Raman spectroscopy of selectively deuterated dimyristoylphosphatidylcholine: studies on dimyristoylphosphatidylcholine-cholesterol bilayers

Dimyristoylphosphatidylcholine (DMPC), selectively deuterated in the sn-2 chain at the 3, 6, and 10 positions is used to probe DMPC-cholesterol interactions in multilamellar dispersions. Using the Raman spectral linewidths of the 2100 cm-1 C2H2 stretching modes as an index of membrane disorder, we demonstrate that cholesterol tends to order, or increase the number of trans carbon-carbon bonds within the DMPC acyl chain near the headgroup region at all temperatures. At low temperatures, cholesterol disorders the acyl chains near the methyl termini by inducing gauche conformers; cholesterol orders the entire chain at higher temperatures. These determinations are qualitatively consistent with conclusions drawn from deuterium nuclear magnetic resonance studies, but specifically reflect acyl chain trans/gauche isomerization on the 10(-12)-10(-13) s vibrational time scale.     

Characterization of DNA structures by Raman spectroscopy: high-salt and low-salt forms of double helical poly(dG-dC) in H2O and D2O solutions and application to B, Z and A-DNA.

Raman spectra of poly(dG-dC) . poly(dG-dC) in D2O solutions of high (4.0M NaCl) and low-salt (0.1M NaCl) exhibit differences due to different nucleotide conformations and secondary structures of Z and B-DNA. Characteristic carbonyl modes in the 1600-1700 cm-1 region also reflect differences in base pair hydrogen bonding of the respective GC complexes. Comparison with A-DNA confirms the uniqueness of C = O stretching frequencies in each of the three DNA secondary structures. Most useful for qualitative identification of B, Z and A-DNA structures are the intense Raman lines of the phosphodiester backbone in the 750-850 cm-1 region. A conformation-sensitive guanine mode, which yields Raman lines near 682, 668, or 625 cm-1 in B (C2'-endo, anti), A (C3'-endo, anti) or Z (C3'-endo, syn) structures, respectively, is the most useful for quantitative analysis. In D2O, the guanine line of Z-DNA is shifted to 615 cm-1, permitting its detection even in the presence of proteins.     

Interaction of ferricytochrome c with zwitterionic phospholipid bilayers: a Raman spectroscopic study

The vibrational Raman spectra of both pure L-alpha-dipalmitoylphosphatidylcholine (DPPC) liposomes and DPPC multilayers reconstituted with ferricytochrome c under varying conditions of pH and ionic strength are reported as a function of temperature. Total integrated band intensities and relative peak height intensity ratios, two spectral scattering parameters used to determine bilayer disorder, are invariant to changes in pH and ionic strength but exhibit a sensitivity to the bilayer concentration of the ferricytochrome c. Protein concentrations were estimated by comparing the 1636 cm-1 resonance Raman line of known ferricytochrome c solutions to intensity values for the reconstituted multilayer samples. Temperature-dependent profiles of the 3100-2800 cm-1 C-H stretching, 1150-1000 cm-1 C-C stretching, 1440 cm-1 CH2 deformation, and 1295 cm-1 CH2 twisting mode regions characteristic of acyl chain vibrations reflect bilayer perturbations due to the weak interactions of ferricytochrome c. The DPPC multilamellar gel to liquid-crystalline phase transition temperature, TM, defined by either the C-H stretching mode I2935/I2880 or the C-C stretching mode I1061/I1090 peak height intensity ratios, is decreased by approximately 4 degrees C for the approximately 10(-4) M ferricytochrome c reconstituted DPPC liposomes. Other spectral features, such as the increase in the 2935 cm-1 C-H stretching mode region and the enhancement of higher frequency CH2 twisting modes, which arise in bilayers containing approximately 10(-4) M protein, are interpreted in terms of protein penetration into the hydrophobic region of the bilayer.     

A Raman study of low frequency intrahelical modes in A-, B-, and C-DNA.

