Helix In Solution Research Articles

The construction of new proteins

AbstractRecent progresses in the elucidation of the folding mechanism and topology of proteins revealed that the formation of folding units with specific topological features is not restricted to a unique primary sequence. This finding presents the basis for the design of polypeptides having the propensity to fold into a tertiary structure that can be achieved by the assembly of peptide blocks exhibiting stable secondary structures. Conformational studies on model peptides show that Aib containing peptides with chain‐lengths of 12‐15 residues are able to form stable amphiphilic helices in solution. On the other hand, oligopeptides with alternating hydrophilic and hydrophobic residues are capable for β‐structure formation for chain‐lengths of 6‐8 residues. Those amphiphilic secondary structures have been used as building‐blocks for the design and synthesis of artificial folding units, their amphiphilic nature acting as major driving force for intramolecular folding. Spectroscopic data obtained for two polypeptides designed as βαβ‐models actually suggest a folded conformation of these molecules in aqueous solution. The implications of these findings for the design of biologically active folded polypeptides are discussed.

Conformational analysis of the octamer d(G-G-C-C-G-G-C-C) in aqueous solution. A one-dimensional and two-dimensional proton NMR study at 300 MHz and 500 MHz.

Proton NMR studies in H2O and 2H2O were carried out on the self-complementary DNA octamer d(G-G-C-C-G-G-C-C) and compared with similar studies on the dimethylated analogue d(G-G-m5C-m5C-G-G-C-C) [Sanderson, M. R., Mellema, J.-R., van der Marel, G. A., Wille, G., van Boom, J. H. & Altona, C. (1983) Nucleic Acids Res. 11, 3333-3346]. Base, H1', H2' and H2" resonances were assigned by means of two-dimensional nuclear Overhauser spectroscopy (NOESY) and correlated spectroscopy (COSY) experiments. Chemical shift vs temperature profiles were used to analyze the temperature-dependent conformational behaviour and to extract thermodynamic parameters for the duplex-to-coil transition. Analysis of proton-proton couplings were used to discriminate between J1'2' and J1'2" and between the H2' and H2" resonances as well as to obtain conformational parameters of the sugar rings. From the NOESY and COSY experiments, the temperature profiles and the analysis of the coupling data it is concluded that: (a) the compound adopts a B-DNA-type helix in solution; (b) the sugar rings in the intact duplex display limited conformational freedom; (c) methylation of the cytosine bases at the C5 position has no measurable effect on the conformational behaviour of the octamer, nor on the conformation of the sugar rings; however, methylation does increase the stability of the duplex in aqueous solution under conditions of low salt concentration.

Sequence-dependent conformation of DNA duplexes. The AATT segment of the d(G-G-A-A-T-T-C-C) duplex in aqueous solution.

The nonexchangeable base and sugar protons of the octanucleotide d(G-G-A-A-T-T-C-C) have been assigned by two-dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) methods in aqueous solution. The assignments are based on distance connectivities of less than 4.5 A established from NOE effects between base and sugar protons on the same strand and occasionally between strands, as well as, coupling connectivities within the protons on each sugar ring. We observe the NOEs to exhibit directionality and are consistent with the d(G-G-A-A-T-T-C-C) duplex adopting a right-handed helix in solution. The relative magnitude of the NOEs between base and sugar H2' protons of the same and 5'-adjacent sugars characterizes the AATT segment to the B-helix type in solution.

Covalent carcinogenic lesions in DNA: NMR studies of O6-methylguanosine containing oligonucleotide duplexes

