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PubMed2001

Native-state conformational dynamics of GART: a regulatory pH-dependent coil-helix transition examined by electrostatic calculations.

Glycinamide ribonucleotide transformylase (GART) undergoes a pH-dependent coil-helix transition with pK(a) approximately 7. An alpha-helix is formed at high pH spanning 8 residues of a 21-residue-long loop, comprising the segment Thr120-His121-Arg122-Gln123-Ala124-Leu125-Glu126-Asn127. To understand the electrostatic nature of this loop-helix, called the activation loop-helix, which leads to the formation and stability of the alpha-helix, pK(a) values of all ionizable residues of GART have been calculated, using Poisson-Boltzmann electrostatic calculations and crystallographic data. Crystallographic structures of high and low pH E70A GART have been used in our analysis. Low pK(a) values of 5.3, 5.3, 3.9, 1.7, and 4.7 have been calculated for five functionally important histidines, His108, His119, His121, His132, and His137, respectively, using the high pH E70A GART structure. Ten theoretical single and double mutants of the high pH E70A structure have been constructed to identify pairwise interactions of ionizable residues, which have aided in elucidating the multiplicity of electrostatic interactions of the activation loop-helix, and the impact of the activation helix on the catalytic site. Based on our pK(a) calculations and structural data, we propose that: (1) His121 forms a molecular switch for the coil-helix transition of the activation helix, depending on its protonation state; (2) a strong electrostatic interaction between His132 and His121 is observed, which can be of stabilizing or destabilizing nature for the activation helix, depending on the relative orientation and protonation states of the rings of His121 and His132; (3) electrostatic interactions involving His119 and Arg122 play a role in the stability of the activation helix; and (4) the activation helix contains the helix-promoting sequence Arg122-Gln123-Ala124-Leu125-Glu126, but its alignment relative to the N and C termini of the helix is not optimal, and is possibly of a destabilizing nature. Finally, we provide electrostatic evidence that the formation and closure of the activation helix create a hydrophobic environment for catalytic-site residue His108, to facilitate catalysis.

Alanine↗
PubMed1995

Conformational dynamics of a mobile loop in the NAD(H)-binding subunit of proton-translocating transhydrogenases from Rhodospirillum rubrum and Escherichia coli.

Transhydrogenase catalyses the reversible transfer of reducing equivalents between NAD(H) and NADP(H) to the translocation of protons across a membrane. Uniquely in Rhodospirillum rubrum, the NAD(H)-binding subunit (called Ths) exists as a separate subunit which can be reversibly dissociated from the membrane-located subunits. We have expressed the gene for R. rubrum Ths in Escherichia coli to yield large quantities of protein. Low concentrations of either trypsin or endoproteinase Lys-C lead to cleavage of purified Ths specifically at Lys227-Thr228 and Lys237-Glu238. Observations on the one-dimensional 1H-NMR spectra of Ths before and after proteolysis indicate that the segment which straddles the cleavage sites forms a mobile loop protruding from the surface of the protein. Alanine dehydrogenase, which is very similar in sequence to the NAD(H)-binding subunit of transhydrogenase, lacks this segment. Limited proteolytic cleavage has little effect on some of the structural characteristics of Ths (its dimeric nature, its ability to bind to the membrane-located subunits of transhydrogenase, and the short-wavelength fluorescence emission of a unique Trp residue) but does decrease the NADH-binding affinity, and does lower the catalytic activity of the reconstituted complex. The presence of NADH protects against trypsin or Lys-C cleavage, and leads to broadening, and in some cases, shifting, of NMR spectral signals associated with amino acid residues in the surface loop. This indicates that the loop becomes less mobile after nucleotide binding. Observation by NMR during a titration of Ths with NAD+ provides evidence of a two-step nucleotide binding reaction. By introducing an appropriate stop codon into the gene coding for the polypeptide of E. coli transhydrogenase cloned into an expression vector, we have prepared the NAD(H)-binding domain equivalent to Ths. The E. coli protein is sensitive to proteolysis by either trypsin or Lys-C in the mobile loop. Judging by the effect of NADH on its NMR spectrum and on the fluorescence of its Trp residues, the protein is capable of binding the nucleotide though it is unable to dock with the membrane-located subunits of transhydrogenase from R. rubrum.

Alanine Dehydrogenase↗
PubMed1993

Protein dynamics: conformational disorder, vibrational coupling and anharmonicity in deoxy-hemoglobin and myoglobin.

In this work we study the temperature dependence of the Soret band lineshape of deoxymyoglobin and deoxyhemoglobin, in the range 300-20 K. To fit the measured spectra we use an approach originally proposed by Champion and coworkers (Srajer et al. 1986; Srajer and Champion 1991). The band profile is modelled as a Voigt function that accounts for the coupling with low frequency vibrational modes, whereas the coupling with high frequency modes is responsible for the vibronic structure of the spectra. Moreover, owing to the position of the iron atom out of the mean heme plane, inhomogeneous broadening brings about a non-Gaussian distribution of 0-0 electronic transition frequencies. The reported analysis enables us to isolate the various contributions to the overall bandwidth, and their temperature dependence points out the relevance of low frequency vibrations and of large scale anharmonic motions starting at temperatures higher than 170 K. Information on the mean iron-heme plane distance and on its temperature dependence, as well as on the heme pocket conformational disorder, is also obtained.

