Showing papers on "Circular dichroism published in 1993"

Spontaneous assembly of a self-complementary oligopeptide to form a stable macroscopic membrane.

Abstract: A 16-residue peptide [(Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys)2] has a characteristic beta-sheet circular dichroism spectrum in water. Upon the addition of salt, the peptide spontaneously assembles to form a macroscopic membrane. The membrane does not dissolve in heat or in acidic or alkaline solutions, nor does it dissolve upon addition of guanidine hydrochloride, SDS/urea, or a variety of proteolytic enzymes. Scanning EM reveals a network of interwoven filaments approximately 10-20 nm in diameter. An important component of the stability is probably due to formation of complementary ionic bonds between glutamic and lysine side chains. This phenomenon may be a model for studying the insoluble peptides found in certain neurological disorders. It may also have implications for biomaterials and origin-of-life research.

Tris(phenanthroline)ruthenium(II) enantiomer interactions with DNA: Mode and specificity of binding

Abstract: Absorbance and fluorescence methods, circular dichroism, UV melting experiments, viscosity, and competition dialysis were used to study the interaction of delta and lambda tris(phenanthroline)ruthenium(II) with DNA. The results of these studies indicated that both isomers bind to DNA by a single mode. The two isomers differ, however, in their effect on the hydrodynamic properties of DNA as measured by viscosity and, therefore, probably differ in their individual binding modes. The optical properties of the fully bound compounds differ from those of the free, but the perturbations of their visible absorbance and fluorescence emission spectra are modest when compared to changes observed for other DNA binding compounds. Binding of both isomers to DNA was found to be weak (in comparison to proven intercalators), with binding constants on the order of 10(4) M-1 determined for their binding to calf thymus DNA. A small, positive enthalpy was found for the binding of each isomer to DNA, suggesting that binding is entropically driven. Both isomers increased the melting temperature of DNA, with little quantitative difference between the two. A modest base specificity was found for each isomer, with the delta isomer preferentially binding to GC base pairs, and the lambda isomer preferentially binding to AT base pairs. Competition dialysis was used to examine the preference of delta and lambda Ru for right-handed B DNA and left-handed Z DNA. Neither isomer exhibits significant selectivity for these radically different DNA secondary structures.

Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network

Abstract: An optimized self-organizing map algorithm has been used to obtain protein topological (proteinotopic) maps. A neural network is able to arrange a set of proteins depending on their ultraviolet circular dichroism spectra in a completely unsupervised learning process. Analysis of the proteinotopic map reveals that the network extracts the main secondary structure features even with the small number of examples used. Some methods to use the proteinotopic map for protein secondary structure prediction are tested showing a good performance in the 200-240 nm wavelength range that is likely to increase as new protein structures are known.

Porphyrin Assemblies On DNA As Studied By A Resonance Light-Scattering Technique

Abstract: Under appropriate conditions of concentration and ionic strength, trans-bis(N-methylpyridinium-4-yl)-diphenylporphine and its copper(II) derivative produce very large, bisignate circular dichroism signals in the Soret region when bound to DNA. The processes leading to these signals are shown to be highly cooperative. A new light-scattering experimental approach is described for detecting such extended aggregates of chomophores in which the radiation used is within an absorption maximum, specifically in the porphyrin Soret absorption region. Results from such resonance light-scattering experiments confirm that extended porphyrin aggregates form on the DNA, which, in contrast, remains dispersed

Formation of a molten globule intermediate early in the kinetic folding pathway of apomyoglobin

Abstract: Hydrogen exchange pulse labeling and stopped-flow circular dichroism were used to establish that the structure of the earliest detectable intermediate formed during refolding of apomyoglobin corresponds closely to that of a previously characterized equilibrium molten globule. This compact, cooperatively folded intermediate was formed in less than 5 milliseconds and contained stable, hydrogen-bonded secondary structure localized in the A, G, and H helices and part of the B helix. The remainder of the B helix folded on a much slower time scale, followed by the C and E helices and the CD loop. The data indicate that a molten globule intermediate was formed on the kinetic folding pathway.

