Energetics of intercalation specificity. I. Backbone unwinding.

An empirical partitioned-potential function with optimized parameters was employed to investigate the basis of intercalation specificity recently observed by experimental techniques. The nature of this specificity is discussed in terms of component interactions for all non-truncated complementary dinucleoside triphosphate mini- (or miniature) helices. Our calculations agree with available evidence indicating the preference of Pyr(3′-5′)Pur sequences over Pur(3′-5′)Pyr sequences to change from the B-DNA to the intercalated conformation. Base–base (stacking) and base–phosphate interactions control the specificity. The extent of net electrostatic charge on the phosphate groups play an important but limited role in establishing the observed specificity. These results should apply for the class of aromatic agents involved in nonspecific intercalation with nucleic acids but not necessarily for aromatic agents involved in specific reactions with a particular nucleotide base.     

Conformational flexibility and structure of creatine kinase

The structural flexibility of creatine kinase has been investigated with the covalent hydrophobic probe 2-[4′-(2″-iodoacetamido) phenyl] aminonaphthalene-6-sulfonic acid (IAANS) which reacts at vastly different rates with the two subunits to give a protein conjugate with fluorescence characteristic of reaction with a site in a hydrophobic cleft. Binding of purine nucleotides greatly enhances the probe fluorescence while pyrimidine nucleotides quench the fluorescence. Small anions bind to nucleotide-free creatine kinase near the location of the transferable phosphoryl group and quench both the IAANS fluorescence of modified creatine kinase and the tryptophan fluorescence of native creatine kinase. Chloride and nitrate non-competitively inhibit MgADP binding both with and without creatine. Fluorescence energy transfer demonstrates that the active sites of creatine kinase are well separated and become further apart after the nucleotide-induced conformational change.     

Conformational flexibility revealed by the crystal structure of a crenarchaeal RadA

Homologous recombinational repair is an essential mechanism for repair of double-strand breaks in DNA. Recombinases of the RecA-fold family play a crucial role in this process, forming filaments that utilize ATP to mediate their interactions with single- and double-stranded DNA. The recombinase molecules present in the archaea (RadA) and eukaryota (Rad51) are more closely related to each other than to their bacterial counterpart (RecA) and, as a result, RadA makes a suitable model for the eukaryotic system. The crystal structure of Sulfolobus solfataricus RadA has been solved to a resolution of 3.2 Å in the absence of nucleotide analogues or DNA, revealing a narrow filamentous assembly with three molecules per helical turn. As observed in other RecA-family recombinases, each RadA molecule in the filament is linked to its neighbour via interactions of a short β-strand with the neighbouring ATPase domain. However, despite apparent flexibility between domains, comparison with other structures indicates conservation of a number of key interactions that introduce rigidity to the system, allowing allosteric control of the filament by interaction with ATP. Additional analysis reveals that the interaction specificity of the five human Rad51 paralogues can be predicted using a simple model based on the RadA structure.     

From triplex to B-form duplex stabilization: reversal of target selectivity by aminoglycoside dimers

Aminoglycosides have been shown to target A-form nucleic acids. Our work has previously shown that neomycin (and other aminoglycosides) bind and stabilize DNA/RNA triplexes and other A-form nucleic acids. We report herein the unexpected B-form duplex stabilization shown by aminoglycoside dimers (neomycin-neomycin and neomycin-tobramycin). The dimers are highly selective for AT rich duplexes and show high affinity (K(a) approximately 10(8)M(-1)) as determined by isothermal titration calorimetry.     

On the flexibility of the boundaries between the A-form and B-form sections in DNA molecule.

The degree of orientation of DNA in a flow has been studied within the interval of the B - A transition induced by ethanol. The orientation of the B DNA (60-65% ethanol, v/v) and that of the A DNA (80-82% ethanol) are nearly identical. This means that both conformations have similar persistence lengths and that there is no aggregation in the course of formation of the A form. Within the transition range (65-78% ethanol) the orientation attains a sharp minimum which coincides with the half-transition point (73% ethanol). The cooperative character of the B - A transition presupposes the existence of boundaries between the alternating sections of the A and B conformations that may entail an increased flexibility of the DNA molecule and a corresponding drop of orientation. Theory predicts an elliptical dependence of the number of boundaries on the proportion of the A form. The experimental degree of orientation follows the same pattern. Quantitative evaluation shows that the flexibility of a boundary is small, so that several dozen of boundaries are required to simulate free rotation.     