We have obtained low frequency (less than 200 cm-1) Raman spectra of calf-thymus DNA and poly(rI).poly(rC) as a function of water content and counterion species and of d(GGTATACC)2 and d(CGCGAATTCGCG)2 crystals. We have found that the Raman scattering from water in the first and second hydration shells does not contribute directly to the Raman spectra of DNA. We have determined the number of strong Raman active modes by comparing spectra for different sample orientations and polarizations and by obtaining fits to the spectra. We have found at least five Raman active modes in the spectra of A- and B-DNA. The frequencies of the modes above 40 cm-1 do not vary with counterion species, and there are only relatively small changes upon hydration. These modes are, therefore, almost completely internal. The mode near 34 cm-1 in A-DNA is mostly internal, whereas the mode near 25 cm-1 is dominated by interhelical interactions. The observed intensity changes upon dehydration were found to be due to the decrease in interhelical distance. Polymer length appears to play a role in the lowest frequency modes.     

Resonance Raman enhancement of phenyl ring vibrational modes in phenyl iron complex of myoglobin.

Resonance Raman spectra are reported for the organometallic phenyl-FeIII complexes of horse heart myoglobin. We observed the resonance enhancement of the ring vibrational modes of the bound phenyl group. They were identified at 642, 996, 1,009, and 1,048 cm-1, which shift to 619, 961, 972, and 1,030 cm-1, respectively, upon phenyl 13C substitution. The lines at 642 and 996 cm-1 are assigned, respectively, as in-plane phenyl ring deformation mode (derived from benzene vibration No. 6a at 606 cm-1) and out-of-plane CH deformation (derived from benzene vibration No. 5 at 995 cm-1). The frequencies of the ring "breathing" modes at 1,009 and 1,048 cm-1 are higher than the corresponding ones in phenylalanine (at 1,004 and 1,033 cm-1) and benzene (at 992 and 1,010 cm-1), indicating that the ring C--C bonds are strengthened (or shortened) when coordinated to the heme iron. The excitation profiles of these phenyl ring modes and a porphyrin ring vibrational mode at 674 cm-1 exhibit peaks near its Soret absorption maximum at 431 nm. This appears to indicate that these phenyl ring modes may be enhanced via resonance with the Soret pi-pi transition. The FeIII--C bond stretching vibration has not been detected with excitation wavelengths in the 406.7-457.9-nm region.     

Local modes in a DNA polymer with hydrogen bond defect.

Vibrations of a homopolymer DNA with localized hydrogen bond defects have been examined using the recently developed decaying mode theory for long-chain polymers with local structural defects. For a poly(dA)-poly(dT) homopolymer having perturbed hydrogen bonds in one base pair, a localized mode at 63.2 cm-1 has been found. This mode has a very nearly pure H-bond stretch or "breathing" character, although the backbones do not separate. This agrees in frequency with a similar result found by other authors using a different approach. We search the full microwave frequency range for other local modes for several models of weakened H bonds. Besides the local mode with breathing characteristics, local modes with other characteristic motions were found, but only for asymmetrically perturbed bonds. We find in general that local modes are not very robust, requiring quite specific, narrow ranges in parameter space. They are also not abundant, there being only three in our most prolific model.     

63/65Cu and 1/2H2O isotope shifts in the low-temperature resonance Raman spectrum of fungal laccase

Resonance Raman spectra are reported for the type 1 Cu site of fungal laccase at 295 and 77 K. The low-temperature spectra show enhanced resolution and reveal several weak bands not previously observed, as well as overtone and combination bands associated with the strong approximately equal to 400 cm-1 fundamentals. A novel low-temperature Raman difference technique has been used to obtain 63/65Cu and 1/2H2O isotope shifts. The strong band at 428 cm-1, and the moderate intensity bands at 408 and 387 cm-1 show small (under 0.6 cm-1 63/65Cu isotope shifts. The aggregate shift is substantially less than that expected for an isolated Cu-S(cys) stretch, implying a high degree of mixing of this coordinate with internal modes of the ligands. 1/2H2O shifts of 1.1 and approximately equal to 0.3 cm-1 are observed for the 387 and 428 cm-1 bands. The isotope shift patterns are quite similar for fungal and tree laccase, as are the frequencies of the dominant bands, indicating that the large differences in relative intensity are primarily associated with differences in the excited state potential. The frequency and isotope shift patterns are appreciably different, however, from those observed for azurin and stellacyanin. In contrast to the other 'blue' Cu proteins, fungal laccase shows no moderate intensity band near 270 cm-1 which can be associated with Cu-imidazole stretching; weak features are seen in this region, but the intensities are too low to determine their 1/2H2O sensitivity. The C-S stretching mode of fungal laccase is identified at 737 cm-1, shifting to 741 cm-1 at 77 K. It is about 10 cm-1 lower than for most 'blue' Cu proteins, and the difference is suggested to reflect smaller kinematic coupling between the C-S and Cu-S coordinates, associated with a smaller Cu-S-C angle. Combination modes of the approx. 400 cm-1 fundamentals are substantially stronger, relative to the overtones, than is predicted by first-order scattering theory, implying changes in the excited-state normal modes (Dushinsky effect) associated with force constant alterations.     

