Studies on the conformational properties of CP-10(42-55), the hinge region of CP-10, using circular dichroism and RP-HPLC.

The conformational properties of CP-10(42-55), a peptide corresponding to the hinge region of CP-10, were investigated using circular dichroism spectroscopy and reverse-phase high-performance liquid chromatography (RP-HPLC). The circular dichroism studies indicated that CP-10(42-55) formed considerable secondary structure in the presence of hydrophobic solution environments including 50% acetonitrile, 50% trifluoroethanol and 200 mM sodium dodecyl sulfate, which comprised a mixture of alpha-helix and beta-sheet. The effect of temperature on the conformation of CP-10(42-55) was investigated between 5 and 40 degrees C, with very small changes in the spectra being observed. RP-HPLC was then used to investigate the effect of temperature on the conformation of CP-10(42-55) in the presence of a hydrophobic surface. Using a C18-adsorbent, CP-10(42-55) exhibited a conformational transition at 25 degrees C, which was associated with an increase in the chromatographic contact area and the binding affinity of the peptide for the stationary phase. In addition, near-planar bandbroadening behaviour indicated that conformational species interconverted with rapid rate constants compared with the chromatographic time scale. These results indicated that the conformational change at 25 degrees C in the RP-HPLC system most likely corresponds to the unfolding of an alpha-helical and/or beta-sheet structure to an extended coil structure. Therefore, the strong chemotactic properties of this peptide may be attributed to its ability to form considerable secondary structure in the presence of a hydrophobic environment.     

Conformational and dynamic differences between N. meningitidis serogroup B and C polysaccharides, using n.m.r. spectroscopy and molecular mechanics calculations

1H-N.m.r. spectroscopy has been used to determine the conformation in aqueous solution of the sialic acid residues of the N. meningitidis serogroup B and non-O-acetylated (O-Ac-)-C polysaccharides, and of N-acetylneuraminic acid (NeuNAc). In all cases, the sugar adopts the 2C5 conformation. The side-chain of NeuNAc adopts a conformation such that H-7 and H-8 are approximately anti-periplanar. This conformation is also found in the (O-Ac-)-C polysaccharide, whereas H-7 and H-8 are gauche in the B polysaccharide. Molecular mechanics calculations have been used to probe the conformational preferences of the variously linked sialic acid residues, and the results are in general agreement with those based on the 1H-n.m.r. data. The 13C-n.m.r. spin-lattice relaxation-times have been interpreted in terms of the molecular dynamics of the B and (O-Ac-)-C polysaccharides. Molecular correlation times have been calculated and details of internal rotational or segmental motion elucidated. The C polysaccharide is characterised by internal or segmental motion in the C-7 to C-9 side-chain of the sialic acid repeating-unit, whereas the B polysaccharide has little or no such movement and tumbles in solution as a rigid species with internal rotation of only the pendant C-9 group. The conformational differences suggest a substantially different three-dimensional structure in solution for these polysaccharides.     

Structure investigation of amphiphilic cyclopeptides in isotropic and anisotropic environments-A model study simulating peptide-membrane interactions.