J‐aggregate formation of a water‐soluble porphyrin in acidic aqueous media

Abstract: J aggregate of a water‐soluble porphyrin, 5,10,15,20‐tetra(4‐sulfophenyl)porphyrin (H2TPPS44−), formed in acidified aqueous solutions, exhibits sharp and intense absorption bands at 491 and 707 nm. These characteristic transitions, J bands, are of linear oscillators polarized in the long axis of rodlike aggregate. The molecules in the aggregate stack so as to lift the degeneracy of the porphyrin planar oscillator excited states. Measurements of flow‐induced linear dichroism, circular dichroism, magnetic circular dichroism, as well as polarized fluorescence excitation spectra provide evidence not only of linear oscillator character of the intense J band at 491 nm, but also of presence of another diffuse absorption band around at 420 nm polarized in the short axis of the aggregate, which is the counterpart of the 491 nm band of porphyrin Soret origin. Extrinsic circular dichroism is induced upon addition of L‐tartaric acid or by mechanical swirling flow in the period of aggregate growth. Resonance Raman spe...

GC base sequence recognition by oligo(imidazolecarboxamide) and C-terminus-modified analogues of distamycin deduced from circular dichroism, proton nuclear magnetic resonance, and methidiumpropylethylenediaminetetraacetate-iron(II) footprinting studies

Abstract: The DNA binding properties of a series of imidazole-containing and C-terminus-modified analogues 4-7 of distamycin are described. These analogues contain one to four imidazole units, respectively. Data from the ethidium displacement assay showed that these compounds bind in the minor groove of DNA, with the relative order of binding constants of 6 (Im3) > 7 (Im4) > 5 (Im2) > 4 (Im1). The reduced binding constants of these compounds for poly(dA-dT) relative to distamycin, while they still interact strongly with poly(dG-dC), provided evidence of GC sequence acceptance. The preferences for GC-rich sequences by these compounds were established from a combination of circular dichroism (CD) titration, proton nuclear magnetic resonance (1H-NMR), and methidiumpropylethylenediaminetetraacetate-iron(II) [MPE.Fe-(II)] footprinting studies. In the CD studies, these compounds produced significantly larger DNA-induced ligand bands with poly(dG-dC) than poly(dA-dT) at comparable ligand concentrations. 1H-NMR studies of the binding of 5 to d-[CATGGCCATG]2 provided further evidence of the recognition of GC sequences by these compounds, and suggested that the ligand was located on the underlined sequence in the minor groove with the C-terminus oriented over the T residue. MPE footprinting studies on a GC-rich BamHI/SalI fragment of pBR322 provided unambiguous evidence for the GC sequence selectivity for some of these compounds. Compounds 4 and 7 produced poor footprints on the gels; however, analogues 5 and 6 gave strong footprints.(ABSTRACT TRUNCATED AT 250 WORDS)

Aromatic side-chain contribution to far-ultraviolet circular dichroism of helical peptides and its effect on measurement of helix propensities.

Abstract: Peptides of the sequence Ac-XKAAAAKAAAAKAAAAK-amide, where X is Tyr, Trp, or Ala, produce circular dichroism spectra that are typical of the alpha-helix; there are, however, significant differences between the Tyr, Trp, or Ala peptides in the magnitudes of the far-ultraviolet bands. A tyrosine or tryptophan residue is needed in each peptide in order to measure accurately the peptide concentration and the mean residue ellipticity. The N- or C-terminal position is chosen because helix fraying is greatest at each end and the Tyr or Trp residue should influence the helix content of the peptide least at these positions. Amide proton exchange measurements by proton nuclear magnetic resonance spectroscopy indicate that the Tyr, Trp, and Ala peptides possess similar amounts of H-bonded secondary structure. Comparison of the far-ultraviolet circular dichroism and absorption spectra of these peptides suggests that the differences in circular dichroism arise in each case from an induced aromatic circular dichroism band, which is positive for Tyr and negative for Trp. Insertion of one to three Gly residues between the aromatic residue and the rest of the helical sequence reduces the induced aromatic band to insignificant levels. Using this procedure of inserting Gly residues between the Tyr and the rest of the helical sequence, we remeasured the helix propensity of Gly. We find that the Ala:Gly ratio of helix propensities is 40, as opposed to our previous estimate of 100 determined using the Tyr peptide without considering the aromatic contribution of Tyr in the analysis [Chakrabartty, A., Schellman, J. A., & Baldwin, R. L. (1991) Nature 351, 586-588].