Conformational flexibility in the molecular structure of rotenone, a naturally occurring insecticide

The crystal and molecular structure of rotenone, a naturally occurring insecticide with mitochondrial and mitotic spindle inhibitory properties, was determined by direct methods. The crystals were orthorhombic, space group, P2_I2_I2_I with two molecules in the asymmetric unit; a = 8.413 (1) Å, b = 19.840(1), c = 23.581(1), V = 3936 ų, Z = 8. The structure was refined by least-squares methods to a final R = 0.067. The two molecules in the asymmetric unit have different conformations about the junction between the nonaromatic rings B and C. Ring B is in a sofa conformation in both molecules, with a slight distortion toward a half-chair in I, but with C8 and C8′ on opposite sides of the planar part of the rings. This difference in conformation results in I having an extended (linear) shape while II is V-shaped. The more elongated conformation of the molecule (I) has not been reported in previous studies. Ring C also has opposite conformations in the two molecules. The angle between the planes formed by rings A and D in molecule I is 64.3°, while in molecule II it is 88.3°. Molecular mechanics techniques were used to determine the energy of the two conformations. These calculations, at room temperature, predict molecule II to be the more stable conformer. The highly flexible site in the B/C ring junction is also chemically very reactive. This flexibility and reactivity are further discussed in terms of rotenone's inhibitory activities.     

Conformational flexibility in DNA structure and its implications in understanding the organization of DNA in chromatin.

X-ray crystallographic studies of drug-nucleic acid crystalline complexes have suggested that DNA first bends or 'kinks' before accepting an intercalative drug or dye. This flexibility in DNA structure is made possible by altering the normal C2' endo deoxyribose sugar puckering in B DNA to a mixed sugar puckering pattern of the type C3' endo (3'-5') C2' endo and partially unstacking base pairs. A kinking scheme such as this would require minimal sterochemical rearrangement and would also involve small energies. This has prompted us to ask more generally if a conformational change such as this could be used by proteins in their interactions with DNA. Here we describe an interesting superhelical DNA structure formed by kinking DNA every ten base pairs. This structure may be used in the organization of DNA within the nucleosome structure in chromatin.     

Conformational flexibility of the acetylcholinesterase tetramer suggested by x-ray crystallography.

Acetylcholinesterase, a polymorphic enzyme, appears to form amphiphilic and nonamphiphilic tetramers from a single splice variant; this suggests discrete tetrameric arrangements where the amphipathic carboxyl-terminal sequences can be either buried or exposed. Two distinct, but related crystal structures of the soluble, trypsin-released tetramer of acetylcholinesterase from Electrophorus electricus were solved at 4.5 and 4.2 A resolution by molecular replacement. Resolution at these levels is sufficient to provide substantial information on the relative orientations of the subunits within the tetramer. The two structures, which show canonical homodimers of subunits assembled through four-helix bundles, reveal discrete geometries in the assembly of the dimers to form: (a) a loose, pseudo-square planar tetramer with antiparallel alignment of the two four-helix bundles and a large space in the center where the carboxyl-terminal sequences may be buried or (b) a compact, square nonplanar tetramer that may expose all four sequences on a single side. Comparison of these two structures points to significant conformational flexibility of the tetramer about the four-helix bundle axis and along the dimer-dimer interface. Hence, in solution, several conformational states of a flexible tetrameric arrangement of acetylcholinesterase catalytic subunits may exist to accommodate discrete carboxyl-terminal sequences of variable dimensions and amphipathicity.     

Conformational flexibility of alpha-lactalbumin related to its membrane binding capacity

Different folding states of the small, globular milk protein bovine alpha-lactalbumin (BLA) induced by the anionic surfactant sodium dodecylsulphate (SDS) have been examined by fluorescence spectroscopy, CD and NMR. The solution structure of the protein in the absence of SDS was also determined, indicating fluidity even under native conditions. BLA is partly denatured to a molten globule (MG)-like state by micromolar concentrations of SDS, and the transitions from native to MG-like state are dependent on pH, the protein being more sensitive to the surfactant at pH 6.5. As indicated by measurements of the intrinsic emission fluorescence, the tertiary structure disappears at lower concentrations of SDS than most of the secondary structure, as estimated from CD data. The MG-like state induced by low concentrations of SDS is not observable by NMR, and is probably fluctuating and/or aggregating. At higher concentrations of SDS above the critic concentration of micelles, an NMR-observable state reappears. This micelle-associated conformer was partially assigned, and found to bear strong resemblance to the acid-tri-fluoroethanol state, retaining weakened versions of the A and C helix of native BLA. We discuss the results in terms of the inherent flexibility of the protein, and its ability to form multiple folding states and to bind to membranes. Also, we propose that proteins with stable MG-like conformers can have these states stabilized by low levels of compounds with surfactant properties in vivo.     