It has been proposed that the membrane allows a much more efficient binding of certain small or medium-sized amphiphilic messenger molecules to their receptor, not only by accumulation of the drug, but also by induction of orientations and conformations that are much more favorable for receptor docking than structures adopted in isotropic phases. A series of eight amphiphilic cyclic peptides containing lipophilic (L-alpha-aminodecanoic acid = Ada, L-alpha-aminohexadecanoic acid = Ahd, Nhdg = N-hexadecylglycine) and hydrophilic (Lys, Asp) amino acids were synthesized and examined by means of NMR spectroscopy and molecular dynamics (MD) simulations in isotropic (CDCl3) and membrane-mimicking anisotropic (SDS/H2O) solvents to study the influence of the environment on their individual conformations. NMR data of cyclo(-Gly1-D-Asp2-Ahd3-Ahd4-Asp5-Gly6+ ++-) (C4), cyclo(-Lys1-D-Pro2-Lys3-Ada4-Pro5-Ada6-) (C5) and cyclo(-Lys1-Pro2-Lys3-Ada4-D-Pro5-Ada6-) (C6) clearly indicate that those compounds are too rigid to perform a conformational change upon transition from an isotropic to an anisotropic environment. On the other hand, the experimental data of cyclo (-Gly1-Asp2-Ahd3-Ahd4-Asp5-Gly6-) (C1), cyclo(-Asp1-Ala2-Nhdg3-Ala4-D-Asp5-) (C7), and cyclo(-D-Asp1-Ala2-Nhdg3-Ala4-Asp5-) (C8) suggest highly flexible unstructured molecules in both environments. However, for cyclo(-Asp1-Asp2-Gly3-Ahd4-Ahd5-Gly6-) (C2) we observed a structure inducing effect of a membrane-like environment. The compound populates three different conformations in SDS/H2O, whereas in CDCI3 no preferred conformation can be detected. cyclo(-D-Asp1-Asp2-Gly3-Ahd4-Ahd5-Gly6-) (C3) clearly exhibits two different conformations with a shifted beta,beta-turn motif in CDCI3 and SDS/H2O solutions. The conformational change could be reproduced in a restraint-free MD simulation using the biphasic membrane mimetic CCl4/H2O. Our results give clear evidence that membrane interactions may not only lead to structure inductions, but can also induce major conformational changes in compounds already exhibiting a defined structure in isotropic solution.     

A quasi-elastic light scattering study of smooth muscle myosin in the presence of ATP.

We have investigated the hydrodynamic properties of turkey gizzard smooth muscle myosin in solution using quasi-elastic light scattering (QELS). The effects of ionic strength (0.05-0.5 M KCl) and light chain phosphorylation on the conformational transition of myosin were examined in the presence of ATP at 20 degrees C. Cumulant analysis and light scattering models were used to describe the myosin system in solution. A nonlinear least squares fitting procedure was used to determine the model that best fits the data. The conformational transition of the myosin monomer from a folded form to an extended form was clearly demonstrated in a salt concentration range of 0.15-0.3 M KCl. Light chain phosphorylation regulates the transition and promotes unfolding of the myosin. These results agree with the findings obtained using sedimentation velocity and electron microscopy (Onishi and Wakabayashi, 1982; Trybus et al., 1982; Trybus and Lowey, 1984). In addition, we present evidence for polymeric myosin coexisting with the two monomeric myosin species over a salt concentration range from 0.05 to 0.5 M KCl. The size of the polymeric myosin varied with salt concentration. This observation supports the hypothesis that, in solution, a dynamic equilibrium exists between the two conformations of myosin monomer and filaments.     

Solid phase synthesis and conformation of sequential glycosylated polytripeptide sequences related to antifreeze glycoproteins

Sequential glycopeptides [Thr(beta-D-galactose)-Ala-Ala]n, with n ranging from 2 to 7, as models of natural antifreeze glycoproteins were synthesized by the continuous flow, solid phase procedure. The conformational properties of these materials in solution were investigated by c.d. and 1H-n.m.r. spectroscopy. In aqueous solution the c.d. pattern is practically independent of chain length and is very similar to that of natural antifreeze glycoproteins. The results are interpreted in terms of random coil structure. The absence of ordered structures is further confirmed by n.m.r. data. A small amount of ordered conformation can be induced either by increasing the temperature of the aqueous solution or by addition of TFE. The c.d. pattern of all glycopeptides in water at temperatures higher than 50 degrees C are compatible with the presence of a small amount of alpha-helix or 3(10) helix. Since the glyco-hexapeptide is too short to form an alpha-helix, the hypothesis is made that in the glycopeptides in water at high temperature a small amount of 3(10) helix is formed. The same is observed for the 21-residue glycopeptide in presence of 85% (v/v) TFE. In this medium, the c.d. data on the glyco-hexapeptide are more compatible with the presence of a small amount of beta-structure.     

Structure of cross-linked rabbit muscle phosphofructokinase in solution.