Circular dichroism spectroscopy at interfaces: a surface second harmonic generation study

Abstract: A new technique which links the phenomenon of surface second harmonic generation (SHG) with circular dichroism (CD) spectroscopy is introduced. SHG spectra of chiral molecules (2,2'-dihydroxy-1,1'-binaphthyl) adsorbed at the air/water interface are dependent on the helicity of the incident circularly polarized beam. Moreover, the magnitude of the circular dichroic effect is 10 3 times greater than that observed in ordinary CD spectroscopy. Several factors including molecular orientation at the surface are considered as possible explanations for this enhancement

A partially folded state of hen egg white lysozyme in trifluoroethanol: structural characterization and implications for protein folding.

Abstract: The effect of 2,2,2-trifluoroethanol (TFE) on the solution conformation of hen egg white lysozyme has been investigated using circular dichroism (CD) and 1H nuclear magnetic resonance (NMR) spectroscopy. Addition of TFE to lysozyme at pH 2.0, 27 degrees C, up to a concentration of 15% (v/v) induces only slight changes in the NMR spectrum. However, above this concentration a cooperative transition to a new but partially structured state of the protein is observed. This state shows no structural cooperativity against further denaturation and is characterized by an ellipticity in the far-UV CD greater than that of the native protein. Near-UV CD intensity is dramatically reduced compared with that of the native state, and 1H NMR studies indicate that side-chain interactions are substantially averaged in this denatured state. Solvent proton/deuterium exchange rates for 66 amide hydrogens were measured site-specifically by a combination of amide trapping experiments and 2D 1H NMR. Significant protection from exchange occurs for about 25 backbone amides, the majority of which are located in regions of the protein that are helical in the native enzyme. By contrast, amides located in a second region of the native protein which contains a beta-sheet and one 3(10)-helix as well as a long loop show little protection. This pattern of protection resembles that found in the stable molten globule state of alpha-lactalbumin and in an early kinetic intermediate detected in the refolding of hen lysozyme.

Oligodeoxynucleotides containing 2'-O-modified adenosine: Synthesis and effects on stability of DNA:RNA duplexes

Abstract: Hybridization thermodynamics were compared for oligonucleotide sequences containing 2'-fluoro dA, 2'-O-methyl A, 2'-O-ethyl A, 2'-O-propyl A, 2'-O-butyl A, 2'-O-pentyl A, 2'-O-nonyl A, 2'-O-allyl A, and 2'-O-benzyl A in place of deoxyadenosine. Although the effect of 2'-modified adenosine on duplex stability is sequence dependent, a clear trend is apparent. For six sequences containing a few 2'-modified adenosines in a background of unmodified deoxynucleotides, the average delta TM per substitution ranged from +1.3 degrees C for 2'-fluoro dA to -2.0 degrees C for 2'-O-nonyl A. For the 2'-O-alkyl series, the average delta TM per substitution correlates well with size of the substituent; the order of stability is 2'-O-methyl A > 2'-O-ethyl A > 2'-O-propyl A > 2'-O-butyl A > 2'-O-pentyl A > 2'-O-nonyl A. This correlation also extends to 2'-fluoro dA, 2'-O-allyl A, and 2'-O-benzyl A if chain length is measured by number of carbon atoms. When examined in the background of 2'-O-methyl ribonucleotides, all 2'-modified adenosines with a substituent no larger than 2'-O-pentyl stabilized the duplex nearly 2 degrees C per substitution compared to unmodified dA. These thermodynamic results and CD spectra of modified and unmodified hybrids support a model of DNA:RNA hybrids in which the geometry is between that of B-form and A-form.