Thermodynamics of DNA containing very stable chemically modified base pairs

DNA chemical modifications caused by the binding of some antitumor drugs give rise to a very strong local stabilization of the double helix. These sites melt at a temperature that is well above the melting temperatures of ordinary AT and GC base pairs. In this work we have examined the melting behavior of DNA containing very stable sites. Analytical expressions were derived and used to evaluate the thermodynamic properties of homopolymer DNA with several different distributions of stable sites. The results were extended to DNA with a heterogeneous sequence of AT and GC base pairs. The results were compared to the melting properties of DNA with ordinary covalent interstrand cross-links. It was found that, as with an ordinary interstrand cross-link, a single strongly stabilized site makes a DNA's melting temperature (T(m)) independent of strand concentration. However in contrast to a DNA with an interstrand cross-link, a strongly stabilized site makes the DNA's T(m) independent of DNA length and equal to T(infinity), the melting temperature of an infinite length DNA with the same GC-content and without a stabilized site. Moreover, at a temperature where more than 80% of base pairs are melted, the number of ordinary (non-modified) helical base pairs (n) is independent of both the DNA length and the location of the stabilized sites. For this condition, n(T) = (2 omega-a)S/(1-S) and S = exp[DeltaS(T(infinity)-T)/(RT)] where omega is the number of strongly stabilized sites in the DNA chain, a is the number of DNA ends that contain a stabilized site, and DeltaS, T, and R are the base pair entropy change, the temperature, and the universal gas constant per mole. The above expression is valid for a temperature interval that corresponds to n<0.2N for omega=1, and n<0.1N for omega>1, where N is the number of ordinary base pairs in the DNA chain.     

The Z-conformation of poly(dA-dT).poly(dA-dT) in solution as studied by ultraviolet resonance Raman spectroscopy

Poly(dA-dT).poly(dA-dT) structures in aqueous solutions with high NaCl concentrations and in the presence of Ni2+ ions have been studied with resonance Raman spectroscopy (RRS). In low water activity the effects of added 95 mM NiCl2 in solution stabilize the syn geometry of the purines and reorganize the water distribution via local interactions of Ni-water charged complexes with the adenine N7 position. It is shown that RRS provides good marker bands for a left-handed helix: i) a purine ring breathing mode around 630 cm-1 coupled to the deoxyribose vibration in the syn geometry, ii) a 1300-1340 cm-1 region characterizing local chemical interactions of the Ni2+ ions with the adenine N7 position, iii) lines at about 1483- and 1582 cm-1 correlated to the anti/syn reorientation of the adenine residues on B-Z structure transition, iv) marker bands of the thymidine carbonyl group couplings at 1680- and 1733 cm-1 due to the disposition of the thymidine residues in the Z helix specific geometry. Hence poly(dA-dT).poly(dA-dT) can adopt a Z form in solution. The Z form observed in alternate purine-pyrimidine sequences does not require G-C base pairs.     

Raman spectroscopic studies of dimyristoylphosphatidic acid and its interactions with ferricytochrome c in cationic binary and ternary lipid-protein complexes.