We report on proton and phosphorus high resolution NMR investigations of the self-complementary dodecanucleotide d(C1-G2-N3-G4-A5-A6-T7-T8-C9-O6meG10-C11-G12) duplexes (henceforth called O6 meG.N 12-mers), N = C, T, A and G, which contain N3.O6meG10 interactions in the interior of the helix. These sequences containing a single modified O6meG per strand were prepared by phosphoamidite synthesis and provide an excellent model for probing the structural basis for covalent carcinogenic lesions in DNA. Distance dependent nuclear Overhauser effect (NOE) measurements and line widths of imino protons demonstrate that the N3 and O6meG.10 bases stack into the duplex and are flanked by stable Watson-Crick base pairs at low temperature for all four O6meG.N 12-mer duplexes. The imino proton of T3 in the O6meG.T 12-mer and G3 in the O6meG.N 12-mer helix, which are associated with the modification site, resonate at unusually high field (8.5 to 9.0 ppm) compared to imino protons in Watson-Crick base pairs (12.5 to 14.5 ppm). The nonexchangeable base and sugar protons have been assigned from two dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) measurements on the O6meG.N 12-mer helices. The directionality of the distance dependent NOEs establish all O6meG.N duplexes to be right-handed helices in solution. The glycosidic torsion angles are in the anti range at the N3.O6meG10 modification site except for O6meG10 in the O6meG.G 12-mer duplex which adopts a syn configuration. This results in altered NOEs between the G3 (anti).O6meG10 (syn) pair and flanking G2.C11 and G4.C9 base pairs in the O6meG.G 12-mer duplex. We observe pattern reversal for cross peaks in the COSY spectrum linking the sugar H1' protons with the H2',2" protons at the G2 and O6meG10 residues in the O6meG.N 12-mer duplexes with the effect least pronounced for the O6meG.T 12-mer helix. The proton chemical shift and NOE data have been analyzed to identify regions of conformational perturbations associated with N3.O6meG10 modification sites in the O6meG.N 12-mer duplexes. The proton decoupled phosphorus spectrum of O6meG.T 12-mer duplex exhibits an unperturbed phosphodiester backbone in contrast to the phosphorus spectra of the O6meG.C 12-mer, O6meG.G 12-mer and O6meG.A 12-mer duplexes which exhibit phosphorus resonances dispersed over 2 ppm characteristic of altered phosphodiester backbones at the modification site. Tentative proposals are put forward for N3.O6meG10 pairing models based on the available NMR data and serve as a guide for the design of future experiments.

Chiral probes for the handedness of DNA helices: enantiomers of tris(4,7-diphenylphenanthroline)ruthenium(II).

The chiral complexes tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) (RuDIP) are shown to be specific chemical probes with which to distinguish right- and left-handed DNA helices in solution. In spectrophotometric titrations of racemic RuDIP with both B-form calf thymus DNA and Z-form poly[d(G-C)], hypochromicity in the intense metal-to-ligand charge-transfer band is found and enhancement in luminescence is observed. The spectrophotometric assay of DNA binding to the well-resolved enantiomers of RuDIP provides a means to determine the helical conformation. Strong chiral specificity is seen in binding experiments with right-handed B-DNA and, on this basis, the absolute configurations are assigned. Although delta-RuDIP can bind by intercalation into the right-handed helix, steric constraints imposed by the helix asymmetry preclude completely binding by the lambda enantiomer. Both isomers, however, are found to bind equally to Z-DNA. Left-handed helices that are more similar structurally to B-DNA would be predicted to display a stereospecific preference for this lambda isomer.

NMR studies of conformational states and dynamics of DNA.

The application of high resolution NMR techniques to the investigation of DNA double helices in solution is currently in a rapid state of change as a result of advances in three different fields. First, new methods (cloning, enzymatic degradation, sonication, and chemical synthesis) have been developed for producing large quantities of short DNA suitable for NMR studies. Second, there have been major advances in the field of NMR in terms of the introduction of new pulse techniques and improvements in instrumentation. Finally, as a result of recent X-ray diffraction studies on short DNA helices and the discovery of left-handed Z-DNA there is heightened interest in the study of DNA structures in solution and the effect of sequence on structure. In the present review, we discuss the way in which NMR techniques have been used to probe various aspects of the DNA properties, including base pairing structure, dynamics of breathing, effect of sequence on DNA structure, internal molecular motions, the effect of environment on the DNA, and the interaction of DNA with small ligands.

Tris (phenanthroline) metal complexes: probes for DNA helicity.