Folding and stability of a tryptophan-containing mutant of ubiquitin.

Abstract: To provide a fluorescence probe for equilibrium and kinetic folding studies on ubiquitin, cassette mutagenesis in an Escherichia coli expression plasmid was used to replace the largely buried Phe 45 by a tryptophan. Under native conditions, the tryptophan fluorescence spectrum of this F45W mutant exhibits a blue-shifted emission maximum at 336 nm indicative of a largely solvent-shielded tryptophan environment. In contrast, the unfolded protein in 6 M guanidine hydrochloride (GuHCl) shows a 4-fold more intense emission band at 353 nm matching that of free tryptophan. The two-dimensional 1H NMR spectrum of F45W ubiquitin was assigned by comparison with published assignments of the wild type. The mutation results in only limited chemical shift changes for residues in the immediate vicinity of residue 45. The structural similarity of F45W with wild-type ubiquitin was confirmed by a preliminary analysis of the nuclear Overhauser spectrum. NMR and circular dichroism measurements of the reversible GuHCl-induced unfolding transition show that the F45W mutation lowers the stability of the folded ubiquitin structure by less than 0.4 kcal/mol. The biological activity of the mutant was found to be indistinguishable from that of wild-type in terms of its reaction with the ubiquitin activating enzyme E1 and an in vitro assay of ATP-dependent protein degradation. The kinetics of folding and unfolding of F45W ubiquitin was studied at two temperatures (8 and 25 degrees C) in a series of fluorescence-detected stopped-flow measurements over a wide range of GuHCl concentrations (0.5-6 M). The measurements at 25 degrees C are consistent with a two-state model with strongly denaturant-dependent folding and unfolding rates above about 2 M GuHCl. However, at lower denaturant concentrations, the rate of the major folding phase becomes GuHCl-independent, and up to 60% of the total fluorescence change occurs during the 2-ms dead time of the stopped-flow measurement. These observations provide clear evidence for the formation of an early folding intermediate during the first few milliseconds of refolding with a partially developed hydrophobic core involving Trp 45. The sigmoid denaturant dependence of the initial amplitude with an apparent midpoint of 1.3 M GuHCl suggests the presence of a discrete state that is destabilized at higher denaturant concentrations. In contrast, there is no evidence for an early intermediate in the folding kinetics at 8 degrees C. The destabilization of the intermediate at low temperature is consistent with a collapsed state stabilized primarily by hydrophobic interactions.

Thermal stability and conformational transitions of scrapie amyloid (prion) protein correlate with infectivity

Abstract: The scrapie amyloid (prion) protein (PrP27-30) is the protease-resistant core of a larger precursor (PrPSc) and a component of the infectious scrapie agent; the potential to form amyloid is a result of posttranslational event or conformational abnormality. The conformation, heat stability, and solvent-induced conformational transitions of PrP27-30 were studied in the solid state in films by CD spectroscopy and correlated with the infectivity of rehydrated and equilibrated films. The exposure of PrP27-30 in films to 60 degrees C, 100 degrees C, and 132 degrees C for 30 min did not change the beta-sheet secondary structure; the infectivity slightly diminished at 132 degrees C and correlated with a decreased solubility of PrP27-30 in sodium dodecyl sulfate (SDS), probably due to cross-linking. Exposing PrP27-30 films to formic acid (FA), trifluoroacetic acid (TFA), trifluoroethanol (TFE), hexafluoro-2-propanol (HFIP), and SDS transformed the amide CD band, diminished the mean residue ellipticity of aromatic bands, and inactivated scrapie infectivity. The convex constraint algorithm (CAA) deconvolution of the CD spectra of the solvent-exposed and rehydrated solid state PrP27-30 identified five common spectral components. The loss of infectivity quantitatively correlated with a decreasing proportion of native, beta-pleated sheet-like secondary structure component, an increasing amount of alpha-helical component, and an increasingly disordered tertiary structure. The results demonstrate the unusual thermal stability of the beta-sheet secondary structure of PrP27-30 protein in the solid state. The conformational perturbations of PrP27-30 parallel the changes in infectivity and suggest that the beta-sheet structure plays a key role in the physical stability of scrapie amyloid and in the ability to propagate and replicate scrapie.