The vibrational Raman spectra of both pure 1-alpha-dimyristoylphosphatidic acid (DMPA) liposomes and DMPA multilayers reconstituted with ferricytochrome c at pH 7 and pH 4, with either sodium or calcium as the cation, are reported as a function of temperature. Multilayers composed of a 1:1 mol ratio DMPA and dimyristoylphosphatidylcholine with perdeuterated acyl chains (DMPC-d54) have also been reconstituted with approximately 10(-4) M ferricytochrome c for Raman spectroscopic observation. Total integrated band intensities and relative peak height intensity ratios, two spectral Raman scattering parameters used to characterize bilayer properties, are sensitive to the presence of both ferricytochrome c and the cation in the reconstituted liposomes. Temperature profiles, derived from the various Raman intensity parameters for the 3,100-2,800 cm-1 lipid acyl chain C-H stretching mode region specifically reflect bilayer perturbations due to the interactions of ferricytochrome c. At pH 4 the calcium DMPA multilamellar gel to liquid crystalline phase transition temperatures Tm, defined by either the C-H stretching mode I2850/I2880 and I2935/I2880 peak height intensity ratios, are 58.5 +/- 0.5 degrees C and 60.0 +/- 0.3 degrees C, respectively. This difference in Tm's resolves the phase transition process into first an expansion of the lipid lattice and then a melting of the lipid acyl chains. At pH 7 the calcium DMPA liposomes show no distinct phase transition characteristics below 75 degrees C. For sodium DMPA liposomes reconstituted with ferricytochrome c at either pH 4.0 or pH 7.0, spontaneous Raman spectra show altered lipid structures at temperatures above 40 degrees C. Resonance Raman spectra indicate that ferricytochrome c reconstituted in either calcium or sodium DMPA liposomes changes irreversibly above Tm. For either the binary lipid or ternary lipid-protein systems reconstituted with DMPC-d54, linewidth parameters of the DMPC-d54 acyl chain CD2 symmetric stretching modes at 2,103 cm-1 provide a sensitive measure of the conformational and dynamic properties of the perdeuterated lipid component, while the 3,000 cm-1 C-H spectral region reflects the bilayer characteristics of the DMPA species in the complex. Although calcium clearly induces a lateral phase separation in the DMPA/DMPC-d54 system at pH 7.5 (Kouaouci, R., J.R. Silvius, I. Grah, and M. Pezolet. 1985. Biochemistry. 24:7132-7140), no distinct lateral segregation of the lipid components is observed in the mixed DMPA/DMPC-d54 lipid system in the presence of either ferricytochrome c or the sodium and calcium cations at pH 4.0.(ABSTRACT TRUNCATED AT 400 WORDS)     

Vibrational fluctuations of hydrogen bonds in a DNA double helix with nonuniform base pairs.

The Green's function technique is applied to a study of breathing modes in a DNA double helix which contains a region of different base pairs from the rest of the double helix. The calculation is performed on a G-C helix in the B conformation with four consecutive base pairs replaced by A-T. The average stretch in hydrogen bonds is found amplified around the A-T base pair region compared with that of poly(dG)-poly(dC). This is likely related to the A-T regions lower stability against hydrogen bond melting. The A-T region may be considered to be the initiation site for melting in such a helix.     

Raman spectroscopy of the thermal properties of reassembled high-density lipoprotein: apolipoprotein A-I complexes of dimyristoylphosphatidylcholine

Isolated complexes of apolipoprotein A-I (apoA-I), the major apoprotein of human plasma high-density lipoproteins, and dimyristoylphosphatidylcholine (DMPC) have been prepared and studied by differential scanning calorimetry (DSC) and Raman spectroscopy. DSC studies establish that complexes having lipid to protein ratios of 200, 100, and 50 to 1 each exhibit a broad reversible thermal transition at Tc = 27 degrees C. The enthalpy of lipid melting for each of the three complexes is about 3 kcal/mol of DMPC. Raman spectroscopy indicates that the physical state of lipid molecules in the complexes is different from that in DMPC multilamellar liposomes. Analysis of the C-H stretching region (2800-3000 cm-1) of the complexes and of the pure components in water suggests that below 24 degrees C (Tc for DMPC) there is considerably less lateral order among lipid acyl chains in the complexes than in DMPC liposomes. Above 24 degrees C, these types of interactions appear to contribute equally or slightly less to the complex structure than in pure DMPC. The temperature dependence of peaks in the C-C stretching region (1000-1180 cm-1) reveals a continuous increase in the number of lipid acyl chain C-C gauche isomers over a broad range with increasing temperature. Compared to liposomes, DMPC in the complexes has more acyl chain trans isomers at temperatures above 24 degrees C; at temperatures above ca. 30 degrees C, trans isomer content is about the same for complexes and liposomes. A large change was observed in a protein vibrational band at 1340 cm-1 for pure vs. complexed apoA-I, indicating that protein hydrocarbon side chains are immobilized by lipid binding. The Raman data indicate that the reduction in melting enthalpy for complexes DMPC (approximately 3 kcal/mol) compared to that for free DMPC (approximately 6 kcal/mol) is due to reduced van der Waals interactions in the low-temperature lipid phase.     