Cross-linked rabbit muscle phosphofructokinase in the active tetrameric and octameric state was studied in solution by hydrodynamic methods and small angle x-ray scattering techniques. The translational diffusion coefficients were determined by means of inelastic light scattering and were found to be 3.60 (+/- 0.02) x 10(-7) cm2 . s-1 for the tetramer and 2.54 (+/- 0.15) x 10(-7) cm2 . s-1 for the octamer. From small angle x-ray scattering measurements the radius of gyration, the specific inner surface area, and the volume were determined for both enzyme forms, revealing that the octameric cross-linked form is approximately spherical, with a diameter of 120.0 A, whereas the tetrameric form is asymmetric having an axial ratio of 2. By comparison of the scattering curves with triaxial geometric bodies which are equivalent in scattering, the tetrameric enzyme is described as a rectangular prism, with overall dimensions of A = 131.0 A, B = 131.0 A, and C = 65.0 A, and the octameric form as that of a cube with A = B = C = 120.0 A. The shape of the protomer, having a radius of gyration of 24.8 A, in the tetramer and octamer is similar to that for the native tetramer at pH 10 in the presence of 5 mM fructose 6-phosphate or 15 mM fructose 1,6-bis-phosphate. From the different shapes of the scattering curves of the native phosphofructokinase at pH 7.5 in the presence of 15 mM ATP and of the cross-linked tetramer or octamer, it can be inferred that the shapes of the protomers are different: in the presence of ATP the protomers are elongated, having an axial ratio of 1.8 to 2.0; the cross-linked state reveals a spherical protomer of radius 33.0 A, similar to that of the native enzyme at pH 7.5 in the presence of fructose 6-phosphate or fructose 1,6-bisphosphate.     

Folding of thermolysin fragments. Hydrodynamic properties of isolated domains and subdomains

Sedimentation analysis in the analytical ultracentrifuge has been used to characterize the size and shape of thermolysin and a number of its fragments obtained by chemical or enzymatic cleavage of the protein. Four fragments (121-316, 206-316, 225/226-316 and 255-316) originate from the C-terminal domain, and two (1-155 and 1-205) from the N-terminal domain of the intact molecule. In aqueous solution at neutral pH the hydrodynamic properties of the C-terminal fragments, except 255-316, are consistent with compact homogeneous monomers. Fragment 255-316 is a monomeric species below 0.08 mg/ml concentration and forms a dimer above this concentration. Dimerization does not lead to changes in fragment conformation, as determined by far-ultraviolet circular dichroic measurements, but to an increase of 5.6 degrees C (to 68.2 degrees C at 1.0 mg/ml) in the temperature for thermal unfolding and a corresponding increase of 4.6 kJ/mol in the free energy of unfolding. Fragments derived from the N-terminal domain show a strong tendency to form high-molecular-mass aggregates. Previous experiments utilizing circular dichroic measurements and antibody binding data suggested that the C-terminal fragments listed above are able to refold in aqueous solution at neutral pH into a stable conformation of native-like characteristics [Dalzoppo, D., Vita, C. & Fontana, A. (1985) J. Mol. Biol. 182, 331-340] (and references cited therein). Present data establish that all these C-terminal fragments are globular monomeric species in solution (at concentrations approximately 0.1 mg/ml) and thus represent 'isolated' domains (or subdomains) with intrinsic conformational stability typical of small globular proteins.     

NMR studies of pH-dependent conformational polymorphism of alternating (C-T)n sequences.

Alternating (C-T)n sequences are involved in the H-DNA structure associated with (GA)n.(CT)n sequences. Low pH values facilitate H-DNA formation. We have undertaken a detailed analysis of the structural consequences of the (C-T)n sequence as a function of pH. The structures of three DNA oligonucleotides, d(CT)4, d(TC)4 and d(TC)15, have been studied by NMR. We found that their conformations are polymorphic and pH dependent. There are at least three major conformational species: an antiparallel-stranded (APS) duplex with entirely C:T base pairs at pH 7, an antiparallel-stranded (APS) duplex with entirely C+:T base pairs at pH 3, and a possible parallel-stranded (PS) duplex with C+:C and T:T base pairs near pH 5. In the intermediate pH range, the APS duplex may have varying numbers of C+:T and C:T base pairs, and there may be a fast exchange going on between APS duplex species involving these two kinds of base pairs. However, the transition between the APS and PS duplexes is slow. Structural refinement of the two octamers, d(TC)4 and d(CT)4, at pH = 6.9 and pH = 3 using 2D-NOE data suggests that the molecules are likely in the duplex form at 5 degrees C. We lack evidence that the structure at pH 3 is a PS structure with T nucleotides residing in the exterior of the helix. Titration of the longer oligonucleotide, d(TC)15, showed a prominent pKa of approximately 6, approaching the value of 7.0 obtained from the titration of poly-(dC).     