The intercalative binding of chiral tris(phenanthroline) metal complexes to DNA is stereo-selective. The enantiomeric selectivity is based upon the differential steric interactions between the two non-intercalating phenanthroline ligands of each isomer with the DNA phosphate backbone. Gel electrophoretic assays of helical unwinding, optical enrichment studies by equilibrium dialysis and luminescence titrations with separated enantiomers of (phen)3Ru2+ all indicate that the delta isomer binds preferentially to the right-handed duplex. The chiral discrimination is governed by the DNA helical asymmetry. Complete stereospecifity is seen with isomers of the bulkier RuDIP (tris-4,7-diphenylphenanthrolineruthenium(II]. While both isomers bind to Z-DNA, a poor template for discrimination, binding of lambda-RuDIP to B-DNA is precluded. These chiral complexes therefore serve as a chemical probe to distinguish left and right-handed DNA helices in solution.

Nuclear Overhauser assignment of the imino protons of the acceptor helix and the ribothymidine helix in the nuclear magnetic resonance spectrum of Escherichia coli isoleucine transfer ribonucleic acid: evidence for costacked helices in solution.

In a previous study we showed that, in the low-field nuclear magnetic resonance (NMR) spectrum of Escherichia coli tRNAVal1, the hydrogen-bonded imino protons of the four Watson-Crick base pairs in the dihydrouridine helix could be assigned on the basis of their proximity to the imino proton of s4U8 by means of sequential Nuclear Overhauser (NOE) connectivity (Hare & Reid, 1982). In the present paper we have used the nearest-neighbor NOE technique to assign all the imino proton resonances of the acceptor helix and the ribothymidine helix of E. coli tRNAIle1. As reference points we used the GU-type base pairs located at positions 5 and 49 in this molecule which are readily identifiable in the NMR spectrum by virtue of containing two imino protons in the same base pair. From UG5 the imino protons of base pairs 4,3,2,1 and 6,7 could be assigned by through-space NOE connectivity. Similarly the imino protons of 50,51,52,53 were assigned by their spatial relationship to G psi 49. NOE connectivity also revealed a base pair stacked on the external side of GC53 which, by analogy with the crystal structure of yeast phenylalanine tRNA, is presumed to be the tertiary pair T54-A58. This was confirmed by NOE connectivity from the thymine methyl resonance. In addition to assigning 17 of the imino resonances in the low-field NMR spectrum of isoleucine tRNA, these NOE studies show that the acceptor helix and the ribothymidine helix are stacked on each other in solution in that base pairs 7 and 49 are directly connected in space.

Kinetics for exchange of imino protons in deoxyribonucleic acid, ribonucleic acid, and hybrid oligonucleotide helices.

The lifetime for opening of individual base pairs in a DNA (dCA5G + dCT5G), and RNA (rCA5G + rCU5G), and a hybrid DNA-RNA (rCA5G + dCT5G) helix have been measured by proton nuclear magnetic resonance. The lifetimes were obtained by saturation recovery experiments performed on the hydrogen-bonding imino protons of the Watson-Crick base pairs. In these oligonucleotide helices the observed relaxation rates were dominated by exchange with water, with the magnetic spin-lattice relaxation time of the imino protons possibly being important only at the lowest temperatures in the DNA helix. It was shown that three interior base pairs in the DNA heptamer dCA5G + dCT5G were in the open-limited region, which means that these imino protons exchange every time the base pair opens. The lifetime of the terminal G X C base pairs in the DNA helix are much shorter than the interior A X T base pairs. The pH dependence of the terminal base pairs indicated that the ends of the helix open and close many times before exchange of the imino protons with water takes place. The temperature dependence of the lifetimes of the interior A X T imino protons in the DNA helix showed that these protons exchange only when the double helix has dissociated into single strands. Thus, these lifetimes measure the rate for dissociation of the double helix. The activation energy for this process was found to be 47 kcal/mol. Comparison of the lifetimes of the interior protons in the DNA, RNA, and hybrid helices showed that the rates of dissociation of the RNA and hybrid helices are very similar at 5 degrees C, whereas the rate for the DNA helix was approximately 1 order of magnitude smaller than that for the other two helices. The reasons for the differences in the kinetics of the three helices are discussed, as are the general dynamics of oligonucleotide helices in solution.

