Biomedical subjects

F G Prendergast

Publications and source records attributed to F G Prendergast.

At least 73 records · Page 4Linked to original sources

PubMed1991

Alterations in the structure, physicochemical properties, and pH of hepatocyte lysosomes in experimental iron overload.

While hemochromatosis is characterized by sequestration of iron-protein complexes in hepatocyte lysosomes, little is known about the effects of excess iron on these organelles. Therefore, we studied the effects of experimental iron overload on hepatocyte lysosomal structure, physicochemical properties, and function in rats fed carbonyl iron. A sixfold increase (P less than 0.0001) in hepatic iron and a fivefold increase in lysosomal iron (P less than 0.01) was observed after iron loading; as a result, hepatocyte lysosomes became enlarged and misshapen. These lysosomes displayed increased (P less than 0.0001) fragility; moreover, the fluidity of lysosomal membranes isolated from livers of iron-loaded rats was decreased (P less than 0.0003) as measured by fluorescence polarization. Malondialdehyde, an end product of lipid peroxidation, was increased by 73% (P less than 0.008) in lysosomal membranes isolated from livers of iron-overloaded rats. While amounts of several individual fatty acids in isolated lysosomal membranes were altered after iron overload, cholesterol/phospholipid ratios, lipid/protein ratios, double-bond index, and total saturated and unsaturated fatty acids remained unchanged. The pH of lysosomes in hepatocytes isolated from livers of iron-loaded rats and measured by digitized video microscopy was increased (control, 4.70 +/- 0.05; iron overload, 5.21 +/- 0.10; P less than 0.01). Our results demonstrate that experimental iron overload causes marked alterations in hepatocyte lysosomal morphology, an increase in lysosomal membrane fragility, a decrease in lysosomal membrane fluidity, and an increase in intralysosomal pH. Iron-catalyzed lipid peroxidation is likely the mechanism of these structural, physicochemical, and functional disturbances.

Animals↗

PubMed1990

Mn(II)-EPR measurements of cation binding by aequorin.

Cation binding at 5 degrees C by aequorin, a bioluminescent protein from the jellyfish Aequorea victoria, was examined by means of Mn(II) EPR. The bioluminescence of aequorin is triggered by Ca(II), as well as by trivalent lanthanides, and is inhibited by Mg(II) and Mn(II). Three EF-hand Ca(II)-binding domains have been identified in the aequorin amino acid sequence. In the work reported here, active native aequorin was found to have a single tight binding site for Mn(II) with an association constant of 0.566 microM-1. Ca(II) and La(III) competed for the Mn(II) site with association constants of 1.92 microM-1 and 1.38 microM-1, respectively. The affinity of Ca(II) and La(III) for their two other (presumed) sites on aequorin was an order of magnitude less than their affinity for the Mn(II) site. Mg(II) competed for the Mn(II) site as well but with a much smaller association constant of 0.0109 microM-1. Ca(II)-independent discharged aequorin did not bind Mn(II) to a significant degree. Conjectures on the location of the Mn(II) site in the aequorin amino acid sequence and on the relationship between the binding parameters of the cations and their influence on aequorin activity are given.

Aequorin↗

PubMed1990

Padé-Laplace method for the analysis of time-resolved fluorescence decay curves.

The interpretation of fluorescence intensity decay times in terms of protein structure and dynamics depends on the accuracy and sensitivity of the methods used for data analysis. The are many methods available for the analysis of fluorescence decay data, but justification for choosing any one of them is unclear. In this paper we generalize the recently proposed Padé-Laplace method to include deconvolution with respect to the instrument response function. In this form the method can be readily applied to the analysis of time-correlated single photon counting data. By extensive simulations we have shown that the Padé-Laplace method provides more accurate results than the standard least squares method with iterative reconvolution under the condition of closely spaced lifetimes. The application of the Padé-Laplace method to several experimental data sets yielded results consistent with those obtained by use of the least squares analysis.

Fluorescence↗

PubMed1990

Tryptophan-47 rotational isomerization in variant-3 scorpion neurotoxin. A combination thermodynamic perturbation and umbrella sampling study.

