Heme-CO as a probe of the conformational state of calmodulin

The interaction of heme-CO with calmodulin, in the presence of calcium, leads to a complex of four heme-CO molecules per protein. No interaction was observed in the absence of calcium. The binding of heme-CO to calmodulin was monitored by the shift in the Soret absorption band from 407 to 420 nm (bound form); the four sites are not spectrally identical. The ligand CO can be photodissociated from the calmodulin-heme-CO complex and the biomolecular recombination kinetics also indicate a heterogeneous mixture. The complex does not bind oxygen reversibly. As calmodulin has only one histidine, the hemes are apparently not bound by the iron atom as in hemoglobin, but are probably loosely associated (Kd = 0.5 microM) in hydrophobic pockets which apparently open when the protein is activated by calcium.     

Cloning of plant cDNAs encoding calmodulin-binding proteins using35S-labeled recombinant calmodulin as a probe

Radiolabelled calmodulin has previously been used to screen cDNA expression libraries to isolate calmodulin-binding proteins. We have modified this technique for the isolation of plant calmodulin-binding proteins. [³⁵S]-methionine was used instead of the inorganic [³⁵S]-sulfate, or¹²⁵I used in previous methods. In addition, theE. coli pET expression system was chosen to obtain high levels of recombinant calmodulin at the time of labelling. The procedure thus takes into account both the specific activity of the probe and the amount of protein necessary for screening a large number of filters. Here we describe in detail a procedure for the production and purification of [³⁵S]-recombinant calmodulin and the use of the radiolabelled protein as a probe to screen plant cDNA expression libraries. The [³⁵S]-labeled calmodulin probe easily detects the λICM-1 phage encoding a partial mouse calmodulin-dependent protein kinase II that was previously isolated using a [¹²⁵I]-calmodulin probe (Sikela and Hahn, 1987). Subsequently, a tobacco root cDNA expression library was screened and a positive clone encoding a calcium-dependent calmodulin-binding protein was isolated.     

Optical and IR absorption as probe of dynamics of heme proteins

The spectroscopy of horseradish peroxidase with and without the substrate analogue benzohydroxamic acid (BHA) was monitored in different solvents as a function of the temperature in the interval from 10 to 300 K. Thermal broadening of the Q(0,0) optical absorption band arises mainly from interaction of the electronic pi --> pi transition with the heme vibrations. In contrast, the width of the IR absorption band of CO bound to heme is controlled by the coupling of the CO transition moment to the electric field of the protein matrix. The IR bandwidth of the substrate free enzyme in the glycerol/H2O solvent hardly changes in the glassy matrix and strongly increases upon heating above the glass transition. Heating of the same enzyme in the trehalose/H2O glass considerably broadens the band. The binding of the substrate strongly diminishes the temperature broadening of the CO band. This result is consistent with the view that the BHA strongly reduces the amplitude of vibrations of the heme pocket environment. Unusually strong thermal broadening of the CO band above the glass transition is interpreted to be caused by thermal population of a very flexible excited conformational substate. The thermal broadening of the same band in the trehalose glass is caused by an increase of the protein vibrational amplitude in each of the conformational substates, their population being independent of the temperature in the glassy matrix.     

Solvent-exposed tryptophans probe the dynamics at protein surfaces.

The dynamics of single tryptophan (W) side chain of protease subtilisin Carlsberg (SC) and myelin basic protein (MBP) were used for probing the surface of these proteins. The W side chains are exposed to the solvent, as shown by the extent of quenching of their fluorescence by KI. Time-resolved fluorescence anisotropy measurements showed that the rotational motion of W is completely unhindered in the case of SC and partially hindered in the case of MBP. The rotational correlation time (phi) associated with the fast local motion of W did not scale linearly with the bulk solvent viscosity (eta) in glycerol-water mixtures. In contrast, phi values of either W side chains in the denatured proteins or the free W scaled almost linearly with eta, as expected by the Stokes-Einstein relationship. These results were interpreted as indicating specific partitioning of water at the surface of the proteins in glycerol-water mixtures.     