Structure and solution conformations of a cyclic trisaccharide from high-resolution n.m.r. spectroscopy and molecular modelling.

The conformational properties of a cyclic trisaccharide: [O-beta-D-glucopyranosyl-(1----6)]3 1,6"-anhydride nonacetate (C36H48O24, 1) have been established by high-resolution 1H- and 13C-n.m.r. spectroscopy in conjunction with potential-energy and molecular-mechanics calculations. The n.m.r. parameters used were nuclear Overhauser enhancements (n.O.e.) and coupling constants. From theoretical models of the trisaccharide, a statistical-mechanics approach was used to compute an ensemble average-relaxation matrix from which the n.O.e. were calculated. The observed nuclear Overhauser enhancements as measured by n.m.r. spectroscopy may be satisfactorily modelled if averaging over two conformational states is considered. In solution, both conformations of the molecule exhibit three-fold symmetry; the beta-linked glucopyranose rings have the 4C1 conformation. In one conformer, the orientation about the (1----6) linkage is characterized by torsion angles phi = 79.5 degrees, psi = 143.5, and omega = -64.3. For the other conformer, these values are phi = -137.7, psi = 68.2, and omega = 45.6. The existence of such a conformer shows that solution behaviour is not dominated by the stabilizing influence of the exoanomeric effect.     

Isoprenylation masks a conformational epitope and enhances trans-dominant inhibitory function of the large hepatitis delta antigen.

Hepatitis delta antigen (HDAg) consists of two species, large (LHDAg) and small (SHDAg), which are identical in sequence except that the large form contains 19 extra amino acids at the C terminus. The large form is prenylated on the Cxxx motif. The small form can trans activate HDV RNA replication, while the large form inhibits it. To determine the molecular basis for their differential functions, we examined the effects of prenylation on the conformation and function of HDAg. We show that the presence of prenylates masks a conformational epitope which is present in SHDAg but hidden in wild-type LHDAg; this epitope becomes exposed in all of the nonprenylated mutant LHDAgs. Prenylation also plays a major role in conferring the trans-dominant negative inhibitory activity of LHDAg, since the loss of prenylation in LHDAg reduced its inhibitory activity. The primary amino acids of the C-terminal sequence also contributed to the maintenance of the HDAg protein conformation; a prenylated LHDAg mutant with a five-amino-acid deletion had an exposed C-terminal epitope. By examining LHDAg mutants which have deletions of various extents of C-terminal sequence, with or without the prenylation motif, we have further shown that all of the prenylated mutants have much higher levels of trans-dominant suppressor activities than do the corresponding nonprenylated mutants. Surprisingly, a few nonprenylated LHDAg mutants were able to trans activate HDV RNA replication, while all of the prenylated ones lost this function. These results suggest that isoprenylates cause the masking of a conformational epitope of HDAg and that conformational differences between the large and small HDAgs account for the differences in their trans-activating and trans-dominant inhibitory biological activities.     

Structural study of poly(beta-benzyl-L-aspartate) monolayers at air-liquid interfaces.

As normally studied, in the solid state or in solution, poly(beta-benzyl-L-aspartate) (PBLA) differs from the other helical polyamino acids in that its alpha-helical conformation is most stable in the left-handed rather than in the right-handed form. The slightly lower energy per residue for the left-handed form in PBLA is easily perturbed, however. The helical screw sense can be inverted in a polar environment and, upon heating above 100 degrees C, a distorted left-handed helix or omega-helix is irreversibly formed. From external reflectance Fourier transform infrared measurements at the air-water interface, the conformation of PBLA in the monolayer state is directly established for the first time. The infrared frequencies of the amide bands suggest that right-handed alpha-helices are formed on the surface of water immediately after spreading the monolayers and independently of the polypeptide conformational distribution in the spreading solution. The right-handed helical form prevails throughout the slow compression of the Langmuir monolayers to collapsed films. The helical screw sense can be reversed by lowering the polarity of the aqueous phase. In addition, an alternate conformation similar to the omega-helix forms on addition of small amounts of isopropanol to the aqueous subphase, and appears to be an intermediate in the helix-helix transition.     