A combination thermodynamic perturbation and umbrella sampling study predicts two free energy wells for the rotational isomerization of the variant-3 scorpion neurotoxin tryptophan-47 indole side chain. One well has the indole side chain in the crystallographic orientation; the other has the indole rotated approximately 220 degrees to form a new conformation with a relative free energy of 3 +/- 2 kcal/mol. The activation barrier is 8.5 kcal/mol from the crystallographic well, from which transition state theory predicts a rate of escape of 2 x 10(5) s-1. Correlations in the displacements of side chains neighboring tryptophan-47 and the isomerization reaction coordinate last up to 20 ps. Favorable conditions of experimental verification are discussed.

Algorithms↗

PubMed1989

Characterization of selectively 13C-labeled synthetic melittin and melittin analogues in isotropic solvents by circular dichroism, fluorescence, and NMR spectroscopy.

The spectroscopic and functional characterization of 13C-labeled synthetic melittin and three analogues is described. Selectively 13C-enriched tryptophan ( [13C delta 1]-L-Trp) and glycine ( [13C alpha]Gly) were incorporated into melittin and three analogues by de novo peptide synthesis. 13C-Labeled tryptophan was incorporated into melittin at position 19 and into single-tryptophan analogues of melittin at positions 17, 11, and 9, respectively. Each of the synthetic peptides contained 13C-labeled glycine at position 12 only. The peptides were characterized functionally in a cytolytic assay, and spectroscopically by CD, fluorescence, and NMR. The behavior of 13C-labeled synthetic melittin was, in all respects, indistinguishable from that of the naturally occurring peptide. All of the analogues were found to be efficient lytic agents and thus were functionally similar to the native peptide, yet no evidence was found for formation of a melittin-like tetramer by any of the analogues in aqueous media, although there was a propensity for apparently nonspecific peptide aggregation, especially for MLT-W9. Since the analogues did exhibit fractional helicities by CD comparable to or even greater than melittin itself in the presence of methanol, we infer that tetramer assembly requires not only the ability to form alpha-helix but also a very precise packing of amino acid side chains of the constituent monomers. The 13C chemical shift of the Gly-12 C alpha was found to be a sensitive marker for helix formation in all of the peptides. For melittin itself, 13C NMR spectra revealed a downfield shift of approximately 1.8 ppm for the Gly-12 13C alpha resonance of the tetramer relative to that observed for the free monomer in D2O. In mixed samples containing melittin monomer and tetramer, two discrete Gly-12 13C alpha peaks were observed simultaneously, suggestive of slow exchange between the two species. We conclude that melittin's ability to form a soluble tetramer is not a prerequisite for cytolytic activity, nor is cytolytic potential precisely correlated with the ability to form an amphiphilic helix.

Amino Acid Sequence↗

PubMed1989

Fluorescence and 13C NMR determination of side-chain and backbone dynamics of synthetic melittin and melittin analogues in isotropic solvents.

The dynamics in isotopic solvents of selectively 13C labeled synthetic melittin and three analogues have been investigated by using NMR and fluorescence techniques both separately and in combination. In conjunction with the "model-free" approach to interpretation of NMR relaxation data [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4570], the availability of steady-state fluorescence anisotropy and lifetime data augment T1, T2, and NOE data to provide quantitative information about fluorophore dynamics in these peptides. A method is presented for using combined fluorescence and NMR data to obtain technique- and model-independent values for parameters describing local motion of 13C-labeled fluorophores in peptides and proteins. The dynamics of melittin and melittin analogues are found to be consistent with structural characteristics inferred from CD, fluorescence, and NMR spectral information presented in the preceding paper (Weaver et al., 1989). In particular, the mobility of the random coil peptide monomers is shown to be quite similar, while side-chain as well as peptide backbone motion in the aggregated or oligomeric species differs markedly among the analogues. For melittin itself, experimentally determined overall rotational correlation times for the monomer and tetramer agree very well with values predicted on the basis of solvent-accessible protein surface area. The local dynamics of selectively 13C-labeled Trp-19 and Gly-12 residues of melittin are also found to be consistent with peptide structure. In random coil melittin monomer, a specific model for the motion indicates that the Trp side chain moves through an approximate angle of +/- 71 degrees about the beta-gamma bond with a correlation time of 159 +/- 24 ps. In melittin tetramer, the indole moiety is spatially more confined with a flip angle of +/- 37 degrees, yet demonstrates an increased rate of motion with a correlation time of 56 +/- 8 ps. The constrained mobility of the Trp-19 side chain is consistent with motional constraints inferred from the X-ray structure of melittin tetramer. These results show that protein side-chain motion, even of moieties as large as indole, can occur on the picosecond time scale and that these motions are reasonably similar to those inferred from molecular dynamics simulations.