Proton transfer dynamics in the nonhomogeneous electric field of a protein.

By adsorption of pyranine (8 hydroxypyrene 1, 3, 6 trisulfonate) to lysozyme we create on the positively charged protein a fluorophoric site with a total charge of -3. Photo dissociation of the dye's hydroxyl proton changes its absorption and fluorescence spectrum, permitting a continuous monitoring of the reprotonation dynamics. Absorbance measurements in the microsecond time scale monitor how the bulk protons penetrate the Coulomb cage of the bound dye. Time-resolved fluorescence monitors how the proton is escaping out of the Coulomb cage of the bound dye. These probe reactions were studied with a series of dye-enzyme complexes where the number of free carboxylate was reduced by amidation, increasing the total charge of the complex from +5 to +12.6. The time-resolved measurements demonstrate the complexity of the electric field in the immediate vicinity of the dye. It is consistent with a negative potential wall (of the anion) surrounded by a positive potential wall of proteinaceous moieties.     

Dipolar couplings as a probe of molecular dynamics and structure in solution.

The introduction of residual dipolar coupling methodology has increased the scope of structural biological problems that can be addressed by NMR spectroscopy. Conformational changes, the relative orientation of domains, and intermolecular complexes can now be characterized accurately and rapidly using NMR. The development of residual dipolar coupling methodology for the rapid recognition of homologous protein folds and for studies of submillisecond timescale dynamics has also seen considerable progress.     

Use of phospholipid transfer protein as a probe to study the lipid dynamics and alkaline phosphatase activity in the brush border membrane of human term placenta

Incubation of placental brush border membrane (BBM) along with sonicated vesicles of exogenous lipids (egg yolk PC) in the presence of phospholipid-transfer protein (PL-TP) showed a decrease in the alkaline phosphatase activity due to the change in the membrane micro-environment, such as fluidity. Effect of substrate concentration was tested by Lineweaver-Burk plot, which showed decreased V(max) and K(M). The effect of temperature was probed by the Arrhenius plot, which showed no change in transition temperature, but a decline in the energy of activation both below and above the transition temperature. The protein-catalyzed transfer of phospholipid from the donor unilamellar vesicles resulted in a substantial increase in the BBM phospholipid and a net decrease in cholesterol/phospholipid molar ratio. The change in membrane fluidity was assessed by translational as well as rotational diffusion of membrane extrinsic fluorescent probes, pyrene and diphenyl-hexatriene. An increased lateral mobility was recorded by the increased pyrene excimer formation. A decrease in fluorescent polarization of diphenyl-hexatriene was observed, which led to the decrease in fluorescence anisotropy and order parameter, and therefore, an increase in membrane fluidity (rotational diffusion). Mean anisotropy parameter was also decreased in the presence of PL-TP. Thus, the placental BBM alkaline phosphatase activity showed a distinct lipid dependence which may have important physiological consequences.     

Protein-polynucleotide scattering centers as a protein structure probe.

When certain basic globular proteins are mixed with nucleic acids near a critical concentration ratio, large, low density scattering centers of about 10(9) particle weight are created. Scattering from these complexes is altered when thermally inactivated proteins are substituted for enzymes in their native, globular conformation. Scattering data from heat-treated ribonuclease and lysozyme mixed with four different synthetic homopolyribonucleotides are reported. The concentration of nucleic acid necessary to produce maximum scattering from a heat-treated protein sample is shown to be a direct indication of the amount of enzyme that remains biologically active after being heated.     

Utility of a fluorescent vitamin E analogue as a probe for tocopherol transfer protein activity