Resonance Raman studies of Escherichia coli sulfite reductase hemoprotein. 2. Fe4S4 cluster vibrational modes

Resonance Raman (RR) spectra from the hemoprotein subunit of Escherichia coli sulfite reductase (SiR-HP) are examined in the low-frequency (200-500 cm-1) region where Fe-S stretching modes are expected. In spectra obtained with excitation in the siroheme Soret or Q bands, this region is dominated by siroheme modes. Modes assignable to the Fe4S4 cluster are selectively enhanced, however, with excitation at 488.0 or 457.9 nm. The assignments are confirmed by observation of the expected frequency shifts in SiR-HP extracted from E. coli grown on 34S-labeled sulfate. The mode frequencies and isotopic shifts resemble those seen in RR spectra of other Fe4S4 proteins and analogues, but the breathing mode of the cluster at 342 cm-1 is higher than that observed in the other species. Spectra of various ligand complexes of SiR-HP reveal only slight sensitivity of the cluster terminal ligand modes to the presence of exogenous heme ligands, at variance with a model of ligand binding in a bridged mode between heme and cluster. Close examination of RR spectra obtained with siroheme Soret-band excitation reveals additional 34S-sensitive features at 352 and 393 cm-1. These may be attributed to a bridging thiolate ligand.     

Phase equilibrium in poly(rA).poly(rU) complexes with Cd2+ and Mg2+ ions, studied by ultraviolet, infrared, and vibrational circular dichroism spectroscopy

Ultraviolet (UV) and infrared (IR) absorption and vibrational circular dichroism (VCD) spectroscopy were used to study conformational transitions in the double-stranded poly(rA). poly(rU) and its components-single-stranded poly(rA) and poly(rU) in buffer solution (pH 6.5) with 0.1M Na+ and different Mg2+ and Cd2+ (10(-6) to 10(-2) M) concentrations. Transitions were induced by elevated temperature that changed from 10 up to 96 degrees C. IR absorption and VCD spectra in the base-stretching region were obtained for duplex, triplex, and single-stranded forms of poly(rA) . poly(rU) at [Mg2+],[Cd2+]/[P] = 0.3. For single-stranded polynucleotides, the kind of conformational transition (ordering --> disordering --> compaction, aggregation) is conditioned by the dominating type of Me2+-polymer complex that in turn depends on the ion concentration range. The phase diagram obtained for poly(rA) . poly(rU) has a triple point ([Cd2+] approximately 10(-4)M) at which the helix-coil (2 --> 1) transition is replaced with a disproportion transition 2AU --> A2U + poly(rA) (2 --> 3) and the subsequent destruction of the triple helix (3 --> 1). The 2 --> 1 transitions occur in the narrow temperature interval of 2 degrees -5 degrees . Unlike 2 --> 1 and 3 --> 1 melting, the disproportion 2 --> 3 transition is a slightly cooperative one and observed over a wide temperature range. At [Me2+] approximately 10(-3) M, the temperature interval of A2U stability is not less than 20 degrees C. In the case of Cd2+, it increases with the rise of ion concentration due to the decrease of T(m) (2-->3). The T(m) (3-->1) value is practically unchanged up to [Cd2+] approximately 10(-3)M. Differences between diagrams for Mg(2+) and Cd2+ result from the various kinds of ion binding to poly(rA).poly-(rU) and poly(rA).     

