Fluorescence spectroscopy protein structure

Enzyme structure may be studied by fluorescence spectroscopy [238-244]. Excitation in the 280-310 nm absorption bands of proteins, usually results in fluorescence from tryptophan (Trp) residues in the 310-390 nm region. The fluorescence from the Trp residues is a convenient marker for protein denaturation and large decreases or red-shifts in fluorescence are observed when proteins are denatured. These changes are most often due to the exposure of the Trp residues that are buried in the protein and may be due to the changes in the proximities of specific residues that may act as fluorescence quenchers. Fluorescence emission characterization of the immobilized... 

The major reasons for using intrinsic fluorescence and phosphorescence to study conformation are that these spectroscopies are extremely sensitive, they provide many specific parameters to correlate with physical structure, and they cover a wide time range, from picoseconds to seconds, which allows the study of a variety of different processes. The time scale of tyrosine fluorescence extends from picoseconds to a few nanoseconds, which is a good time window to obtain information about rotational diffusion, intermolecular association reactions, and conformational relaxation in the presence and absence of cofactors and substrates. Moreover, the time dependence of the fluorescence intensity and anisotropy decay can be used to test predictions from molecular dynamics.(167) In using tyrosine to study the dynamics of protein structure, it is particularly important that we begin to understand the basis for the anisotropy decay of tyrosine in terms of the potential motions of the phenol ring.(221) For example, the frequency of flips about the C -C bond of tyrosine appears to cover a time range from milliseconds to nanoseconds.(222)... 

Such ambiguity and also the low structural resolution of the method require that the spectroscopic properties of protein fluorophores and their reactions in electronic excited states be thoroughly studied and characterized in simple model systems. Furthermore, the reliability of the results should increase with the inclusion of this additional information into the analysis and with the comparison of the complementary data. Recently, there has been a tendency not only to study certain fluorescence parameters and to establish their correlation with protein dynamics but also to analyze them jointly, to treat the spectroscopic data multiparametrically, and to construct self-consistent models of the dynamic process which take into account these data as a whole. Fluorescence spectroscopy gives a researcher ample opportunities to combine different parameters determined experimentally and to study their interrelationships (Figure 2.1). This opportunity should be exploited to the fullest. 

As shown above, the intrinsic fluorescence spectra of proteins as well as coenzyme groups and probes shift within very wide ranges depending on their environment. Since the main contribution to spectral shifts is from relaxational properties of the environment, the analysis of relaxation is the necessary first step in establishing correlations of protein structure with fluorescence spectra. Furthermore, the study of relaxation dynamics is a very important approach to the analysis of the fluctuation rates of the electrostatic field in proteins, which is of importance for the understanding of biocatalytic processes and charge transport. Here we will discuss briefly the most illustrative results obtained by the methods of molecular relaxation spectroscopy. 

Thus, at present, fluorescence spectroscopy is capable of providing direct information on molecular dynamics on the nanosecond time scale and can estimate the results of dynamics occurring beyond this range. The present-day multiparametric fluorescence experiment gives new opportunities for interpretation of these data and construction of improved dynamic models. A further development of the theory which would provide an improved description of the dynamics in quantitative terms with allowance for the structural inhomogeneity of protein molecules and the hierarchy of their internal motions is required. 

Fluorescence spectroscopy and its applications to the physical and life sciences have evolved rapidly during the past decade. The increased interest in fluorescence appears to be due to advances in time resolution, methods of data analysis and improved instrumentation. With these advances, it is now practical to perform time-resolved measurements with enough resolution to compare the results with the structural and dynamic features of macromolecules, to probe the structures of proteins, membranes, and nucleic acids, and to acquire two-dimensional microscopic images of chemical or protein distributions in cell cultures. Advances in laser and detector technology have also resulted in renewed interest in fluorescence for clinical and analytical chemistry. 

In both the far- and near-UV regions, CD spectra can be used empirically as fingerprints of a particular protein, with the spectrum resulting from the aromatic residues being rather more specific and hence diagnostic. The far-UV spectra, however, can provide information about the protein conformation in terms of its secondary structure. As for fluorescence spectroscopy or any spectroscopic method, the sample needs to be chemically pure and homogeneous. 

Structure, of proteins CD spectral analysis, 219-243 for classification, 274 fluorescence spectroscopy, 245-265... 

Mazerrolles, G., Devaux, M. F., Duboz, G., Duployer, M. H., Riou, N. M., and Dufour, E. (2001). Infrared and fluorescence spectroscopy for monitoring protein structure and interaction changes during cheese ripening. Lait 81, 509-527. 

Conformational changes are detected in the near-UV CD spectrum if they affect the conformation or environment of side chain aromatic residues. On the other hand, they affect the far-UV CD spectrum only if substantial changes in secondary structure content occur. Larger amounts of proteins are required for near-UV CD than for far-UV CD spectroscopy given that absorptions in the former are at least an order of magnitude smaller. Because the pH-dependent conformational changes in BmPBP involve aromatic side chains, possibly tryptophan, they were also mirrored in fluorescence spectroscopy. This is fortuitous given that fluorescence requires smaller amounts of protein, which can consequently be obtained from natural sources. It is important to point out that for such experiments, however, proteins must be scrupulously pure and devoid of other chromophores. 

Fluorescence spectroscopy offers several inherent advantages for the characterization of molecular interactions and reactions. Firstly, it is 100-1000 times more sensitive than other spectrophotometric techniques. Secondly, fluorescent compounds are extremely sensitive to their environment. For example, vitamin A that is buried in the hydrophobic interior of a fat globule has fluorescent properties different from molecules that are in an aqueous solution. This environmental sensitivity enables characterization of viscosity changes such as those attributable to the thermal modifications of triglyceride structure, as well as the interactions of vitamin A with proteins. Third, most fluorescence methods are relatively rapid (less than 1 s with a Charge Coupled Device detector). One particularly advantageous property of fluorescence is that one can actually see it since it involves the emission of photons. The technique is suitable for at-line and on/in-line process control. 

The study by Determan et al. [224] focuses on the effects of polymer degradation products on the primary, secondary, and tertiary structure of TT, OVA, and lysozyme after incubation for 0 or 20 days in the presence of ester (lactic acid and glycolic acid) and anhydride [sebacic acid and l,6-bis(p-carboxyphenoxy)hexane] monomers. The structure and antigenicity or enzymatic activity of each protein in the presence of each monomer was quantified. SDS-PAGE, circular dichroism, and fluorescence spectroscopy were used to assess/evaluate the primary, secondary, and tertiary structures of the proteins, respectively. ELISA was used to measure changes in the antigenicity of TT and OVA and a fluorescence-based assay was used to determine the enzymatic activity of lysozyme. TT toxoid was found to be the most stable in the presence of anhydride monomers, while OVA was most stable in the... 

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