The tocopherol transfer protein (TTP) is a member of the CRAL-TRIO family of lipid binding proteins that facilitates vitamin E transfer between membrane vesicles in vitro. In cultured hepatocytes, TTP enhances the secretion of tocopherol to the media; presumably, tocopherol transfer is at the basis of this biological activity. The mechanism underlying ligand transfer by TTP is presently unknown, and available tools for monitoring this activity suffer from complicated assay procedure and poor sensitivity. We report the characterization of a fluorescent vitamin E analogue, (R)-2,5,7,8-tetramethylchroman-2-[9-(7-nitrobenz[1,2,5]oxadiazol-4-ylamino)nonyl]chroman-6-ol (NBD-TOH), as a sensitive and convenient probe for the ligand binding and transfer activities of TTP. Upon binding to TTP, NBD-TOH fluorescence is blue shifted, and its intensity is greatly enhanced. We used these properties to accurately determine the affinity of NBD-TOH to TTP. The analogue binds to TTP reversibly and with high affinity (K(d) = 8.5 +/- 6 nM). We determined the affinity of NBD-TOH to a TTP protein in which lysine 59 is replaced with a tryptophan. When occurring in humans, this heritable mutation causes the ataxia with vitamin E deficiency (AVED) disorder. We find that the affinity of NBD-TOH to this mutant TTP is greatly diminished (K(d) = 71 +/- 19 nM). NBD-TOH functioned as a sensitive fluorophore in fluorescent resonance energy transfer (FRET) experiments. Using the fluorescent lipids TRITC-DHPE or Marina Blue-DHPE as a donor or an acceptor for NBD-TOH fluorescence, we obtained high-resolution kinetic data for tocopherol movement out of lipid bilayers, a key step in the TTP-facilitated ligand transfer reaction.     

The tryptophan phosphorescence of porcine and mutant bovine odorant-binding proteins: a probe for the local protein structure and dynamics

Vertebrate odorant-binding proteins (OBPs) are small extracellular proteins belonging to the lipocalin superfamily. They have been supposed to play a role in events of odorant molecules detection by carrying, deactivating, and/or selecting odorant molecules. The OBPs share a conserved folding pattern, an eight-stranded beta-barrel flanked by an alpha-helix at the C-terminal end of the polypeptide chain. The beta-barrel creates a central nonpolar cavity whose role is to bind and transport hydrophobic odorant molecules. These proteins reversibly bind odorant molecules with dissociation constants ranging from nanomolar to micromolar range. In this work, we have studied the structural features of the OBP from pig and from cow by phosphorescence spectroscopy. The obtained results demonstrate that the indolic phosphorescence of the two studied proteins can be readily detected at ambient temperature solutions and that it is owed exclusively to the internal tryptophan residue located next to the ligand binding cavity, which is generally conserved in the mammalian OBPs. In addition, while both the phosphorescence spectrum and the lifetime yield a picture of the fold of the studied protein in good agreement with the protein crystallographic structures, the triplet probe points out that in solution the polypeptide structure of the both investigated OBPs exists as a multiplicity of slowly interconverting protein conformations. Finally, this work also demonstrates that it is possible to directly detect the binding of the ligands to OBPs as variations of the protein luminescence features, thus, representing the very first observation reported in the literature so far that a fast and direct assay can be used for monitoring the binding of ligands to OBPs.     

Structure and dynamics of calmodulin in solution.

To characterize the dynamic behavior of calmodulin in solution, we have carried out molecular dynamics (MD) simulations of the Ca2+-loaded structure. The crystal structure of calmodulin was placed in a solvent sphere of radius 44 A, and 6 Cl- and 22 Na+ ions were included to neutralize the system and to model a 150 mM salt concentration. The total number of atoms was 32,867. During the 3-ns simulation, the structure exhibits large conformational changes on the nanosecond time scale. The central alpha-helix, which has been shown to unwind locally upon binding of calmodulin to target proteins, bends and unwinds near residue Arg74. We interpret this result as a preparative step in the more extensive structural transition observed in the "flexible linker" region 74-82 of the central helix upon complex formation. The major structural change is a reorientation of the two Ca2+-binding domains with respect to each other and a rearrangement of alpha-helices in the N-terminus domain that makes the hydrophobic target peptide binding site more accessible. This structural rearrangement brings the domains to a more favorable position for target binding, poised to achieve the orientation observed in the complex of calmodulin with myosin light-chain kinase. Analysis of solvent structure reveals an inhomogeneity in the mobility of water in the vicinity of the protein, which is attributable to the hydrophobic effect exerted by calmodulin's binding sites for target peptides.     

