Investigation of chimerical and tagged forms of recombinant rat nucleoside diphosphate kinases alpha and beta. Interaction with rhodopsin-transducin complex and thermal stability.

To elucidate the physicochemical basis of differences between the isoforms of mammalian multifunctional nucleoside diphosphate kinase (NDP), we investigated the recombinant rat homohexameric NDP kinases alpha and beta, consisting of highly homologous alpha or beta subunits of 152 residues each and differing only in variable regions V1 and V2, and their chimerical forms (NDP kinase alpha(1-130)beta(131-152) and NDP kinase beta(1-130)alpha(131-152)) and tagged derivatives (NDP kinase HA-alpha(1-130)beta(131-152), NDP kinase HA-beta(1-130)alpha(131-152), and NDP kinase HA-beta). The thermal stability of these proteins and the ability of some of them to interact with the rhodopsin-transducin (R*Gt) complex have been studied. It was found that NDP kinase alpha, NDP kinase alpha(1-130)beta(131-152), and NDP kinase HA-alpha(1-130)beta(131-152) were similar in their thermal stability (T(1/2) = 61-63 degrees C). NDP kinase beta, NDP kinase beta(1-130)alpha(131-152), NDP kinase HA-beta(1-130)alpha(131-152), and NDP kinase HA-beta were inactivated at a lower temperature (T(1/2) = 51-54 degrees C). NDP kinase HA-alpha(1-130)beta(131-152) interacted with the R*Gt complex in the same manner as NDP kinase alpha, whereas the interaction of NDP kinase HA-beta(1-130)alpha(131-152) and NDP kinase beta with the photoreceptor membranes under the same conditions was very weak. It is suggested that the variability of the region V1 is a structural basis for the multifunctionality of NDP kinase hexamers in the cell.

Decomposition of protein tryptophan fluorescence spectra into log-normal components. I. Decomposition algorithms.

Two algorithms of decomposition of composite protein tryptophan fluorescence spectra were developed based on the possibility that the shape of elementary spectral component could be accurately described by a uniparametric log-normal function. The need for several mathematically different algorithms is dictated by the fact that decomposition of spectra into widely overlapping smooth components is a typical incorrect problem. Only the coincidence of components obtained with various algorithms can guarantee correctness and reliability of results. In this paper we propose the following algorithms of decomposition: (1) the SImple fitting procedure using the root-Mean-Square criterion (SIMS) operating with either individual emission spectra or sets of spectra measured with various quencher concentrations; and (2) the pseudo-graphic analytical procedure using a PHase plane in coordinates of normalized emission intensities at various wavelengths (wavenumbers) and REsolving sets of spectra measured with various Quencher concentrations (PHREQ). The actual experimental noise precludes decomposition of protein spectra into more than three components.

Decomposition of protein tryptophan fluorescence spectra into log-normal components. II. The statistical proof of discreteness of tryptophan classes in proteins.

The physical causes for wide variation of Stokes shift values in emission spectra of tryptophan fluorophores in proteins have been proposed in the model of discrete states (Burstein, E. A., N. S. Vedenkina, and M. N. Ivkova. 1973. Photochem. Photobiol. 18:263-279; Burstein, E. A. 1977a. Intrinsic Protein Luminescence (The Nature and Application). In Advances in Science and Technology (Itogi Nauki i Tekhniki), Biophysics Vol. 7. VINITI, Moscow [In Russian]; Burstein, E. A. 1983. Molecular Biology (Moscow) 17:455-467 [In Russian; English translation]). It was assumed that the existence of the five most probable spectral classes of emitting tryptophan residues and differences among the classes were analyzed in terms of various combinations of specific and universal interactions of excited fluorophores with their environment. The development of stable algorithms of decomposition of tryptophan fluorescence spectra into log-normal components gave us an opportunity to apply two mathematically different algorithms, SImple fitting with Mean-Square criterion (SIMS) and PHase-plot-based REsolving with Quenchers (PHREQ) for the decomposition of a representative set of emission spectra of proteins. Here we present the results of decomposition of tryptophan emission spectra of >100 different proteins, some in various structural states (native and denatured, in complexes with ions or organic ligands, in various pH-induced conformations, etc.). Analysis of the histograms of occurrence of >300 spectral log-normal components with various maximum positions confirmed the statistical discreteness of several states of emitting tryptophan fluorophores in proteins.

Decomposition of protein tryptophan fluorescence spectra into log-normal components. III. Correlation between fluorescence and microenvironment parameters of individual tryptophan residues.