Bee Venoms↗

PubMed1989

Photochemically-induced dynamic nuclear polarization proton-NMR of aequorin discharged by calcium-independent light emission.

Photochemically-induced dynamic nuclear polarization was used to identify exposed amino-acid residues and to assign resonances in the 1H-NMR spectrum of Ca(II)-independent discharged (inactivated) aequorin. A previous nuclear magnetic resonance, circular dichroism and fluorescence study [Ray, B.D., Ho, S., Kemple, M. D., Prendergast, F. G. and Nageswara Rao, B.D. (1985) Biochemistry 24, 4280-4287] indicated that as the Ca(II)-activated bioluminescent protein aequorin from jellyfish spontaneously emits light in the absence of Ca(II), it changes from a rigid, fully active form to a discharged form in which a number of amino-acid residues are more mobile than in the native protein. Laser-photochemically-induced dynamic nuclear polarization experiments identified tryptophan and tyrosine residues, but not histidine residues, in Ca(II)-independent discharged aequorin to be accessible to the flavin dye used. These exposed residues are also among the mobile residues of the Ca(II)-independent discharged protein. Resonances of all the protons (including the alpha protons) of the accessible tryptophan and tyrosine residues were assigned with the aid of two-dimensional photochemically-induced dynamic nuclear polarization J-correlated spectroscopy. The oxidized chromophore, from which light is emitted in aequorin, was not accessible to the dye in the Ca(II)-independent discharged protein. No exposed residue was detected in the photochemically-induced dynamic nuclear polarization spectrum of Ca(II)-independent discharged aequorin from which the oxidized chromophore was removed, corroborating the previous finding that in this apo-discharged form the protein partially refolds and thereby loses some of the mobility acquired in the formation of the Ca(II)-independent discharged protein.

Aequorin↗

PubMed1989

Molecular dynamics of tryptophan in ribonuclease-T1. II. Correlations with fluorescence.

The interactions of tryptophan-59 (TRP-59) and its protein environment in ribonuclease-T1 (RNAse-T1) were examined in a 50-ps molecular dynamics simulation. The simulation used was previously shown to demonstrate a fluorescence anisotropy decay that closely agreed with the experimentally determined limiting anisotropy for RNAse-T1 (Axelsen, P. H., C. Haydock, and F. G. Prendergast. 1988. Biophys. J. 54:249-258). Further characterization of TRP-59 side chain dynamics and its protein environment has now been completed and correlated to other photophysical properties of this protein. Angular fluctuations of the side chain occur at rates of 1-10 cycles/ps and are limited to +/- 0.3 radians in all directions. Side chain motions are primarily limited by nonpolar collisions, although most side chain atoms have some collisional contact with polar atoms in the adjacent protein matrix or water. The steric relationship between PRO-39 and TRP-59 changes abruptly at 16 ps into the simulation. Two types of interaction with water are observed. First, a structural water appears to H-bond with the greater than N-H group of TRP-59. Second, water frequently contacts the six-atom ring. The electrostatic field experienced by the TRP-59 rings appears to be relatively constant and featureless regardless of ring orientation. We make the following interferences from our data: The fluorescent emission of TRP-59 may be red-shifted relative to TRP in nonpolar solvents either as a result of specific interactions with the structural water or relaxations of proximal bulk water and polar protein moieties. The quenching efficiency of polar interactions with TRP-59 must be extremely low given their frequency and the high quantum yield of RNAse-T1. This low efficiency may be due to restricted and unfavorable interaction geometries. PRO-39 is located near two titratable HIS residues in RNAse-T1 and may be involved in pH-dependent fluorescence phenomena by virtue of a metastable interaction with TRP-59. The interaction of bulk water with TRP-59 illustrates features of the gated transition state model for transient exposure to exogenously added collisional quenching agents. The restrictive environment of TRP-59 suggests that extrinsic quenching can only occur via interactions with the edge of the indole six-atom ring and that the efficiency of a quencher in a protein environment is likely to be a function of molecular symmetry.