The role of nonspecific hydrophobic interactions in the biological activity of N epsilon-acyl derivatives of glucagon. Studies of conformation, receptor binding, and adenylate cyclase activation.

Glucagon was acylated at position 12 using conditions favoring reaction with the epsilon-amino group of lysine. The N epsilon-acetyl, N epsilon-hexanoyl, and N epsilon-decanoyl derivatives were prepared and purified. Secondary structure as measured by circular dichroism was lower in all derivatives than in glucagon, both in 95% methanol and in 25 mM sodium dodecyl sulfate at pH 2 and pH 12. N epsilon-Acetyl glucagon was less active than the native hormone in a radioreceptor assay and higher concentrations of this derivative were required to stimulate the adenylate cyclase activity of rat liver plasma membranes. The maximal extent of cyclase activation by this derivative was less than that found with the native hormone. N epsilon-Hexanoyl glucagon and N epsilon-decanoyl glucagon had greater activity than N epsilon-acetyl glucagon in receptor binding as well as in adenylate cyclase activation, although these two derivatives were not as active as the native hormone. N epsilon-hexanoyl glucagon and N epsilon-decanoyl glucagon were more potent in receptor binding than in adenylate cyclase activation. From these results it appears that the positive charge of the epsilon-amino groups may have a specific role in obtaining maximal biological activity, while the acyl groups contribute to the nonspecific hydrophobic interactions between the hormone and its receptor. In addition, a possible relationship between stabilization of the amphipathic helix in solution and the activity of these and other N epsilon-derivatives of glucagon is discussed.

Left-handed deoxyribonucleic acid double helix in solution.

Magnetic shielding constants were calculated for the synthetic deoxyribonucleic acid (DNA) double helix poly(dG-dC).poly(dG-dC) from the x, y, and z coordinates of Z-DNA of Rich and co-workers [Wang, A. H-J., Quigley, G. J., Kolpak, F. J., Crawford, J. L., van Boom, J. H., van der Marel, G., & Rich, A. (1979) Nature (London) 282, 680-686)] and B-DNA of Arnott & Hukins [Arnott, S., & Hukins, D. W. L. (1972) Biochem. Biophys. Res. Commun. 47, 1504-1509], taking into account the contribution to shielding from ring current effects and effects from the diamagnetic and paramagnetic components of the atomic magnetic anisotropy. Comparison of the calculated shielding values with the experimentally observed nuclear magnetic resonance shift data for poly(dG-dC).poly(dG-dC) in high salt solution shows striking agreement for Z-DNA and considerable deviation for B-DNA, indicating that this synthetic DNA double helix is high salt solution can assume the spatial configuration of the left-handed Z-DNA double helix known to occur in crystals.

Conformational characteristics of the trinucleoside diphosphate dApdApdA and its constituents from nuclear magnetic resonance and circular dichroism studies. Extrapolation to the stacked conformers.

Proton NMR studies at 360 MHz were carried out on the trinucleoside diphosphate dApdApdA, the dinucleoside monophosphate dApdA and the methylphosphate esters of the monomers pdA, pdAp and dAp. The compounds were also investigated by means of circular dichroism at varying temperatures. Complete NMR spectral assignments are given. The thermodynamics of stacking derived from the circular dichroic spectra was used to extrapolate the observed coupling constants, measured at a range of temperatures, to values appropriate to the pure stacked states of the dimer and the trimer. The data were interpreted in terms of the N and S deoxyribose pseudorotational ranges [Altona, C. and Sundaralingam, M. (1972) J. Am. Chem. Soc. 94, 8205--8212] and it is shown that the right-handed single helix of the trimer occurs in two distinct forms: the major one corresponds to a B-DNA-like structure, S-S-S; the minor one (30%) has the sugar rings in a hitherto unsuspected S-S-N conformational sequence. The dimer behaves quite similarly, a mixture of 70% S-S and 30% S-N stacks being indicated. The detailed geometry of teh S-type sugar rings is not invariable but appears to undergo a slight shift when another base stacks on top of a given nucleotide unit. The backbone angles of the fully stacked B-DNA-like single helix in solution are (starting the notation at P) beta = 187 degrees, gamma = 50 degrees, delta = 138 degrees, epsilon = 186 degrees.