In our previous paper (Reshetnyak, Ya. K., and E. A. Burstein. 2001. Biophys. J. 81:1710-1734) we confirmed the existence of five statistically discrete classes of emitting tryptophan fluorophores in proteins. The differences in fluorescence properties of tryptophan residues of these five classes reflect differences in interactions of excited states of tryptophan fluorophores with their microenvironment in proteins. Here we present a system of describing physical and structural parameters of microenvironments of tryptophan residues based on analysis of atomic crystal structures of proteins. The application of multidimensional statistical methods of cluster and discriminant analyses for the set of microenvironment parameters of 137 tryptophan residues of 48 proteins with known three-dimensional structures allowed us to 1) demonstrate the discrete nature of ensembles of structural parameters of tryptophan residues in proteins; 2) assign spectral components obtained after decomposition of tryptophan fluorescence spectra to individual tryptophan residues; 3) find a correlation between spectroscopic and physico-structural features of the microenvironment; and 4) reveal differences in structural and physical parameters of the microenvironment of tryptophan residues belonging to various spectral classes.

The identification of tryptophan residues responsible for ATP-induced increase in intrinsic fluorescence of myosin subfragment 1.

ATP binding to myosin subfragment 1 (S1) induces an increase in tryptophan fluorescence. Chymotryptic rabbit skeletal S1 has 5 tryptophan residues (Trp113, 131, 440, 510 and 595), and therefore the identification of tryptophan residues perturbed by ATP is quite complex. To solve this problem we resolved the complex fluorescence spectra into log-normal and decay-associated components, and carried out the structural analysis of the microenvironment of each tryptophan in S1. The decomposition of fluorescence spectra of S1 and S1-ATP complex revealed 3 components with maxima at ca. 318, 331 and 339-342 nm. The comparison of structural parameters of microenvironment of 5 tryptophan residues with the same parameters of single-tryptophan-containing proteins with well identified fluorescence properties applying statistical method of cluster analysis, enabled us to assign Trp595 to 318 nm, Trp440 to 331 nm, and Trp 13, 131 and 510 to 342 nm spectral components. ATP induced an almost equal increase in the intensities of the intermediate (331 nm) and long-wavelength (342 nm) components, and a small decrease in the short component (318 nm). The increase in the intermediate component fluorescence most likely results from an immobilization of some quenching groups (Met437, Met441 and/or Arg444) in the environment of Trp440. The increase in the intensity and a blue shift of the long component might be associated with conformational changes in the vicinity of Trp510. However, these conclusions can not be extended directly to the other types of myosins due to the diversity in the tryptophan content and their microenvironments.

Comparative study of recombinant rat nucleoside diphosphate kinases alpha and beta by intrinsic protein fluorescence.

Nucleoside diphosphate (NDP) kinases of mammals are hexamers of two sorts of randomly associated highly homologous subunits of 152 residues each and, therefore exist in cell as NDP kinase isoforms. The catalytic properties and three-dimensional structures of the isoforms are very similar. The physiological meaning of the existence of the isoforms in cells remained unclear, but studying recombinant rat NDP kinases alpha and beta, each containing only one sort of subunits, we discovered that, in contrast to the isoenzyme beta, NDP kinase alpha is able to interact with the complex between bleached rhodopsin and G-protein transducin in retinal rod membranes at lowered pH values (Orlov et al. FEBS Lett. 389, 186-190, 1996). In order to search for possible molecular basis of such differences between these isoenzymes, a detailed comparative study of their intrinsic fluorescence properties in a large range of solvent conditions was performed in this work. The isoenzymes alpha and beta both contain the same three tryptophan (Trp78, 133, Ind 149) and four tyrosine (Tyr 52, 67, 147, and 151) residues per subunit, but exhibit pronounced differences in their fluorescence properties (both in spectral positions and shape and quantum yield values) and behave differently under pH titration. Whereas NDP kinase alpha undergoes spectral changes in the pH range 5-7 with the mid-point at 6.2, no unequivocal indication of a structural change of NDP kinase beta under pH titration from 9 to 5 was obtained. Since the pH dependencies obtained for fluorescence of isoenzyme alpha resembles the dependence of its binding to the rhodopsin-transducin complex it was suggested that the differences between the NDP kinase isoenzymes alpha and beta in the pH-induced behavior, revealed by the fluorescence spectroscopy, and the differences in their ability to interact with rhodopsin-transducin complex may have the same physical nature, that would be a physico-chemical reason of possible functional dissimilarity of NDP kinase isoforms in cell. An additional analysis of three-dimensional structure of homologous NDP kinases revealed that the source of the differences in fluorescence properties and pH-titration behavior between the isoenzymes alpha and beta may be due to the difference in their global electrostatic charges, rather than to any structural differences between them at neutral pH. The unusually high positive electrostatic potential at he deeply buried active site Tyr52 makes possible that it exists in deprotonated tyrosinate form at neutral and moderately acidic solution. Such a possibility may account for rather unusual fluorescence properties of NDP kinase alpha: (i) rather long-wavelength emission of NDP kinase alpha at ca. 340 nm at pH ca. 8 at extremely low accessibility to external quenchers and, possibly, (ii) an unusually high quantum yield value (ca. 0.42).