Attempts to mimic docking processes of the immune-system - recognition-induced formation of protein multilayers

Abstract: The assemblage of protein multilayers induced by molecular recognition, as seen, for example, in the immune cascade, has been mimicked by using streptavidin as a docking matrix. For these experiments, this protein matrix was organized on liposomes, monolayers at the air-water interface, and self-assembled layers on gold, all three containing biotin lipids. The docking of streptavidin to biotin at liposomal surfaces was confirmed by circular dichroism. Mixed double and triple layers of streptavidin, concanavalin A, antibody Fab fragments, and hormones are prepared at the air-water interface and on gold surfaces and were characterized by fluorescence microscopy and plasmon spectroscopy. With the use of biotin analogs that have lower binding constants it has been possible to achieve multiple formation and competitive replacement of the oriented protein assemblages.

Pigments induce folding of light-harvesting chlorophyll a/b-binding protein.

Abstract: The conformational behaviour of the light-harvesting chlorophyll a/b-binding protein (LHCP), the apoprotein of the major light-harvesting complex (LHCII) of photosystem II in plants, has been studied. According to the circular dichroism in the ultraviolet range measured with isolated LHCII, the protein in the complex adopts a folded structure with a high content of alpha helix (about 60%), whereas the non-pigmented, solubilized protein has a less ordered structure (about 20% alpha helix). LHCP-pigment complexes that have been reconstituted from the overexpressed protein and isolated pigments in the presence of detergents display a protein CD signal similar to that of authentic LHCII, indicating that LHCP folds into the native structure during the reconstitution procedure. Renaturation of LHCP in these experiments is dependent on the presence of pigments and the formation of stable LHCP-pigment complexes. Pigment-induced engagement of LHCP in a compact structure has also been shown by two additional experimental approaches. (a) Upon complex formation, LHCP or its precursor (pLHCP) becomes resistant to trypsin digestion with the exception of an N-terminal segment of the protein; the same protection of LHCP is known to occur in intact thylakoids. (b) Pigment binding renders a cysteine residue within the N-proximal hydrophobic domain of the protein as well as a newly introduced cysteine four amino acid positions from the C terminus inaccessible to modification with a sulfhydryl-specific label whereas the N terminus stays susceptible to specific labelling. These observations support the notion that only the N terminus protrudes from a compact protein-pigment structure in LHCII. The fact that the major part of LHCP is trypsin-resistant in pigmented complexes reconstituted in the absence of a membrane or even lipids justifies caution in using protection against trypsin as a criterion for the integration of LHCP into the thylakoid membrane.

Peptide models of protein folding initiation sites. 1. Secondary structure formation by peptides corresponding to the G- and H-helices of myoglobin.

Abstract: Myoglobin has been extensively studied as a model system for protein folding in vitro. As part of an ongoing study of myoglobin folding, we have synthesized a series of peptide fragments corresponding to portions of the sequence of the sperm whale protein. The conformational preferences of these peptides have been investigated by circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution. In this paper we describe the folding propensities of two peptides (Mb-G and Mb-H), corresponding to the G- and H-helix segments of the myoglobin sequence. The Mb-G peptide shows evidence of a very small population of helical conformations in aqueous solution, both by CD and NMR. By contrast, the monomeric Mb-H peptide is found by CD to adopt a significant population (ca. 30%) of ordered helix and by NMR to populate helical conformations in rapid dynamic equilibrium with unfolded states. The Mb-H peptide undergoes a well-characterized, concentration-dependent monomer-tetramer equilibrium. At peptide concentrations greater than 1 mM there is an increase in the population of helix, to approximately 85% according to the CD spectrum, through self-association to form a tetramer. Both medium-range NOE connectivities and a CD spectrum characteristic of ordered helix are observed at low peptide concentrations, establishing that helical conformations are present in the monomeric state of Mb-H. The relative helicity at various sites throughout the Mb-H peptide has been estimated using a novel method for assessing the distribution of helical populations based on the relative magnitudes of medium-range d alpha beta (i,i+3) NOE connectivities. The population of ordered helix is seen to be highest in the center of the peptide sequence; the ends of the peptide show evidence of pronounced fraying.