Animals↗
PubMed2002

Excited state conformational dynamics of semiflexibly bridged electron donor-acceptor systems: a semiempirical CI-study including solvent effects.

Semiempirical (AM1) molecular orbital theory with configuration interaction has been used to investigate the harpooning mechanism proposed from experimental studies on semiflexibly bridged electron donor-acceptor systems. Our calculations on the charge-transfer state of N-phenyl-4-(4-cyano-naphth-1-ylmethyl)-piperidine, 1, confirm the proposed harpooning mechanism including an intermediate loosely folded charge-transfer state and reproduce the thermodynamics obtained from our spectroscopic studies closely. The structural details of the extended (ECT), intermediate (ICT) and compact (CCT) charge-transfer states are discussed, as are the transition states connecting them. Solvent effects have been modeled using self-consistent reaction field (SCRF) calculations within the polarized continuum model. The effect of solvent polarity on the stabilities of the three charge-transfer states is discussed.

Computer Simulation↗
PubMed1994

The dynamic conformation of plant cytoplasmic 5S rRNAs.

Recently we have proposed a new three-dimensional model of plant 5S rRNAs structure. To verify this proposal we present here new data on RNase T1 digestion and hydroxyl radical hydrolysis of lupin and wheat germ 5S rRNAs at various buffer and temperature conditions. Interestingly, the guanosine residues 85-87 in the loop D of these RNAs, are resistant to RNase T1 at native but not at denaturating conditions. On the other hand, the reaction of 5S rRNA with the hydroxyl radicals showed different reactivity of many nucleotides in various parts of the molecule and suggest conformational changes, which occur mostly in the loops. The experimental data clearly support involvement of the nucleotides occupying conserved positions in the loops in the tertiary interactions in plant 5S rRNA structure.

Base Sequence↗
PubMed1996

Synthesis, Structure, and Conformational Dynamics of Bridgehead-Substituted Nitrosamines. Di-1-adamantylnitrosamine and Di-1-norbornylnitrosamine.

The synthesis of two bridgehead-substituted nitrosamines, di-1-adamantylnitrosamine (1) and di-1-norbornylnitrosamine (2), is described, and their solid state crystal structures are reported. Large bridgehead substituents increase the NNO angle of the nitrosamine (compared to that found for dimethylnitrosamine) without deconjugating the NNO pi system significantly. This structural change correlates with a red-shifted optical absorption, a diminished N,N rotational barrier, and a greater ease of oxidation of these hindered nitrosamines than is observed for dimethylnitrosamine. The electronic basis of these structure/function correlations is examined. It is concluded that 1 is more strained than N-nitroso-2,2,6,6-tetramethylpiperidine (3) which is more strained than 2 and that a raised (in energy) NO n orbital is primarily responsible for the extreme properties of the former.

Journal Article↗
PubMed1992

Static and dynamic conformational properties of AT sequences in B-DNA.

A theoretical study of the optimal conformations of nucleic acid oligomers containing tracts of AT base pairs is presented. The oligomers are studied in isolation and complexed with netropsin, a minor groove binding ligand. The flexibility of the oligomers and of their complexes is calculated by adiabatic mapping with respect to the total winding angle. The results of this study show that in uncomplexed oligomers the dinucleotide junctions AA, AT and TA have very different structural parameters and different responses to winding stress. The TA junction is clearly the most flexible and is the principal site for accommodating the imposed overwinding. Complexation by netropsin leads to two important effects: firstly, the three junctions adopt more uniform structures, the largest changes again being observed for TA, secondly, the differences in flexibility as a function of sequence are strongly attenuated.

Base Composition↗
PubMed1988

Comparative calorimetric studies on the dynamic conformation of plant 5S rRNA. I. Thermal unfolding pattern of lupin seeds and wheat germ 5S rRNAs, also in the presence of magnesium and sperminium cations.

An attempt has been made to correlate differential scanning calorimetry melting profiles of 5S rRNAs from lupin seeds (L.s.) and wheat germ (W.g.) with their structure. It is suggested that the observed differences in thermal unfolding are due to differences in RNA nucleotide sequence and as a consequence in higher order structures. Interesting effects induced by magnesium cation, perprotonated and permethylated sperminium tetracations are discussed. It is suggested that the difference in the stabilizing effect of the three cations results from different mode of their interactions with RNA. "Pure" electrostatic interactions expected for permethylated tetracations are rather weak due to the steric hindrance around each positively charged nitrogen atom. Electrostatic interactions of the other two cations are significantly enhanced by coordination bonding for magnesium and by hydrogen bonding for protonated sperminium cation.

Calorimetry, Differential Scanning↗
PubMed1991

Comparative calorimetric studies on the dynamic conformation of plant 5S rRNA: II. Structural interpretation of the thermal unfolding patterns for lupin seeds and wheat germ.