Amino Acid Sequence↗

PubMed1989

Padé-Laplace method for analysis of fluorescence intensity decay.

This novel approach to the analysis of multiexponential functions is based on the combined use of the Laplace transform and Padé approximants (Yeramian, E., and P. Claverie. 1987. Nature (Lond.). 326:169-174). It is similar in principle to the well-known Isenberg method of moments (Isenberg, I. 1983. Biophys. J. 43:141-148) traditionally applied to the analysis of fluorescence decay. The advantage of the Padé-Laplace method lies in its ability to detect the number of components in a multiexponential function as well as their parameters. In this paper we modified the original method so that it can be applied to the analysis of multifrequency phase/modulation measurements of fluorescence decay. The method was tested first on simulated data. It afforded recovery up to four distinct lifetime components (and their fractional contributions). In the case of simulated data corresponding to continuous lifetime distributions (nonexponential decay), the results of the analysis by the Padé-Laplace method indicated the absence of discrete exponential components. The method was also applied to real phase/modulation data gathered on known fluorophores and their mixtures and on tryptophan fluorescence in phospholipase A2. The lifetime and fraction recoveries were consistent with those obtained from standard methods involving nonlinear least-square fitting.

Mathematics↗

PubMed1988

Biochemical and amino acid sequence analysis of human eosinophil granule major basic protein.

Eosinophil granule major basic protein (MBP) is a relatively low molecular weight cationic (pI greater than 10) protein present in the crystalloid core of the eosinophil granule. Amino acid sequence analysis of this protein was undertaken as part of an analysis of the structural basis of the potent cytotoxic activities of MBP on parasites and mammalian cells. Many conventional sequencing strategies were unworkable because of the unusual amino acid composition of MBP and its insolubility in solutions buffered at neutral pH. Less conventional chemical reactions, including cyanogen bromide-induced cleavage at tryptophan and acid-induced cleavage at aspartic acid, were used successfully to obtain peptides which allowed definition of the amino acid sequence of MBP. Characterization of MBP by reverse-phase high pressure liquid chromatography and two-dimensional gel analysis showed no microheterogeneity that might be attributed to post-translational modifications. Comparison of the MBP sequence with a protein sequence data base showed that MBP has no significant sequence homology with other characterized proteins. The basicity (pI 10.9) and hydrophobicity predicted from the MBP sequence are likely responsible for the observed affinity of this cytotoxic molecule for cell surfaces and some serum proteins.

Amino Acid Sequence↗

PubMed1988

Measurements of fluorescence lifetimes by use of a hybrid time-correlated and multifrequency phase fluorometer.

Measurements of homogeneous and heterogeneous fluorescence intensity decays using a hybrid time-correlated single photon counting/multifrequency phase fluorometer are reported. A trio of fluorophores exhibiting a range of decay profiles was selected. p-Terphenyl, 1,4-bis[2-(4-methyl-5-phenyloxazolyl)]benzene [(Me)2POPOP], and p-bis[2-(5-phenyloxazolyl)]benzene (POPOP), commonly used reference fluorophores, were analyzed initially; their emissions were characterized by monoexponential decay functions. Additionally, emissions from two single tryptophan proteins with different decay profiles were measured. Scorpion neurotoxin variant 3 required three exponentials to fit the emission decay properly (average lifetime approximately 500 ps). At pH 5.5, the fluorescence emission of ribonuclease T1 showed a monoexponential decay with a measured lifetime of approximately 4.0 ns. Thus, in each case, the results from both measurements were consistent between the two detection systems, confirming the view that the two approaches for measuring fluorescence lifetimes are equivalent.