Local structural plasticity of the prion protein. Analysis of NMR relaxation dynamics

A template-assisted conformational change of the cellular prion protein (PrP(C)) from a predominantly helical structure to an amyloid-type structure with a higher proportion of beta-sheet is thought to be the causative factor in prion diseases. Since flexibility of the polypeptide is likely to contribute to the ability of PrP(C) to undergo the conformational change that leads to the infective state, we have undertaken a comprehensive examination of the dynamics of two recombinant Syrian hamster PrP fragments, PrP(29-231) and PrP(90-231), using (15)N NMR relaxation measurements. The molecular motions of these PrP fragments have been studied in solution using (15)N longitudinal (T(1)) and transverse relaxation (T(2)) measurements as well as [(1)H]-(15)N nuclear Overhauser effects (NOE). These data have been analyzed using both reduced spectral density mapping and the Lipari-Szabo model free formalism. The relaxation properties of the common regions of PrP(29-231) and PrP(90-231) are very similar; both have a relatively inflexible globular domain (residues 128-227) with a highly flexible and largely unstructured N-terminal domain. Residues 29-89 of PrP(29-231), which include the copper-binding octarepeat sequences, are also highly flexible. Analysis of the spectral densities at each residue indicates that even within the structured core of PrP(C), a markedly diverse range of motions is observed, consistent with the inherent plasticity of the protein. The central portions of helices B and C form a relatively rigid core, which is stabilized by the presence of an interhelix disulfide bond. Of the remainder of the globular domain, the parts that are not in direct contact with the rigid region, including helix A, are more flexible. Most significantly, slow conformational fluctuations on a millisecond to microsecond time scale are observed for the small beta-sheet. These results are consistent with the hypothesis that the infectious, scrapie form of the protein PrP(Sc) could contain a helical core consisting of helices B and C, similar in structure to the cellular form PrP(C). Our results indicate that residues 90-140, which are required for prion infectivity, are relatively flexible in PrP(C), consistent with a lowered thermodynamic barrier to a template-assisted conformational change to the infectious beta-sheet-rich scrapie isoform.     

Further evidence for the absence of polyproline II stretch in the XAO peptide

It has been suggested that the alanine-based peptide with sequence Ac-XX-[A](7)-OO-NH(2), termed XAO where X denotes diaminobutyric acid and O denotes ornithine, exists in a predominantly polyproline-helix (P(II)) conformation in aqueous solution. In our recent work, we demonstrated that this "polyproline conformation" should be regarded as a set of local conformational states rather than as the overall conformation of the molecule. In this work, we present further evidence to support this statement. Differential scanning calorimetry measurements showed only a very small peak in the heat capacity of an aqueous solution of XAO at 57 degrees C, whereas the suggested transition to the P(II) structure should occur at approximately 30 degrees C. We also demonstrate that the temperature dependence of the (3)J(HNHalpha) coupling constants of the alanine residues can be explained qualitatively in terms of Boltzmann averaging over all local conformational states; therefore, this temperature dependence proves that a conformational transition does not occur. Canonical MD simulations with the solvent represented by the generalized Born model, and with time-averaged NMR-derived restraints, demonstrate the presence of an ensemble of structures with a substantial amount of local P(II) conformational states but not with an overall P(II) conformation.     

Redox-induced conformational changes in plastocyanin: an infrared study.

