.

Constrained Analysis of Fluorescence Anisotropy Decay:Application to Experimental Protein Dynamics

Hydrodynamic properties as well as structural dynamics of proteins can be investigated by the well-established experimental method of fluorescence anisotropy decay. Successful use of this method depends on determination of the correct kinetic model, the extent of cross-correlation between parameters in the fitting function, and differences between the timescales of the depolarizing motions and the fluorophores fluorescence lifetime. We have tested the utility of an independently measured steady-state anisotropy value as a constraint during data analysis to reduce parameter cross correlation and to increase the timescales over which anisotropy decay parameters can be recovered accurately for two calcium-binding proteins. Mutant rat F102W parvalbumin was used as a model system because its single tryptophan residue exhibits monoexponential fluorescence intensity and anisotropy decay kinetics. Cod parvalbumin, a protein with a single tryptophan residue that exhibits multiexponential fluorescence decay kinetics, was also examined as a more complex model. Anisotropy decays were measured for both proteins as a function of solution viscosity to vary hydrodynamic parameters. The use of the steady-state anisotropy as a constraint significantly improved the precision and accuracy of recovered parameters for both proteins, particularly for viscosities at which the proteins rotational correlation time was much longer than the fluorescence lifetime. Thus, basic hydrodynamic properties of larger biomolecules can now be determined with more precision and accuracy by fluorescence anisotropy decay.

Kinetic models and data analysis methods for fluorescence anisotropy decay

Publisher Summary This chapter describes the kinetic models and data analysis methods for fluorescence anisotropy decay. Time-resolved fluorescence anisotropy is a powerful technique, for investigating macromolecular dynamics. In a time-resolved anisotropy measurement, a sample is excited, by linearly polarized light, and the anisotropy (or polarization) decay of the resulting emission is evaluated, by observing the fluorescence decay at polarizations parallel and perpendicular to the excitation. The emission can be depolarized, by a variety of dynamic and photophysical processes. Photophysical mechanisms of depolarization include the vibrational relaxation of the excited-state fluorophore as well as more unusual events, such as resonance energy transfer. Each of these depolarizing processes occurs with a characteristic correlation time and the influence of a given process on the anisotropy decay depends on the relative values of that correlation time and the lifetime of the excited state, as determined by a fluorescence intensity decay experiment. Time-resolved fluorescence anisotropy data may be analyzed, by several algorithms, including the method of moments, the Laplace transform, and nonlinear least squares (NLLS).

Fluorescence Determination of Tryptophan Side-Chain Accessibility and Dynamics in Triple-Helical Collagen-Like Peptides

We report tryptophan fluorescence measurements of emission intensity, iodide quenching, and anisotropy that describe the environment and dynamics at X and Y sites in stable collagen-like peptides of sequence (Gly-X-Y)(n). About 90% of tryptophans at both sites have similar solvent exposed fluorescence properties and a lifetime of 8.5-9 ns. Analysis of anisotropy decays using an associative model indicates that these long lifetime populations undergo rapid depolarizing motion with a 0.5 ns correlation time; however, the extent of fast motion at the Y site is considerably less than the essentially unrestricted motion at the X site. About 10% of tryptophans at both sites have a shorter ( approximately 3 ns) lifetime indicating proximity to a protein quenching group; these minor populations are immobile on the peptide surface, depolarizing only by overall trimer rotation. Iodide quenching indicates that tryptophans at the X site are more accessible to solvent. Side chains at X sites are more solvent accessible and considerably more mobile than residues at Y sites and can more readily fluctuate among alternate intermolecular interactions in collagen fibrils. This fluorescence analysis of collagen-like peptides lays a foundation for studies on the structure, dynamics, and function of collagen and of triple-helical junctions in gelatin gels.

Refining Hydrodynamic Shapes of Proteins: The Combination of Data From Analytical Ultracentrifugation and Time-Resolved Fluorescence Anisotropy Decay

Classical physical biochemical techniques are currently experiencing a renaissance as the result of several technological advances made over the past decade. One major reason for this renaissance is the need to understand the structures and functional characteristics of wild-type and mutant proteins that are now readily available through recombinant-DNA methods. Mutations of interest include alterations at specific functional sites, truncations, and switched domains. Another major reason for this renaissance is the availability of cheap, accessible computing power to facilitate data reduction. It is now possible and appropriate to combine the data from different physical techniques to obtain information which would not be obtainable from any single technique alone. This chapter examines a way in which the combination of analytical ultracentrifugation and time-resolved fluorescence anisotropy data permits knowledge of the hydrodynamic shape of a protein to be refined.