Enzymic unwinding of DNA. 2. Chain separation by an ATP-dependent DNA unwinding enzyme.

The DNA-stimulated ATPase characterized in the accompanying paper is shown to be a DNA unwinding enzyme. Substrates employed were DNA, RNA hybrid duplexes and DNA-DNA partial duplexes prepared by polymerization on fd phage single-stranded DNA template. The enzyme was found to denature these duplexes in an ATP-dependent reaction, without detectably degrading. EDTA, an inhibitor of the Mg2+-requiring ATPase, was found to prevent denaturation suggesting that dephosphorylation of the ATP and not only its presence is required. These results together with those from enzyme-DNA binding studies lead to ideas regarding the mode of enzymic action. It is proposed that the enzyme binds, in an initial step, to a single-stranded part of the DNA substrate molecule and that from here, energetically supported by ATP dephosphorylation, it invades double-stranded parts separating base-paired strands by processive, zipper-like action. It is further proposed that chain separation results from the combined action of several enzyme molecules and that a tendency of the enzyme to aggregate with itself reflects a tendency of the molecules to cooperate. Various functions are conceivable for the enzyme.     

Conformational flexibility in the apolipoprotein E amino-terminal domain structure determined from three new crystal forms: implications for lipid binding

An amino-terminal fragment of human apolipoprotein E3 (residues 1-165) has been expressed and crystallized in three different crystal forms under similar crystallization conditions. One crystal form has nearly identical cell dimensions to the previously reported orthorhombic (P2(1)2(1)2(1)) crystal form of the amino-terminal 22 kDa fragment of apolipoprotein E (residues 1-191). A second orthorhombic crystal form (P2(1)2(1)2(1) with cell dimensions differing from the first form) and a trigonal (P3(1)21) crystal form were also characterized. The structures of the first orthorhombic and the trigonal form were determined by seleno-methionine multiwavelength anomalous dispersion, and the structure of the second orthorhombic form was determined by molecular replacement using the structure from the trigonal form as a search model. A combination of modern experimental and computational techniques provided high-quality electron-density maps, which revealed new features of the apolipoprotein E structure, including an unambiguously traced loop connecting helices 2 and 3 in the four-helix bundle and a number of multiconformation side chains. The three crystal forms contain a common intermolecular, antiparallel packing arrangement. The electrostatic complimentarity observed in this antiparallel packing resembles the interaction of apolipoprotein E with the monoclonal antibody 2E8 and the low density lipoprotein receptor. Superposition of the model structures from all three crystal forms reveals flexibility and pronounced kinks in helices near one end of the four-helix bundle. This mobility at one end of the molecule provides new insights into the structural changes in apolipoprotein E that occur with lipid association.     

Conformational flexibility of angiotensin II. A carbon-13 spin-lattice relaxation study.

Carbon-13 spin-lattice relaxation times (T1) have been determined for the carbon in the octapeptide hormone [5-isoleucine]-angiotensin II in aqueous solution. Two possible models for molecular motion are considered: isotropic overall motion of the hormone with internal motion of some residues and anisotropic overall molecular motion. The data are interpreted in detail using the former model. The alpha carbons of the peptide backbone are all equally restricted in their motion. The correlation time for overall molecular reorientation, calculated from an everage T1 value of 95 msec for the alpha carbons in the peptide backbone, is ca. 5 times 10-10 sec. The carbons in the side chains are more mobile than those in the peptide backbone, with the exception of the side chain of the Tyr residue which does not undergo rapid segmental motion. We propose that [5-isoleucine]-angiotensin II has a restricted backbone conformation and that the alpha carbons of the N- and C-terminal residues are constrained to nearly the same extent as the remaining alpha carbons in the peptide backbone. Chemical shift data indicate that the Pro residue adopts the trans conformation about the His-Pro bond and that the imidazole ring of His has a strong preference for the N-tau -H tautomer.     