CH-.pi. interaction as an important driving force of host-guest complexation in apolar organic media. Binding of monools and acetylated compounds to resorcinol cyclic tetramer as studied by proton NMR and circular dichroism spectroscopy

Abstract: The CH-π interaction plays an important role in the complexation involving resorcinol cyclic tetramer 1 in chloroform. Thus, host I binds simple monools 4-10 via a cooperation of CH-π and hydrogen-bonding interactions, where the binding constants at 298 K (up to 13 M -1 ) increase with increasing chain lengths (from ethyl through propyl to butyl) as well as branching in the aliphatic moiety of the guests. In addition, the largest complexation-induced 1 H NMR upfield shift for a bound guest occurs at the terminal methyl group, which must therefore be deeply incorporated in the aromatic cavity of the host

Conformational behavior of Escherichia coli OmpA signal peptides in membrane mimetic environments.

Abstract: Nuclear magnetic resonance and circular dichroism (CD) studies of isolated peptides corresponding to WT and mutant OmpA signal sequences are reported; all of the peptides adopt substantial amounts of alpha-helical structure both in 1:1 (v/v) trifluoroethanol (TFE)/water and in sodium dodecyl sulfate (SDS) micelles. In TFE/water, the helix begins after the positively charged N-terminal residues and is most stable in the hydrophobic core, which correlates with results obtained previously for other signal sequences. The helix is weaker between the hydrophobic core and the C-terminus; such a break in the helix appears to be common to other signal peptides studied previously and could be of functional importance. No clear correlation could be established between the helicity of the peptides in TFE/water and their in vivo activities. All the peptides have a higher alpha-helix content in SDS than in TFE/water, and there is a good correlation between helix content in SDS and in vivo activity. Helicity in SDS for the functional peptides increases both at the N-terminus and in the hydrophobic core, and is driven by a strong association of the core with the hydrophobic chains of the detergent. The extension of the helix toward the N-terminus may be a result of neutralization of the N-terminal positive charges by the headgroups of the micelles, which removes unfavorable electrostatic interactions with the helix dipole. All these comparisons were facilitated by the use of upfield shifts of Halpha protons in helical regions relative to random coil chemical shifts, which also yielded estimates of helical content that correlated well with the CD results.

Thermodynamic characterization of the structural stability of the coiled-coil region of the bZIP transcription factor GCN4.

Abstract: The thermal stability of a 56 amino acid fragment of GCN4 has been studied by high-sensitivity differential scanning calorimetry and circular dichroism spectroscopy. This fragment contains the leucine zipper and part of the basic region. The thermal unfolding of GCN4-56 is a reversible process and can be well represented by a reaction of the form N2 2U, indicating that the unfolding of the leucine zipper is a two-state process in which the helices are only stable when they are in the coiled-coil conformation. As expected, the transition temperature is concentration dependent. At pH 7.06 and a protein concentration of 5 x 10(-4) M the transition temperature is close to 70 degrees C while at 5 x 10(-6) M it is close to 50 degrees C. The enthalpy change for unfolding is 31.5 kcal mol-1 at 70 degrees C. Since the isolated helices are unstable, interactions at the interface between the two helices play a key role in the stabilization of the native dimer. These interactions primarily involve the burial of apolar surface from the solvent (hydrophobic effect) and electrostatic interactions. Structural thermodynamic calculations have permitted a dissection of the magnitude of the various contributions to the total Gibbs free energy of stabilization.