Fluorescent Dyes↗

PubMed1988

Tyrosine hydrogen-bonding and environmental effects in proteins probed by ultraviolet resonance Raman spectroscopy.

Ultraviolet resonance Raman spectra with 229-nm excitation are reported for aqueous tyrosine and for ovomucoid third domain proteins from chicken [OMCHI3(-)] and from chachalaca [OMCHA(-)], as well as alpha 1-, alpha 2-, and beta-purothionin. At this excitation wavelength interference from phenylalanine is minimized, and it is possible to determine the frequencies of the Tyr ring modes nu 8a and nu 8b. The nu 8b frequency decreases with the degree of Tyr H-bond donation, reaching a limiting value for deprotonated tyrosine. This spectroscopic indicator of H-bond strength was calibrated by using the model compound p-cresol in H-bond acceptor solutions for which the enthalpy of H-bond formation can be obtained from the literature. With this calibration it is possible to estimate Tyr H-bond enthalpies in proteins for which Tyr is a H-bond donor; values of 13.7, 9.6, and 11.2 kcal/mol were found for OMCHA3(-) and for alpha 1- (or alpha 2-) and beta-purothionin, respectively. The intensity of the 1176-cm-1 nu 9a band of Tyr excited at 229 nm and also the intensity ratio of the Tyr 830/850-cm-1 Fermi doublet excited at 200 nm both correlate strongly with the estimated H-bond enthalpies, but large deviations are seen for the purothionins, reflecting a special environment for the Tyr residue of these proteins, which is believed to be constrained in a hydrophobic pocket. The molar intensity of the strong approximately 1000-cm-1 nu 12 band of phenylalanine in aqueous solution is about half the value observed in most proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

Electronic transitions in molecules in static external fields. I. Indole and Trp-59 in ribonuclease T1.

Electronic transition properties of indole perturbed by its environment were calculated by use of quantum-mechanical semi-empirical numerical methods. The environment was represented by a discrete set of charges placed at different positions around the indole ring. Wavelength shifts and transition intensity changes in indole were evaluated for several, specifically modeled geometries of external charges. This methodology was employed to estimate the extent of spectroscopic changes induced by small nonprotein polar species on the Trp-59 residue in the anisotropic environment of the protein ribonuclease T1. The geometry of the residue environment was obtained from dynamically equilibrated X-ray crystallographic data of the protein.

Endoribonucleases↗

PubMed1988

Tryptophan sidechain dynamics in hydrophobic oligopeptides determined by use of 13C nuclear magnetic resonance spectroscopy.