Electron transfer and protein dynamics in the photosynthetic reaction center.

We have measured the kinetics of electron transfer (ET) from the primary quinone (Q(A)) to the special pair (P) of the reaction center (RC) complex from Rhodobacter sphaeroides as a function of temperature (5-300 K), illumination protocol (cooled in the dark and under illumination from 110, 160, 180, and 280 K), and warming rate (1.3 and 13 mK/s). The nonexponential kinetics are interpreted with a quantum-mechanical ET model (Fermi's golden rule and the spin-boson model), in which heterogeneity of the protein ensemble, relaxations, and fluctuations are cast into a single coordinate that relaxes monotonically and is sensitive to all types of relaxations caused by ET. Our analysis shows that the structural changes that occur in response to ET decrease the free energy gap between donor and acceptor states by 120 meV and decrease the electronic coupling between donor and acceptor states from 2.7 x 10(-4) cm(-1) to 1.8 x 10(-4) cm(-1). At cryogenic temperatures, conformational changes can be slowed or completely arrested, allowing us to monitor relaxations on the annealing time scale (approximately 10(3)-10(4) s) as well as the time scale of ET (approximately 100 ms). The relaxations occur within four broad tiers of conformational substates with average apparent Arrhenius activation enthalpies of 17, 50, 78, and 110 kJ/mol and preexponential factors of 10(13), 10(15), 10(21), and 10(25) s(-1), respectively. The parameterization provides a prediction of the time course of relaxations at all temperatures. At 300 K, relaxations are expected to occur from 1 ps to 1 ms, whereas at lower temperatures, even broader distributions of relaxation times are expected. The weak dependence of the ET rate on both temperature and protein conformation, together with the possibility of modeling heterogeneity and dynamics with a single conformational coordinate, make RC a useful model system for probing the dynamics of conformational changes in proteins.     

Identification of a new serum protein polymorphism as transferrin

Summary By isoelectric focusing at pH 3.5–9.5, Kühnl and Spielmann (1977) recently demonstrated a new genetically determined serum protein polymorphism designated Hp^a because of an apparently specific reaction with antihaptoglobin. In this study the polymorphism was reproduced, but the components were found to focus at pH 5.8, which is different from the pI of haptoglobin, and immunologic relation to haptoglobin could not be comfirmed. Using pure transferrin as a reference, the results of isoelectric focusing, crossed immunoelectrophoresis, and immunofixation indicated that the polymorphic components were identical to transferrin. This polymorphism does not correspond to the already known transferrin polymorphism, as the two usual genes, tentatively designated Tf¹ and Tf², in my population sample (n=132) were 0.19 and 0.81, and, further, all individuals except three in the sample belong to type Tf-C.     

Energy transfer between fluorescent proteins using a co-expression system in Mycobacterium smegmatis.

The goal of this study was to establish a two-plasmid co-expression system for Mycobacterium smegmatis. Two vectors with compatible origins of replication and a polylinker, which allows modular cloning of promoters and genes, were constructed and used to clone genes encoding a blue fluorescent protein (BFP) and a green fluorescent protein (GFP). A 160-fold variation of GFP expression levels in M. smegmatis was achieved by combining three promoters with different copy numbers of the vectors. An efficient energy transfer between BFP and GFP in M. smegmatis was observed by fluorescence measurements and demonstrated that these genes were simultaneously expressed from both vectors. Thus, these vectors will be valuable for all strategies where co-expression of proteins in M. smegmatis is needed, e.g. for constructing a two-hybrid system or for deleting essential genes.     

Fluorescence energy transfer as a probe for nucleic acid structures and sequences.

The primary or secondary structure of single-stranded nucleic acids has been investigated with fluorescent oligonucleotides, i.e., oligonucleotides covalently linked to a fluorescent dye. Five different chromophores were used: 2-methoxy-6-chloro-9-amino-acridine, coumarin 500, fluorescein, rhodamine and ethidium. The chemical synthesis of derivatized oligonucleotides is described. Hybridization of two fluorescent oligonucleotides to adjacent nucleic acid sequences led to fluorescence excitation energy transfer between the donor and the acceptor dyes. This phenomenon was used to probe primary and secondary structures of DNA fragments and the orientation of oligodeoxynucleotides synthesized with the alpha-anomers of nucleoside units. Fluorescence energy transfer can be used to reveal the formation of hairpin structures and the translocation of genes between two chromosomes.     