Kinetics of folding of the all-beta sheet protein interleukin-1 beta

Abstract: The folding of the all-beta sheet protein, interleukin-1 beta, was studied with nuclear magnetic resonance (NMR) spectroscopy, circular dichroism, and fluorescence. Ninety percent of the beta structure present in the native protein, as monitored by far-ultraviolet circular dichroism, was attained within 25 milliseconds, correlating with the first kinetic phase determined by tryptophan and 1-anilinonaphthalene-8-sulfonate fluorescence. In contrast, formation of stable native secondary structure, as measured by quenched-flow deuterium-hydrogen exchange experiments, began after only 1 second. Results from the NMR experiments indicated the formation of at least two intermediates with half-lives of 0.7 to 1.5 and 15 to 25 seconds. The final stabilization of the secondary structure, however, occurs on a time scale much greater than 25 seconds. These results differ from previous results on mixed alpha helix-beta sheet proteins in which both the alpha helices and beta sheets were stabilized very rapidly (less than 10 to 20 milliseconds).

Circular Dichroism Studies of Barnase and Its Mutants: Characterization of the Contribution of Aromatic Side Chains

Abstract: The circular dichroism spectrum of barnase has been analyzed by examining the spectra of a series of mutants in which every single aromatic residue has been replaced. The spectrum of wild-type barnase is quite atypical for a protein of the alpha + beta class, with very low intensities and a minimum in the far-UV at 231 nm. The minimum at 231 nm is associated with the presence of Trp-94. Many other mutations involving aromatic residues have an effect on the spectral features in the far-UV. The major features in the near-UV spectra arise from essentially additive contributions of the three tryptophan residues Trp-35, Trp-71, and Trp-94. Tyrosine contributions are less prominent, with Tyr-78 and Tyr-97 contributing the most to the CD spectrum. The close charge-aromatic interaction between Trp-94 and His-18, which is important for the fluorescence properties of the protein, contributes little to the CD spectrum, as does the close aromatic-aromatic interaction between Tyr-13 and Tyr-17. However, the observed near-UV spectrum of wild-type barnase could not be simulated by the sum of the contributions of aromatic residues defined by difference spectra of protein variants carrying aromatic residues. Aromatic residues play an important role in determining the circular dichroism spectrum of proteins not only in the near-UV but also in the far-UV region.

Structure of (rGGCGAGCC)2 in solution from NMR and restrained molecular dynamics

Abstract: The duplex (rGGCGAGCC)2 contains tandem G x A mismatches--a common motif in the secondary structures of biological RNAs. The three-dimensional structure of (rGGCGAGCC)2 was derived using molecular dynamics and energy minimization with NMR-derived restraints for 78 interproton distances (per strand), 18 hydrogen bonds for the six Watson-Crick G x C pairs, and 26 dihedral angles (per strand). The G x A mismatch structures are similar to those observed in a DNA duplex [Li, Y., Zon, G., & Wilson, W. D. (1991) Proc. Natl. Acad. Sci. U.S.A. 80, 26-30] and an RNA hairpin [Heus, H. A., & Pardi, A. (1991) Science 253, 191-193], with hydrogen bonds from guanine 2-amino and N3 to adenine N7 and 6-amino, respectively. The other G 2-amino and A 6-amino protons are within hydrogen-bonding distance of a phosphate oxygen and 2'-oxygen, respectively. Strong interstrand A-A and G-G stacking is observed between the G x A mismatches. This contrasts with the poor stacking observed between the G x A mismatches and closing G x C base pairs. The stems are basically A-form with all bases in the anti conformation and all nonterminal sugars in the C3'-endo conformation. The structure rationalizes previous thermodynamic, circular dichroism, and imino proton NMR results and suggests tandem G x A mismatches in RNA may provide a contact site for tertiary interactions.

SecA, the peripheral subunit of the Escherichia coli precursor protein translocase, is functional as a dimer.