Helical periodicity of DNA determined by enzyme digestion.

The periodicity of DNA has been determined by binding short, stiff pieces of DNA to a flat surface and using DNase I to probe the accessibility of the phosphodiester bonds. We have found that the distance between successive DNase I cutting sites is 10.6 +/- 0.1 bases for the DNA immobilized on three different surfaces. We identify this value with the number of base pairs per turn of the DNA double helix in solution.

Generation of a third-order folded structure for chromatin

A model for the initiation of the diffuse-condensed transition of chromatin induced by a change in the conformation of lysine-rich histones is proposed. Three levels of folded structures are discussed. The first-order folded structure refers to the structure of the repeat unit of chromatin, which is called the nucleosome. The nucleosome contains a nuclease resistant region in which 140 base pairs of DNA are wrapped around the surface of a histone aggregated of two copies each of the histones H2A, H2B, H3 and H4. This DNA-histone aggregate is called a core particle. The nuclease accessible region of the nucleosome is approximately 60 base pairs of DNA which link the core particle, hence the terminology “linker DNA.” The lysine-rich histones, (Hl, H5), which are more loosely bound than the core histones, are associated with the linker DNA. The second-order folded structure refers to the conformation of a polynucleosome. Based on neutron scattering and quasielastic light scattering studies the second-order folded structure is assumed to be an extended helix in solution with 5–7 nucleosome units per turn. The third-order folded structure is defined as that structure resulting from the first stage in the condensation process induced by a conformational change in the lysine-rich histones. Generation of the third-order folded structure in the proposed model is effected by an increased affinity of the lysine-rich histones for super-helical DNA in the core particles in adjacent turns of the second-order folded structure. Since the lysine-rich histones preferentially bind to A-T rich regions in DNA, the distribution of these regions would determine the third-order folded structure. The net effect of a non-random distribution of A-T rich regions as in the proposed model is the generation of a helix for the third-order folded structure. The assumption of a non-random distribution of A-T rich regions is indirectly supported by proflavine binding studies reported herein and by the existence of repetitive and non-repetitive DNA regions inferred from renaturation studies. One consequence of the proposed mechanism is that the majority of the A-T rich regions are in the interior of the third-order folded structure. Promoter sites of high A-T content would then be inaccessible to polymerases. The proposed model also suggests a role for spacer DNA in the genome. Higher order folded structures must also be present in the final state of condensed chromatin since the three orders of folded structures considered in this communication accounts for only 2% of that required in the diffuse-condensed transition.

1H nuclear magnetic resonance investigation of flexibility in DNA.

In this paper we report successful observations of proton NMR spectra of native double helical salmon sperm and calf thymus DNA of various lengths. Measurements of the linewidths arising from proton--proton dipolar interactions are used to obtain information about the dynamic behavior of DNA helices in solution. Depending upon which protons are used to monitor the local internal motions of the DNA, different results are obtained. The lowfield resonances from hydrogen-bonded imino protons in the base pairs indicate that the correlation time for reorientation of base pairs is less than 3 x 10(-7) sec, whereas correlation times for motion of neighboring sugar protons relative to the aromatic protons are less than 5 x 10(-8) sec for DNA that is over 200 base pairs long. These observations indicate that there is considerably more internal flexibility in the DNA molecues, especially in the backbone, than is indicated by classic hydrodynamic studies of DNA.

Structural Investigations on Poly(4-hydroxy-L-proline). 2. Physicochemical Studies

We report experimental studies which confirm our prediction, namely that the ordered structure of poly(hydroxypro1ine) in solution corresponds to a left-handed helical structure with intrachain hydrogen bonds. The CD studies show that the poly(hydroxypro1ine) molecule has essentially the same conformation in aqueous solution and in the film obtained subsequently by evaporation. X-ray diffraction patterns of the sample in this form (B form) have been recorded at different relative humidities. The patterns recorded at relative humidities over 66% can be interpreted in terms of a helical structure with intrachain hydrogen bonds. These results lead us to conclude that the ordered conformation of poly(hydroxypro1ine) in solution is form B and not form A. This offers a simple explanation for the greater stability of the poly(hydroxypro1ine) helix in solution as compared to the poly(pro1ine) form I1 helix and also for the absence of mutarotation for poly(hydroxypro1ine).