Structure and dynamics of calcium-activated calmodulin in solution.

Two 4-ns molecular dynamics simulations of calcium loaded calmodulin in solution have been performed, using both standard nonbonded cutoffs and Ewald summation to treat electrostatic interactions. Our simulation results are generally consistent with solution experimental studies of calmodulin structure and dynamics, including NMR, cross-linking, fluorescence and x-ray scattering. The most interesting result of the molecular dynamics simulations is the detection of large-scale structural fluctuations of calmodulin in solution. The globular N- and C-terminal domains tend to move approximately like rigid bodies, with fluctuations of interdomain distances within a 7 A range and of interdomain angles by up to 60 deg. Essential dynamics analysis indicates that the three dominant types of motion involve bending of the central helix in two perpendicular planes and a twist in which the domains rotate in opposite directions around the central helix. In the more realistic Ewald trajectory the protein backbone remains mostly within a 2-3 A root-mean-square distance from the crystal structure, the secondary structure within the domains is conserved and middle part of the central helix becomes disordered. The central helix itself exhibits limited fluctuations, with its bend angle exploring the 0-50 degrees range and the end-to-end distance falling in 39-43 A. The results of the two simulations were similar in many respects. However, the cutoff trajectory exhibited a larger deviation from the crystal, loss of several helical hydrogen bonds in the N-terminal domain and lack of structural disorder in the central helix.     

Brownian dynamics simulations of probe and self-diffusion in concentrated protein and DNA solutions.

We have developed a Brownian dynamics algorithm for simulating probe and self-diffusion in concentrated solutions of DNA and protein. In these simulations, proteins are represented as spheres with radii given by their hydrodynamic radii, while DNA is modeled as a wormlike chain of hydrodynamically equivalent spherical frictional elements. The molecular interaction potentials employed by the program allow for intramolecular stretching and bending motions of the DNA chains, short-range Lennard-Jones interactions, and long-range electrostatic interactions. To test the program, we have carried out simulations of bovine serum albumin (BSA) probe diffusion and DNA self-diffusion in solutions of short-chain DNA as a function of both DNA concentration and solution ionic strength. In addition, we report on simulations of BSA self-diffusion as a function of BSA concentration and ionic strength. Based on a comparison to available experimental data, we find that our simulations accurately predict these transport properties under conditions of physiological salt concentration and predict the stronger concentration dependence observed at lower salt concentrations. These results are discussed in light of the nature of the intermolecular interactions in such systems and the approximations and limitations of the simulation algorithm.     

Water dynamics simulation as a tool for probing proton transfer pathways in a heptahelical membrane protein

The proton transfer pathway in a heptahelical membrane protein, the light-driven proton pump bacteriorhodopsin (BR), is probed by a combined approach of structural analysis of recent X-ray models and molecular dynamics (MD) simulations that provide the diffusion pathways of internal and external water molecules. Analyzing the hydrogen-bond contact frequencies of the water molecules with protein groups, the complete proton pathway through the protein is probed. Beside the well-known proton binding sites in the protein interior-the protonated Schiff base, Asp85 and Asp96, and the H(5)O(2) (+) complex stabilized by Glu204 and Glu194-the proton release and uptake pathways to the protein surfaces are described in great detail. Further residues were identified, by mutation of which the proposed pathways can be verified. In addition the diffusion pathway of water 502 from Lys216 to Asp96 is shown to cover the positions of the intruding waters 503 and 504 in the N-intermediate. The transiently established water chain in the N-state provides a proton pathway from Asp96 to the Schiff base in the M- to N-transition in a Grotthus-like mechanism, as concluded earlier from time-resolved Fourier transform infrared experiments [le Coutre et al., Proc Nat Acad Sci USA 1995;92:4962-4966].