Conformational flexibility of avidin: the influence of biotin binding

Ligand binding to proteins is a key process in cell biochemistry. The interaction usually induces modifications in the unfolding thermodynamic parameters of the macromolecule due to the coupling of unfolding and binding equilibria. In addition, these modifications can be attended by changes in protein structure and/or conformational flexibility induced by ligand binding. In this work, we have explored the effect of biotin binding on conformation and dynamic properties of avidin by using infrared spectroscopy including kinetics of hydrogen/deuterium exchange. Our results, along with previously thermodynamic published data, indicate a clear correlation between thermostability and protein compactness. In addition, our results also help to interpret the thermodynamic binding parameters of the exceptionally stable biotin:AVD complex.     

Conformational flexibility of the regulatory site of phosphoribulokinase as demonstrated with bifunctional reagents.

Phosphoribulokinase (PRK) is one of several chloroplastic enzymes whose activity is regulated by thiol-disulfide exchange via thioredoxin. Activation entails reduction of an active-site disulfide bond between Cys16 and Cys55. Bifunctional cross-linking reagents have been used to approximate the interresidue distance between Cys16 and Cys55, an issue which impinges on the relative conformational states of the activated and deactivated forms of the enzyme. Spinach PRK is rapidly inactivated by stoichiometric levels of 4,4'-difluoro-3,3'-dinitrodiphenyl sulfone (FNPS) or 1,5-difluoro-2,4-dinitrobenzene (DFNB), which span 9 and 3.5 A, respectively. ATP, but not ribulose 5-phosphate, retards the rate of inactivation, suggesting that modification has occurred at the nucleotide binding domain of the active site. Sulfhydryl modification is indicated by partial reversibility of inactivation as effected by exogenous thiols. Tryptic mapping by reverse-phase chromatography of [14C]carboxymethylated enzyme, subsequent to its reaction with either FNPS or DFNB, demonstrates modification of Cys16 and Cys55 by both reagents, and formation of only one major chromophoric peptide in each case. On the basis of the sequence analysis of the purified chromophoric peptides, Cys16 and Cys55 are cross-linked by both FNPS and DFNB. Thus, the intrasubunit distance between the beta-sulfhydryls of Cys16 and Cys55 is dynamic rather than static. Diminished conformational flexibility upon oxidation of the regulatory sulfhydryls to a disulfide may be partially responsible for the concomitant loss of enzymatic activity.     

Conformational flexibility of the N-terminal domain of apolipoprotein a-I bound to spherical lipid particles

Lipid binding of human apolipoprotein A-I (apoA-I) occurs initially through the C-terminal alpha-helices followed by conformational reorganization of the N-terminal helix bundle. This led us to hypothesize that apoA-I has multiple lipid-bound conformations, in which the N-terminal helix bundle adopts either open or closed conformations anchored by the C-terminal domain. To investigate such possible conformations of apoA-I at the surface of a spherical lipid particle, site-specific labeling of the N- and C-terminal helices in apoA-I by N-(1-pyrene)maleimide was employed after substitution of a Cys residue for Val-53 or Phe-229. Neither mutagenesis nor the pyrene labeling caused discernible changes in the lipid-free structure and lipid interaction of apoA-I. Taking advantage of a significant increase in fluorescence when a pyrene-labeled helix is in contact with the lipid surface, we monitored the behaviors of the N- and C-terminal helices upon binding of apoA-I to egg PC small unilamellar vesicles. Comparison of the binding isotherms for pyrene-labeled apoA-I as well as a C-terminal helical peptide suggests that an increase in surface concentration of apoA-I causes dissociation of the N-terminal helix from the surface leaving the C-terminal helix attached. Consistent with this, isothermal titration calorimetry measurements showed that the enthalpy of apoA-I binding to the lipid surface under near saturated conditions is much less exothermic than that for binding at a low surface concentration, indicating the N-terminal helix bundle is out of contact with lipid at high apoA-I surface concentrations. Interestingly, the presence of cholesterol significantly induces the open conformation of the helix bundle. These results provide insight into the multiple lipid-bound conformations that the N-terminal helix bundle of apoA-I can adopt on a lipid or lipoprotein particle, depending upon the availability of space on the surface and the surface composition.     

Anisotropic flexibility of DNA and the nucleosomal structure.

Potential energy calculations of the DNA duplex dimeric subunit show that the double helix may be bent in the direction of minor and major grooves much more easily than in other directions. It is found that the total winding angle of DNA decreases upon such bending. A new model for DNA folding in the nucleosome is proposed on the basis of these findings according to which the DNA molecule is kinked each fifth base pair to the side of the minor and major grooves alternatively. The model explains the known contradiction between a C-like circular dichroism for the nucleosomal DNA and the nuclease digestion data, which testify to the B-form of DNA.