13C magnetic resonance evidence for anisotropic molecular motion in collagen fibrils

Relaxation times and integrated intensities have been obtained from dipolar decoupled 13C magnetic resonance spectra of reconstituted fibrils of chick calvaria collagen enriched at the glycine C a and C′ positions. The data obtained are consistent with a model in which collagen molecules reorient about the long axis of the helix with a rotational diffusion constant ( R 1) of ~10 7 s −1, a value similar to that expected for the helix in solution. Data obtained from natural abundance 13C spectra of native (crosslinked) calf achilles tendon and rat tail tendon provide evidence of rapid anisotropic reorientation for at least 75% of the carbons in these tissues. Hence, our preliminary data indicate that, in these materials, the intermolecular interactions in the fibrilar collagen lattice can accommodate rapid reorientation at a majority of carbon sites.

Polypeptide models of collagen: Properties of (Pro-Pro-betaAla)n.

AbstractWe have synthesized (Pro‐Pro‐βAla)n as a model for collagen. The synthetic polytripeptide, mol wt 6500, exhibits a large negative optical rotation with a very strong negative Cotton effect centered at 216 nm. The optical rotatory dispersion of (Pro‐Pro‐βAla)n followed a single‐term Drude equation and the λc was 195 nm. The rotation decreased markedly on heating with the midpoint of the broad transition at 55°C. Preliminary studies also showed loss of structure in guadinine HCl. The circular dichroism spectrum of the polymer exhibited a deep trough at 190 nm. The marked similarities of solution properties of (Pro‐Pro‐βAla)n to (Pro‐Pro‐Gly)n suggest that β‐alanine can replace glycine in generating collagen‐like helix in solution.

Dissymmetric recognition of the helical sense of deoxyribonucleic acid and evidence for binding of reporter molecules from the minor groove of DNA

Through the utilization of optically active DNP-derivatives of l- and d-proline, evidence is presented which suggests that nucleic acids exist as right-handed helices in solution. The results of ultraviolet absorption, circular dichroism, proton magnetic resonance (pmr), T m of the helix-coil transition, viscometric, and binding studies are consistent with the above interpretation. It is shown that several types of DNA (i.e., salmon sperm, calf thymus, Micrococcus luteus, poly d(A-T)-poly d(A-T) and poly d(I-C)-poly d(I-C)) exist in a right-handed helical structure in solution. In addition, evidence is presented which strongly indicates that the 2,4-dinitroaniline ring of DNP-proline is intercalated between base-pairs of DNA and the prolyl side chain situated in the minor groove. Moreover, it is shown that the more sterically hindered DNP-derivatives exhibit a higher selectivity for A-T binding sites.

ChemInform Abstract: TOPOGRAPHY OF NUCLEIC ACID HELICES IN SOLUTION PART 29, STERIC EFFECTS ON THE INTERCALATION OF AROMATIC CATIONS TO DEOXYRIBONUCLEIC ACID

Chemischer InformationsdienstVolume 5, Issue 4 Physical Organic Chemistry ChemInform Abstract: TOPOGRAPHY OF NUCLEIC ACID HELICES IN SOLUTION PART 29, STERIC EFFECTS ON THE INTERCALATION OF AROMATIC CATIONS TO DEOXYRIBONUCLEIC ACID E. J. GABBAY, E. J. GABBAYSearch for more papers by this authorR. E. SCOFIELD, R. E. SCOFIELDSearch for more papers by this authorC. S. BAXTER, C. S. BAXTERSearch for more papers by this author E. J. GABBAY, E. J. GABBAYSearch for more papers by this authorR. E. SCOFIELD, R. E. SCOFIELDSearch for more papers by this authorC. S. BAXTER, C. S. BAXTERSearch for more papers by this author First published: January 29, 1974 https://doi.org/10.1002/chin.197404090Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume5, Issue4January 29, 1974 RelatedInformation