Two oligopeptides, t-boc-LAWAL-OMe and t-boc-LALALW-OMe, were synthesized for the purpose of examining the sidechain dynamics of the tryptophan residue in hydrophobic environments by 13C nuclear magnetic resonance and fluorescence spectroscopy. In both peptides, the tryptophan sidechain was greater than 95% enriched with 13C at the C delta 1 position. Spin-lattice relaxation time (T1) and steady-state nuclear Overhauser effect (NOE) data were obtained at 50.3 and 75.4 MHz for both peptides in CD3OD, and at 75.4 MHz for t-boc-LALALW-OMe in lysolecithin-D2O micelles. We have adapted the model-free approach of G. Lipari and A. Szabo (1982, J. Am. Chem. Soc. 104:4546) to interpret the 13C-NMR data. Computer-generated curves based on experimental data obtained at a single frequency demonstrate relationships between an effective correlation time for tryptophan sidechain motion (tau e), a generalized order parameter (sigma) describing the extent of motional restriction, and an overall correlation time for the peptide (tau m). Assuming predominantly dipolar relaxation, least-squares fits of the dual frequency relaxation data provide values for these parameters for both peptides. The contribution of chemical shift anisotropy (CSA), however, is also explicitly assessed in the data analysis, and is shown to perturb the predicted sigma, tau e, and tau m values and to decrease chi(2) values observed in nonlinear least-squares analysis of the data. Because of uncertainty in the contribution of CSA to the relaxation of the indole ring 13C delta 1 atom, nonlinear least-squares analysis of the relaxation data were performed with and without inclusion of a CSA term in the appropriate relaxation equations. Neglecting CSA, an overall peptide correlation time of 0.69 ns is predicted for t-boc-LAWAL-OMe in CD3OD at 20 degrees C compared with 1.28 ns for t-boc-LALALW-OMe. Given these tau m values and taking into account the effect of measurement error in the T1 and NOE data, the internal dynamics of the tryptophan residue of t-boc-LAWAL-OMe in this isotropic environment are described by a range of tau e values from 70 to 112 ps and sigma values between 0.22 and 0.36. Similarly, for t-boc-LALALW-OMe, 68 less than or equal to tau e less than or equal to 93 ps and 0.09 less than or equal to sigma less than or equal to 0.17. The Ch-terminal position of the tryptophan residue in the hexapeptide may account for its lower order parameter. In lysolecithin micelles, the model-free approach applied tot-boc-LALALW-OMe predicts a Te between 0.87 and 1.08 ns, and an order parameter range of 0.72-0.80, assuming an average Tm of 14 ns (Saunders, L. 1966. Biochim. Biophys. Acta. 125:70) for a typical peptide-micelle complex. In this case, measurement of only two 13C relaxation parameters at a single frequency yields sufficient information, plotted in the form of a composite T1-NOE solution curve, to constrain the allowed values of the model-free motional parameters within a relatively narrow range. The predicted range of eV and Te values for the peptide-micelle complex demonstrate that both the rate and spatial mobility of the indole moiety are markedly restrained in the anisotropic micelle environment relative to free methanol solution. Steady-state fluorescence anisotropy measurements made on the peptides dissolved in methanol or with synthetic lysolecithins in water were used to calculate apparent order parameters for tryptophan motion; these values agree well with order parameters calculated from 13C NMR data. The reported results are relevant to the issue of protein dynamic events occurring on the picosecond time scale predicted by molecular dynamics simulations.

Carbon Isotopes↗

PubMed1988

Molecular dynamics of tryptophan in ribonuclease-T1. I. Simulation strategies and fluorescence anisotropy decay.

Molecular dynamics simulations of Ribonuclease-T1 (RNAse-T1) were performed using x-ray crystal coordinates for the enzyme and various simulation strategies. From each of the simulations, a predicted fluorescence anisotropy decay for the single-tryptophan residue was derived and compared with experimental values for the limiting anisotropy of this protein. Simulations conducted in vacuo demonstrated large displacements among some of the residues adjacent to the tryptophan side chain. As a consequence, the ring system rotates relatively unhindered through an angle far in excess of that implied by experimental data. In contrast, the explicit simulation of solvent within a stochastic boundary led to excellent agreement between simulation and experiment. In the case of RNAse-T1, the experimentally-determined limiting anisotropy is useful as a criterion of simulation accuracy in the vicinity of the tryptophan side chain.

Computer Simulation↗

PubMed1987

Attenuated total reflectance Fourier transform infrared studies of the interaction of melittin, two fragments of melittin, and delta-hemolysin with phosphatidylcholines.