Assignment of fingerprint vibrations in the resonance Raman spectra of rhodopsin, isorhodopsin, and bathorhodopsin: implications for chromophore structure and environment

13C- and 2H-labeled retinal derivatives have been used to assign normal modes in the 1100-1300-cm-1 fingerprint region of the resonance Raman spectra of rhodopsin, isorhodopsin, and bathorhodopsin. On the basis of the 13C shifts, C8-C9 stretching character is assigned at 1217 cm-1 in rhodopsin, at 1206 cm-1 in isorhodopsin, and at 1214 cm-1 in bathorhodopsin. C10-C11 stretching character is localized at 1098 cm-1 in rhodopsin, at 1154 cm-1 in isorhodopsin, and at 1166 cm-1 in bathorhodopsin. C14-C15 stretching character is found at 1190 cm-1 in rhodopsin, at 1206 cm-1 in isorhodopsin, and at 1210 cm-1 in bathorhodopsin. C12-C13 stretching character is much more delocalized, but the characteristic coupling with the C14H rock allows us to assign the "C12-C13 stretch" at approximately 1240 cm-1 in rhodopsin, isorhodopsin, and bathorhodopsin. The insensitivity of the C14-C15 stretching mode to N-deuteriation in all three pigments demonstrates that each contains a trans (anti) protonated Schiff base bond. The relatively high frequency of the C10-C11 mode of bathorhodopsin demonstrates that bathorhodopsin is s-trans about the C10-C11 single bond. This provides strong evidence against the model of bathorhodopsin proposed by Liu and Asato [Liu, R., & Asato, A. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 259], which suggests a C10-C11 s-cis structure. Comparison of the fingerprint modes of rhodopsin (1098, 1190, 1217, and 1239 cm-1) with those of the 11-cis-retinal protonated Schiff base in methanol (1093, 1190, 1217, and 1237 cm-1) shows that the frequencies of the C-C stretching modes are largely unperturbed by protein binding. In particular, the invariance of the C14-C15 stretching mode at 1190 cm-1 does not support the presence of a negative protein charge near C13 in rhodopsin. In contrast, the frequencies of the C8-C9 and C14-C15 stretches of bathorhodopsin and the C10-C11 and C14-C15 stretches of isorhodopsin are significantly altered by protein binding. The implications of these observations for the mechanism of wavelength regulation in visual pigments and energy storage in bathorhodopsin are discussed.     

Redox-induced conformational changes in myoglobin and hemoglobin: electrochemistry and ultraviolet-visible and Fourier transform infrared difference spectroscopy at surface-modified gold electrodes in an ultra-thin-layer spectroelectrochemical cell.

Using suitable surface-modified electrodes, we have developed an electrochemical system which allows a reversible heterogeneous electron transfer at high (approximately 5 mM) protein concentrations between the electrode and myoglobin or hemoglobin in an optically transparent thin-layer electrochemical (OTTLE) cell. With this cell, which is transparent from 190 to 10,000 nm, we have been able to obtain electrochemically-induced Fourier-transform infrared (FTIR) difference spectra of both proteins. Clean protein difference spectra between the redox states were obtained because of the absence of redox mediators in the protein solution. The reduced-minus-oxidized difference spectra are characteristic for each protein and arise from redox-sensitive heme modes as well as from polypeptide backbone and amino acid side chain conformational changes concomitant with the redox transition. The amplitudes of the difference bands, however, are small as compared to the total amide I absorbance, and correspond to approximately 1% (4%) of the reduced-minus-oxidized difference absorbance in the Soret region of myoglobin (hemoglobin) and to less than 0.1% of the total amide I absorbance. Some of the bands in the 1560-1490-cm-1 spectral regions could be assigned to side-chain vibrational modes of aromatic amino acids. In the conformationally sensitive spectral region between 1680 and 1630 cm-1, bands could be attributed to peptide C = O modes because of their small (2-5 cm-1) shift in 2H2O. A similar assignment could be achieved for amide II modes because of their strong shift in 2H2O.(ABSTRACT TRUNCATED AT 250 WORDS)     

The hydrogen-bonding structure in parallel-stranded duplex DNA is reverse Watson-Crick