Abstract: SecA, the peripheral ATPase domain of the Escherichia coli precursor protein translocase, was denatured in 6 M guanidine hydrochloride. Circular dichroism and intrinsic tryptophan fluorescence spectra revealed that the protein is transformed into a random-coil configuration. Upon dilution of the chaotropic agent, SecA refolds into its native, functional conformation as a homodimer. As structural criteria, the native dimeric state was assayed by size-exclusion chromatography, chemical cross-linking, tryptophan fluorescence, and circular dichroism. Functional SecA heterodimers were formed of which the individual subunits were tagged with fluorescent dyes to allow measurements of the association state of the monomers by resonance energy transfer using steady-state and time-resolved fluorescence spectroscopy. SecA retained its dimeric structure during translocation, while energy transfer was abolished only by denaturation. The ''half-of-the-sites activity'' was investigated by constructing heterodimers formed from native and 8-azido-ATP-inactivated SecA. Heterodimers have lost the ability to support translocation of the precursor protein proOmpA in an in vitro translocation system. It is concluded that the dimeric structure is maintained during translocation and required for functionality.

The DNA strand in DNA.RNA hybrid duplexes is neither B-form nor A-form in solution.

Abstract: The structure of the DNA.RNA hybrid (GTCACATG).(caugugac), where lowercase letters designate RNA residues, has been determined on the basis of J-coupling analysis and 2D-NOE studies. The central hexamer in this sequence has been previously studied [Reid, D. G., Salisbury, S. A., Brown, T., Williams, D. H., Vasseur, J.-J., Rayner, B., & Imabach, J.-L. (1983) Eur. J. Biochem. 135, 307-314] via one-dimensional NOE methods and circular dichroism studies. Contrary to their results, we find that this duplex does not assume a B-form conformation in solution. Instead, the RNA residues retain their C3'-endo (A-form) conformation, as indicated by the absence of H1'-H2' couplings and by strong H6/H8 to (n-1) H2'NOEs. The sugars of the DNA residues, on the other hand, do not assume an A-form (or a B-form) conformation but an intermediate conformation in the O4'-endo range (P approximately 72-110 degrees), as indicated by the presence of strong H1'-H4' NOEs, medium-strength H2"-H3' COSY cross peaks, strong H3'-H4' DQF-COSY cross peaks, and H1'-H2' coupling constants that are of approximately the same magnitude as the H1'-H2" coupling constants. These results suggest that the RNA strand not only retains its N-type structure but also exerts an influence on the conformation of the DNA strand. Our results provide strong evidence that DNA.RNA hybrid duplexes do not assume an all-C2'-endo B-type conformation; neither do they assume an all-C3'-endo A-type conformation in solution. Furthermore, although not the main focus of this study, a comparison of the longitudinal relaxation times of the DNA and RNA residues indicates the need for extended relaxation delays in two-dimensional NMR spectra of hybrid duplexes, as has been previously observed for DNA.RNA chimeric duplexes (Wang, A. C., Kim, S.-G., Chou, S.-H., Orban, J., Flynn, P., & Reid, B. R. (1992) Biochemistry 31, 3940-3946).

Bacteriorhodopsin encapsulated in transparent sol-gel glass: a new biomaterial

Abstract: The photosynthetic membrane protein of bacteriorhodopsin (bR) was encapsulated in an optically transparent and porous silica matrix using a modified sol-gel procedure. The absorption spectra and the kinetics of the photocycle characteristic of the proton pumping function of bR were studied systematically throughout the different stages of the glass formation process. This new biomaterial was characterized by means of its optical absorption, circular dichroism (CD), and Raman spectra; its photocycle kinetics; the characteristic activation parameters of its photocycle; and its deionization and cation regeneration properties. The global trimeric bR structure, the local structure of the retinal chromophore, and the proton pumping function of bR were not affected by the encapsulation process. It was also found that the bR glass formed allowed transport of small ions such as Ca[sup 2+] in to and out of the glass medium, and those ions were found to affect the properties of the protein just as they do in aqueous suspensions. The bR protein was found to bleach if delipidated prior to encapsulation. These observations as well as analysis of the CD spectrum suggest that the bR is encapsulated along with its membrane lipids. These results taken together suggest that this optically transparent system offersmore » a potentialy useful new bR-containing material for optical imaging and optically based ion-sensoring devices as developed and proposed for other bR-based systems. 24 refs., 8 figs., 2 tabs.« less