The conformational changes associated with the redox transition of plastocyanin (PC) were investigated by absorption and reaction-induced infrared spectroscopy. In addition to spectral features readily ascribed to beta and turn protein secondary structures, the amide I band shows a major component band at 1647 cm(-1) in both redox states of the protein. The sensitivity of this component to deuteration and increasing temperature suggests that PC adopts an unusual secondary structure in solution, which differs from those described for other type I copper proteins, such as azurin and halocyanin. The conformations of oxidized and reduced PC are different, as evidenced (1) by analysis of their amide I band contour and the electrochemically induced oxidized-minus-reduced difference spectrum and (2) by their different thermal stability. The redox-induced difference spectrum exhibits a number of difference bands within the conformationally sensitive amide I band that could be assigned to peptide C=O modes, in light of their small shift upon deuteration, and to signals attributable to side chain vibrational modes of Tyr residues. Lowering the pH to 4.8 induces destabilization of both redox states of the protein, more pronounced for reduced PC, without significantly affecting their secondary structure. Besides the conformational differences obtained at neutral pH, the oxidized-minus-reduced difference spectrum shows two broad and strong negative bands at 1405 and 1571 cm(-1), assigned to COO(-) vibrations, and a broad positive band at 1710 cm(-1), attributed to the C=O vibration of a COOH group(s). These bands are indicative of a protonation of (an) Asp or Glu side chain(s) upon plastocyanin oxidation at acidic pH.     

The nature of stacking interations in polynucleotides. Molecular states in Oligo- and polyribocytidylic acids by relaxation analysis.

The dynamics of the helix-coil transition of single-stranded poly(C) (polyribocytidylate) and CpC (cytidyly(3'-5')cytosine) was investigated by an improved cable temperature-jump technique. The single-strand relaxation was characterized by following the ultraviolet (uv) absorbance changes at 248 and 280 nm. Poly(C) and CpC showed single relaxation processes with amplitudes corresponding to those expected from equilibrium melting curves. The relaxation time contants in the range of 25-100 ns were independent of the nucleotide concentration, but strongly dependent upon temperature. Using thermodynanic parameters obtained from circular dichroism (CD) and uv absorbance melting curves, the following rate constants k (at 20 degrees C, 1.05 M ionic strength, pH 7) and activation enthalpies EA were calculated for poly (C): helix formation kR = 1.11 X 10(-7) s-1 (EAR = 2.6 kcal); helix dissociation kD = 2.1 X 10(6) s-1 (EAD = 11.9 kcal). The rate constants obtained for CpC were higher by a factor of about 2 in kR and 12 in kD, whereas the activation enthalpies closely corresponded to those found for the polymer. In addition to the single-stranded helix-coil relaxation, poly(C) and CpC exhibit a relaxation process with a time constant below 25 ns and maximum amplitudes at wavelengths lambda greater than or equal to 285 nm. The same process is found in cytidine and is attributed to hydration equilibria. The hydration reaction can be considered to be in equilibrium during the entire time range of the helix-coil transition and thus the data obtained for the helix-coil transition can be described by a simple two-state model. The rate parameters indicate the existence of relatively high energy barriers in the helix-coil transition and provide strong evidence evidence against an oscillating dimer model. If there is an ensemble of substates for one of the states (as may be expected for the coil form), the energy difference between the populated substates is small compared with the energy difference between the major conformational states.     

The conformation of glucagon: predictions and consequences.

It is proposed that glucagon, a polypeptide hormone, is delicately balanced between two major conformational states. Utilizing a new predictive model [Chou, P.Y., and Fasman, G.D. (1974), Biochemistry 13, 222] which considers all the conformational states in proteins (helix, beta sheet, random coil, and beta turns), the secondary structural regions of glucagon are computed herein. The conformational sensitivity of glucagon may be due to residues 19-27 which have both alpha-helical potential (mean value of Palpha = 1.19) as well as beta-sheet potential (mean value of Pbeta = 1.25). Two conformational states are predicted for glucagon. In predicted form (a), residues 5-10 form a beta-sheet region while residues 19-27 form an alpha-helical region (31% alpha, 21% beta) agreeing well with the circular dichroism (CD) spectra of glucagon. The similarity in the CD spectra of glucagon and insulin further suggests the presence of beta structure in glucagon, since X-ray analysis of insulin showed 24% beta sheet. In predicted form (b), both regions, residues 5-10 and residues 19-27, are beta sheets sheets (0% alpha, 52% beta) in agreement with the infrared spectral evidence that glucagon gels and fibrils have a predominant beta-sheet conformation. Since three reverse beta turns are predicted at residues 2-5, 10-13, and 15-18, glucagon may possess tertiary structure in agreement with viscosity and tritium-hydrogen exchange experiments. A proposal is offered concerning an induced alpha yields beta transition at residues 22-27 in glucagon during receptor site binding. Amino acid substitutions are proposed which should disrupt the beta sheets of glucagon with concomitant loss of biological activity. The experimental findings that glucagon aggregates to form dimers, trimers, and hexamers can be explained in terms of beta-sheet interactions as outlined in the present predictive model. Thus the conflicting conclusions of previous workers, concerning the conformation of glucagon in different environments, can be rationalized by the suggested conformational transition occurring within the molecule.     