Raman spectra of the parallel-stranded duplex formed from the deoxyoligonucleotides 5'-d-[(A)10TAATTTTAAATATTT]-3' (D1) and 5'-d[(T)10ATTAAAATTTATAAA]-3' (D2) in H2O and D2O have been acquired. The spectra of the parallel-stranded DNA are then compared to the spectra of the antiparallel double helix formed from the deoxyoligonucleotides D1 and 5'-d(AAATATTTAAAATTA-(T)10]-3' (D3). The Raman spectra of the antiparallel-stranded (aps) duplex are reminiscent of the spectra of poly[d(A)].poly[d(T)] and a B-form structure similar to that adopted by the homopolymer duplex is assigned to the antiparallel double helix. The spectra of the parallel-stranded (ps) and antiparallel-stranded duplexes differ significantly due to changes in helical organization, i.e., base pairing, base stacking, and backbone conformation. Large changes observed in the carbonyl stretching region (1600-1700 cm-1) implicate the involvement of the C(2) carbonyl of thymine in base pairing. The interaction of adenine with the C(2) carbonyl of thymine is consistent wtih formation of reverse Watson-Crick base pairing in parallel-stranded DNA. Phosphate-furanose vibrations similar to those observed for B-form DNA of heterogenous sequence and high A,T content are observed at 843 and 1092 cm-1 in the spectra of the parallel-stranded duplex. The 843-cm-1 band is due to the presence of a sizable population of furanose rings in the C2'-endo conformation. Significant changes observed in the regions from 1150 to 1250 cm-1 and from 1340 to 1400 cm-1 in the spectra of the parallel-stranded duplex are attributed to variations in backbone torsional and glycosidic angles and base stacking.     

Intrapleural fluid movements described by a porous flow model.

We injected technetium-labeled albumin (at a concentration similar to that of the pleural fluid) in the costal region of anesthetized dogs (n = 13) either breathing spontaneously or apneic. The decay rate of labeled activity at the injection site was studied with a gamma camera placed either in the anteroposterior (AP) or laterolateral (LL) projection. In breathing animals (respiratory frequency approximately 10 cycles/min), 10 min after the injection the activity decreased by approximately 50% on AP and approximately 20% on LL imaging; in apneic animals the corresponding decrease in activity was reduced to approximately 15 and approximately 3%, respectively. We considered label translocation from AP and LL imaging as a result of bulk flows of liquid along the costomediastinal and gravity-dependent direction, respectively. We related intrapleural flows to the hydraulic pressure gradients existing along these two directions and to the geometry of the pleural space. The pleural space was considered as a porous medium partially occupied by the mesh of microvilli protruding from mesothelial cells. Solution of the Kozeny-Carman equation for the observed flow velocities and pressure gradients yielded a mean hydraulic radius of the pathways followed by the liquid ranging from 2 to 4 microns. The hydraulic resistivity of the pleural space was estimated at approximately 8.5 x 10(5) dyn.s.cm-4, five orders of magnitude lower than that of interstitial tissue.     

Binding of E.coli lac repressor to non-operator DNA*

It is shown by melting profile analysis of lac repressor-DNA complexes that repressor binds tightly and preferentially (relative to single-stranded DNA) to double-stranded non-operator DNA. This binding stabilizes the DNA against melting and the repressor against thermal denaturation. Analysis of the extent of stabilization and the rate of dissociation of repressor from non-operator DNA as a function of sodium ion concentration shows, in confirmation of other studies,(3,4) that the binding constant (K(RD)) is very ionic strength dependent; K(RD) increases from approximately 10(6) M(-1) at approximately 0.1 M Na(+) to values in excess of 10(10) M(-1) at 0.002 M Na(+). Repressor bound to non-operator DNA is not further stabilized against thermal denaturation by inducer binding, indicating that the inducer and DNA binding sites probably represent separately stabilized local conformations. Transfer melting experiments are used to measure the rate of dissociation of repressor from operator DNA. These experiments show that most of the ionic strength dependence of the binding constant is in the dissociation process; the estimated dissociation rate constant decreases from greater than 10(-1) sec(-1) at [Na(+)] >/= 0.02 M to less than 10(-4) sec(-1) at [Na(+)] 相似文献   

Predicted role of particular phonons in the strand separation melting of the DNA double helix

The frequency-dependent vibrational fluctuation of the hydrogen bonds around a nucleation defect for the strand separation melting of a DNA polymer poly(dG)-poly(dC) is studied using a modified self-consistent phonon theory. There are two critical frequency bands around the defect at 340 K which is near the temperature at which the hydrogen bonds in neighboring cells melt. The first band is between 60 cm-1 and 120 cm-1 which is essential for the melting proceeding in +z direction(3'----5' in the G backbone). The second is the band under 20 cm-1 which is important for the melting proceeding in -z direction.