Interaction of recombinant rat nucleoside diphosphate kinase alpha with bleached bovine retinal rod outer segment membranes: a possible mode of pH and salt effects.

An attempt was made to reveal the mode of action of protons and salts on the recently discovered GTP gamma S-dependent interaction of bovine retinal rod outer segments (ROS)1 nucleoside diphosphate kinase (NDP kinase) with the complex between bleached visual receptor rhodopsin and retinal G-protein transducin in bovine ROS membranes. The properties of recombinant rat NDP kinase alpha, that is immunologically similar to the soluble NDP kinase from bovine ROS preparation, have been studied in solution by means of protein fluorescence at different pH and salt concentrations and results were compared with pH and salt effects on the binding of NDP kinase alpha to bleached bovine ROS membranes. The results suggest that NDP kinase alpha itself may serve as a target for protons and salts and mediates their effects on the interaction between the enzyme and ROS membranes.

The impact of managed care on the practice of occupational therapy by hand therapists.

Current Occupational Therapy (OT) literature on managed care is limited to opinion or anecdotal experience. Questionnaires were mailed to 214 OTs practicing as hand therapists nationwide to test the null hypothesis that there was no difference in reimbursement or barriers to practice for OT services between patients enrolled in managed care organizations and those enrolled in fee-for-service insurance plans. Statistically significant results were obtained indicating greater barriers to reimbursement for OT under managed care. The primary effect has been the increased administrative demands it places on both therapists and patients. [Article copies available for a fee from The Haworth Document Delivery Service: 1-800-342-9678. E-mail address: getinfo@haworth.com].

Conformational changes in the muscle proteins of cured beef during heating.

Structural changes caused by heating in the proteins of cured beef longissimus dorsi muscle were examined by fluorescence and differential scanning calorimetry. Denaturation occurred in four temperature ranges-40-55°C, 55-61°C, 62-70°C and above 70°C, in contrast to the three endothermic transitions reported for non-cured meats.

Parvalbumin conformers revealed by steady-state and time-resolved fluorescence spectroscopy.

The binding of calcium to whiting (one tryptophan residue) and pike (one tyrosine residue) parvalbumins has been studied by means of kinetic and steady-state fluorescence techniques. The decay curves of the tryptophan and tyrosine fluorescence of the parvalbumins are best fitted by a sum of two exponents for any metal state of the proteins. The data can be interpreted as a nonexponential decay of the fluorescence of a single-type chromophore or in terms of equilibria between compact and relaxed conformers of the parvalbumins in each metal state. Fluorescence quenching by I-ions and effects of H2O/D2O substitution confirm the second interpretation. The constants of the equilibria have been evaluated.

Cation binding effects on the pH, thermal and urea denaturation transitions in alpha-lactalbumin.

The binding of monovalent (Na+, K+) and divalent (Ca2+, Mg2+) cations to bovine alpha-lactalbumin at 20 and 37 degrees C has been studied by means of intrinsic protein fluorescence. The values of apparent binding constants for these ions obtained at 37 degrees C are about one order of magnitude lower than those measured at 20 degrees C. Urea and alkali (pH greater than 10) induce unfolding transitions which involve stable partially unfolded intermediates for all metal ion-bound forms of alpha-lactalbumin. Heating induces similar partially unfolded states. Nevertheless, the partially unfolded states induced by heating, urea, alkaline or acidic treatments are somewhat different in their tryptophan residue environment properties. The results have been interpreted in terms of a simple scheme of equilibria between metal-free and metal-bound forms in their native, partially unfolded and unfolded states. The scheme provides an approach to the quantitative interpretation of any transition equilibrium shift induced by a low molecular mass species able to be bound by a protein.

Some aspects of studies of thermal transitions in proteins by means of their intrinsic fluorescence.