Thermal unfolding of 5S rRNA from wheat germ (WG) and lupin seeds (LS) was studied in solution. Experimental curves of differential scanning calorimetry (DSC) were resolved into particular components according to the thermodynamic model of two-state transitions. The DSC temperature profiles for WG and LS differ significantly in spite of very high similarities in the sequence of both molecules. Those results are interpreted according to a model of the secondary and tertiary molecular structure of 5S rRNA. A comparison of the 'nearest neighbour' model of interaction with the experimental thermodynamic results enables a complete interpretation of the process of the melting of its structures. In light of our observations, the crucial differences between both DSC melting profiles are mainly an outcome of different thermodynamic properties of the first helical fragment 'A' made up of 9 complementary base pairs. It contains 6 differences in the nucleotide sequence of both types of molecules, which still retain 9-meric double helixes. The temperature stability of his helix in WG is much lower than of the LS one. Moreover, the results supply evidence for a strong specific tertiary interaction between the two hairpin loops 'c' and 'e' in both 5S rRNA molecules, modulated by small differences in the thermodynamic properties of both 5S rRNA.

Base Sequence↗
PubMed2005

Microsecond timescale backbone conformational dynamics in ubiquitin studied with NMR R1rho relaxation experiments.

NMR spin relaxation experiments are used to characterize the dynamics of the backbone of ubiquitin. Chemical exchange processes affecting residues Ile 23, Asn 25, Thr 55, and Val 70 are characterized using on- and off-resonance rotating-frame 15N R1rho relaxation experiments to have a kinetic exchange rate constant of 25,000 sec(-1) at 280 K. The exchange process affecting residues 23, 25, and 55 appears to result from disruption of N-cap hydrogen bonds of the alpha-helix and possibly from repacking of the side chain of Ile 23. Chemical exchange processes affecting other residues on the surface of ubiquitin are identified using 1H-15N multiple quantum relaxation experiments. These residues are located near or at the regions known to interact with various enzymes of the ubiquitin-dependent protein degradation pathway.

Magnetic Resonance Spectroscopy↗
PubMed1992

1H-15N-NMR studies of bacteriorhodopsin Halobacterium halobium. Conformational dynamics of the four-helical bundle.

Series of uniformly and selectively 15N-labeled bacteriorhodopsins of Halobacterium halobium (strain ET 1001) were obtained and a 1H-15N-NMR study was performed in methanol/chloroform (1:1) and 0.1 M NH4CHOO, medium which mimics that in the membrane in vivo. Less than half of the cross-peaks expected from the amino acid sequence of uniformly 15N-labeled bacteriorhodopsin were observed, using heteronuclear 1H-15N coherence spectroscopy. In order to assign the observed cross-peaks, a selective 15N-labeling of amino acid residues (Tyr, Phe, Trp, Lys, Gly, Leu, Val or Ile) was carried out and 1H-15N-NMR spectra of bacteriorhodopsin and its fragments C1 (residues (72-231), C2 (residues 1-71), B1 (residues 1-155) and BP2 (residues 163-231) were investigated. By this procedure, all observed 1H-15N cross-peaks of the entire bacteriorhodopsin were found to belong to the transmembrane segments A, B and G. The cross-peaks from four (C, D, E and F) helical bundles (79-189 residues) were missed. These results clearly indicate that dynamic processes occur in the four helice bundle. The significance of this, in respect to bacteriorhodopsin functioning, is discussed.

Amino Acid Sequence↗
PubMedDate not supplied

Tomotherapy: a new concept for the delivery of dynamic conformal radiotherapy.

Tomotherapy, literally "slice therapy," is a proposal for the delivery of radiation therapy with intensity-modulated strips of radiation. The proposed method employs a linear accelerator, or another radiation-emitting device, which would be mounted on a ring gantry like a CT scanner. The patient would move through the bore of the gantry simultaneously with gantry rotation. The intensity modulation would be performed by temporally modulated multiple independent leaves that open and close across the slit opening. At any given time, any leaf would be (1) closed, covering a portion of the slit, (2) open, allowing radiation through, or (3) changing between these states. This method would result in the delivery of highly conformal radiation. Overall treatment times should be comparable with contemporary treatment delivery times. The ring gantry would make it convenient to mount a narrow multisegmented megavoltage detector system for beam verification and a CT scanner on the treatment unit. Such a treatment unit could become a powerful tool for treatment planning, conformal treatment, and verification using tomographic images. The physical properties of this treatment delivery are evaluated and the fundamental design specifications are justified.

Humans↗
PubMed1992

Dynamical conformational change of DNA induced by synthetic polymers: direct observation by fluorescence microscopy.

Brownian motion and Higher-order structure of DNA molecules complexed with synthetic polymers: poly-L-arginine (pArg) and polyacrylamide (pAAm), were observed using fluorescence microscopy. Quantitative analyses of the conformation and motion of the complexes demonstrate that these two polymers influence the structure of DNA molecules in an opposite way. The electrostatic binding of pArg induces contraction of DNA, whereas the intercalative pseudo-grafting of pAAm extends DNA.

Acrylic Resins↗