Attenuated total reflectance Fourier transform infrared spectroscopy (ATR FT-IR) has been used to monitor alterations in phospholipid organization in thin layers of 1,2-dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), induced by the membrane lytic peptide melittin, its fragments 1-15 (hydrophobic fragment) and 16-26 (hydrophilic fragment), and delta-hemolysin. In addition, the secondary structures of the peptides and the orientation of helical fragments were determined with respect to the bilayer. The insertion of melittin into POPC caused large perturbations in the order and increased rates of motion of the acyl chains, as monitored by the frequency and half-width of the symmetric CH2 stretching vibration near 2850 cm-1, as well as by the ATR dichroic ratio for this mode. Changes in DPPC organization were less and were consistent with peptide-induced static disordering (gauche rotamer formation) in the acyl chains. Melittin adopted primarily an alpha-helical secondary structure, although varying small proportions of beta and/or aggregated forms were noted. The helical segments were preferentially oriented perpendicular to the bilayer plane. Several modes of melittin/lipid interaction were considered in an attempt to semiquantitatively understand the observed dichroic ratios. By considering the peptide as a bent rigid rod, a plausible model for its lytic properties has been developed. The hydrophilic fragment in DPPC showed a secondary structure with little alpha-helix present. As judged by its effect on phospholipid acyl chain organizational parameters, the fragment did not penetrate the bilayer substantially. The hydrophobic fragment in DPPC gave amide I spectral patterns consistent with a mixture of predominantly beta-antiparallel pleated sheet with a smaller fraction of alpha-helix.(ABSTRACT TRUNCATED AT 250 WORDS)

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1987

Tryptophan fluorescence studies of subunit interaction and rotational dynamics of human luteinizing hormone.

Human luteinizing hormone (hLH) has a single tryptophan residue occurring in the beta-subunit (beta hLH). This provides an intrinsic fluorescent probe, in native hLH and beta hLH, that is unambiguously assigned. The fluorescence intensities of hLH and beta hLH are, however, significantly different. This difference has been utilized in studying the interaction of fluorescent beta hLH with the nonfluorescent alpha-subunit. The accessibility of the tryptophan residue in native hLH and beta hLH has been assessed by measuring the rate of collisional fluorescence quenching and by solvent perturbation (D2O/H2O) of fluorescence. Fluorescence anisotropy measurements have been used in studying the intramolecular dynamics and segmental tryptophan mobility in hLH and beta hLH. Lifetime-resolved anisotropy, measured by the technique of oxygen quenching of fluorescence, has revealed the presence of segmental tryptophan motion. These data can be satisfactorily explained in terms of fast segmental tryptophan motion and rotational diffusion of the whole protein and do not require that intersubunit motion be invoked for intact hLH as it was suggested earlier on the basis of fluorescence depolarization of fluorescein-labeled hLH [Bishop, W. H., & Ryan, R. J. (1975) Biochem. Biophys. Res. Commun. 65, 1184-1190].

Fluorescence Polarization↗

PubMed1987

Site-specific mutagenesis of the alpha-helices of calmodulin. Effects of altering a charge cluster in the helix that links the two halves of calmodulin.

Alteration of residues 82-84 in the alpha-helix that links the two halves of calmodulin results in a differential effect on activator activity. Previous studies (Lukas, T. J., Burgess, W. H., Prendergast, F. G., Lau, W., and Watterson, D. M. (1986) Biochemistry 25, 1458-1464) indicated the importance of positive charge clusters in the calmodulin-binding protein, myosin light chain kinase. This suggested the possible importance of complementary negative charge clusters in calmodulin. By using an efficient cassette mutagenesis approach and a synthetic calmodulin gene (Roberts, D. M., Crea, R., Malecha, M., Alvarado-Urbina, G., Chiarello, R. H., and Watterson, D. M. (1985) Biochemistry 24, 5090-5098), this possibility was directly addressed by engineering a new calmodulin, VU-8 calmodulin, in which the glutamate cluster at residues 82-84 in the synthetic gene product (VU-1 calmodulin) was replaced by three lysines. VU-8 calmodulin activated phosphodiesterase to the same maximal extent as VU-1 calmodulin, although there was an alteration in the concentration of calmodulin required for half-maximal stimulation. In contrast, myosin light chain kinase was activated to only 30% of maximal activity and NAD kinase was not activated. These results provide insight into the functional role of the unusual central helix structure found in the calmodulin family of proteins and indicate that different, although possibly overlapping, chemical complementarities are employed in the interaction between calmodulin and its various physiological targets.

Amino Acid Sequence↗