The changes in intrinsic fluorescence parameters induced by thermal transitions in proteins are developed on the background of the common thermal fluorescence quenching due to an activation of collisions between the excited chromophores and neighbouring quenching groups. Two methods of separation of the thermal quenching and conformational change contributions to the temperature dependence of the fluorescence parameters are presented. One is based on the use of the linearity of the plots of the reciprocal fluorescence quantum yield, 1/q, vs. the T/eta ratio (T, temperature; eta, solvent viscosity) for native proteins containing a single fluorescing chromophore (T.L. Bushueva , E.P. Busel and E.A. Burstein , Biochim. Biophys, Acta 534 (1978) 141). The other method is based on a consideration of the phase plots for the tryptophan fluorescence of proteins (fluorescence intensity at a fixed wavelength vs. intensity at any other fixed wavelength). The methods have been used for a study of the thermal transitions in Mg2+-loaded whiting parvalbumin (tryptophan fluorescence), Mg2+-loaded pike parvalbumins pI 4.2 (tyrosine fluorescence) and pI 5.0 (phenylalanine fluorescence), and Ca2+-loaded bovine alpha-lactalbumin (tryptophan fluorescence). The thermal denaturation curves for the parvalbumins show two-stepped character. The main change of the protein conformation occurs at the higher temperature step. Comparison of the fluorescence data with the microcalorimetry results shows that the maxima of the asymmetric heat sorption peaks for pike parvalbumins correlate with the mid-points of the higher temperature steps of the fluorimetric curves.

Sodium and potassium binding to parvalbumins measured by means of intrinsic protein fluorescence.

The binding of Na+ and K+ to whiting parvalbumin (pI 4.4) and pike parvalbumins (pI 4.2 and 5.0) results in a shift of the tryptophan fluorescence spectrum towards shorter wavelengths by 2-4 nm for the whiting protein and in a rise of the tyrosine and phenylalanine fluorescence quantum yield for the pike proteins. The effective binding constants of Na+ and K+ to parvalbumins are within the range of 10 M-1 to 100 M-1. Physiological concentrations of Na+ and K+ lower the affinity of whiting parvalbumin for Ca2+ and Mg2+ by almost an order of magnitude.

Comparative study of physiochemical properties of two pike parvalbumins by means of their intrinsic tyrosyl and phenylalanyl fluorescence.

Physicochemical properties of two pike parvalbumins (pI 5.0 and 4.2), belonging to two different gene lineages, have been studied by their intrinsic tyrosine and phenylalanine fluorescence. The CD sites of these paravalbumins have similar affinities to Ca2+ (and Mg2+) ions, but the EF sites of the proteins have very different affinities to these ions. This results in differing stabilities of these parvalbumins to pH-, urea-, and temperature-induced denaturation. The structure of pike parvalbumin pI 5.0, which binds Ca2+, and Mg2+ ions more tightly, is more stable than that of parvalbumin pI 4.2. Both proteins have higher affinities for Na+ ions than for K+ ions.

Intrinsic fluorescence spectra of a tryptophan-containing parvalbumin as a function of thermal, pH and urea denaturation.

The thermal, pH and urea denaturation of the calcium-loaded protein from whiting has been studied by means of the intrinsic fluorescence of the single tryptophan residue. pH denaturation of the protein takes place at a pH greater than 11.5 and lower than 5.5. Thermal denaturation of the protein occurs at temperatures above 55 degrees C. Urea initiates the denaturation of the calcium-loaded protein at rather low concentrations (1.0 M). In all cases, whether pH, thermal or urea denaturation, intermediate states of the protein were recorded. The fluorescence spectra of these intermediates are similar to that of the protein with one equivalent of calcium bound. Whiting parvalbumin binds calcium in the presence of 7.5 M urea but under these conditions, calcium-binding constants of the protein have been shown to be 10(2)-10(3)M(-1) (in comparison with 5 X 10 (8) and 6 X 10(6)M(-1) in the absence of any denaturing agents).

Fluorescence studies of the calcium binding to whiting (Gadus merlangus) parvalbumin.

The calcium binding by parvalbumin of whiting (Gadus merlangus) has been studied using tryptophanyl fluorescence characteristics. Titration of Ca2+-free parvalbumin with Ca2+ leads to a very pronounced blue shift, narrowing and intensification of the fluorescence spectrum. These spectral changs proceed in two stages reflecting the existence of at least three forms which can be interpreted as (a) the protein without Ca2+, (b) with one Ca2+ and (c) with two bound Ca2+ ions/molecule. The fluorescence of these forms has been identified and the fluorescence spectra measured at varied Ca2+ concentrations were resolved into three components corresponding to these spectral forms. The dependence of the relative concentration of the three fomrs on Ca2+ concentrations agree well with the two-step binding of Ca2+ to parvalbumin: Protein + Ca in equilibrium K1 protein x Ca; Protein x Ca + Ca in equilibrium K2 Ca x protein x Ca. The equilibrium binding constants K1 and K2 obtained by the computer fit are approximately 5 X 10(8) M-1 and 6 X 10(6) M-1. This scheme and the K1 and K2 value are in a good agreement with the independent experimental data resulting from EGTA titration of Ca2+-saturated parvalbumin and pH titratin of parvalbumin in the presence of EGTA and CA2+.