Comparison of protein structure in crystals and in solution by laser raman scattering. I. Lysozyme

The laser Raman-scattering technique was employed to examine the question of whether the structure of a globular protein is the same in crystals as in solution. Lysozyme was selected as a model system for this study. In the amide I and amide III regions we found a good agreement between the Raman spectra of lysozyme chloride crystals (in 100% relative humidity) and lysozyme solution (at pH 4.50), indicating that the main-chain conformation is the same between two phases. However, small but definite spectral differences were observed near 464, 622, 644, 934, 960, 978, 1032, 1129, and 1196 cm^?1. Some of these spectral differences may be interpreted in terms of side-chain conformational changes. Additionally, we present Raman spectrum of lysozyme in the lyophilized form and compare it to those of crystals and solution. It was concluded that lyophilization caused conformational changes appreciably, both in the main chain and side chain.     

Electron paramagnetic resonance of Cu2+:Mb single crystal. Conformational changes.

Copper introduced into met-myoglobin crystals occupies various sites as indicated by electron paramagnetic resonance parameters. Cu2+ (A) is probably liganded to histidine A10, lysine A14, and asparagine GH4 (Banaszak et al., 1965) and shows superhyperfine interaction with a single (imidazole) nitrogen. Cu2+ (B) and Cu2+ (C) correspond to other anisotropic sites described in less detail. Cu2+ (A) exhibits a transition to an isotropic form with a transition temperature of 40.5 degrees C. This transition indicates a conformational change in myoglobin and could correspond to a motion of A helix away from the GH section. The transition temperature is 7 degrees C higher than the one previously reported (Atanasov, 1971) for myoglobin in solution.     

Solution NMR studies of colicin E1 C-terminal thermolytic peptide. Structural comparison with colicin A and the effects of pH changes

The aqueous solution structure of the C-terminal thermolytic peptide of colicin E1 has been investigated using both one- and two-dimensional NMR techniques. The NMR data are consistent with a fold for the peptide very similar to that reported for the colicin A C-terminal peptide in the crystalline state, although some differences have been noted. The one-dimensional NMR spectrum of the peptide has been used to follow changes in both the structure and dynamics of the peptide on changing pH. The in vitro functionally competent form of the peptide (present in solution only below pH 6) does not differ in structure significantly from the higher pH form. However, small local conformational changes are observed together with an increase in mobility in some of the more hydrophilic regions. This suggests that the effect of lower pH is to change the ease with which the major conformational changes during insertion into a membrane can occur.     

A duplex of the oligonucleotides d(GGGGGTTTTT) and d(AAAAACCCCC) forms an A to B conformational junction in concentrated salt solutions

The coexistence of both A form and B form tracts and formation of an A-B junction in the oligomer d(GGGGGTTTTT).d(AAAAACCCCC) in saturated sodium chloride solution have been detected by Raman spectroscopy. The entire duplex adopts the familiar B-form conformation in aqueous solution at low salt concentrations (0.1M NaCl). In 6M NaCl the adoption of an A form is observed within the G,C tract while a B-form is maintained in the A.T tract. The experimental results indicate that two different helical forms can co-exist in a rather short oligonucleotide and that formation of an A-B junction can occur over a fairly small span of bases. This is in agreement with recent rules governing the relation between base sequence and secondary structure of DNA published from this laboratory. The conformational preferences of each of the individual oligomers d(AAAAACCCCC) and d(GGGGGTTTTT) have also been investigated. The oligomer d(AAAAACCCCC) is single stranded but some evidence for base stacking is observed at 2 degrees C. In contrast, a double stranded B-form structure characterized by wobble G-T base pairing is observed for d(GGGGGTTTTT) in 0.1M and 6M NaCl.