The spatial configuration of ordered polynucleotide chains. I. Helix formation and base stacking.

A single virtual bond scheme set forth previously for the treatment of average properties of randomly coiling polynucleotides is here applied to the calculation of helical parameters which characterize a regularly repeating polynucleotide molecule. Only a fraction of the enormous number of conformationally feasible helixes fulfill the geometric criteria of vertical base stacking usually associated with ordered polynucleotide chains. Detailed examination of the nature and mode of base stacking feasible in a single helical backbone structure indicates that the handedness of a base stacking arrangement does not correlate either quantitatively or qualitatively with the handedness of the polymer backbone. A number of polynucleotide chains which exhibit lefthanded base stacking patterns in nmr and CD studies may, in fact, be righthanded helixes.     

Information contained in the amino acid sequence of theα1(I)-chain of collagen and its consequences upon the formation of the triple helix,of fibrils and crosslinks

The molecule of type I collagen from skin consists of two alpha1(I)-chains and one alpha2-chain. The sequence of the entire alpha1-chain comprising 1052 residues is summarily presented and discussed. Apart from the 279 residues of alpha1(I)-CB8 whose sequence has been established for rat skin collagen, all sequences have been determined for calf skin collagen. In order to facilitate sequence analysis, the alpha1-chain was cleaved into defined fragments by cyanogen bromide or hydroxylamine or limited collagenase digestion. Most of the sequence was established by automated stepwise Edman degradation. The alpha1-chain contains two basically different types of sequences: the triple helical region of 1011 amino acid residues in which every third position is occupied by glycine and the N- and C-terminal regions not displaying this type of regularity. Both of these non-triple helical regions carry oxidizable lysine or hydroxylysine residues as functional sites for the intermolecular crosslink formation. Implications of the amino acid sequence for the stability of the triple helix and the fibril as well as for formation of crosslinks are discussed. Evaluation of the sequence in connection with electron microscopical investigations yielded the parameters of the axial arrangement of the molecules within the fibrils. Axial stagger of the molecules by a distance D = 670 angstrom = 233 amino acid residues results in maximal interaction of polar sequence regions of adjacent molecules and similarly of regions of hydrophobic residues. Ordered aggregation of molecules into fibrils is, therefore, regulated by electrostatic and electrophobic forces. Possible loci of intermolecular crosslinks between the alpha1-chains of adjacent molecules may be deduced from the dimensions of the axial aggregation of molecules.     

Atomic resolution analysis of a 2:1 complex of CpG and acridine orange.

Cytidylyl-3', 5'-guanosine and acridine orange crystallize in a highly-ordered triclinic lattice which diffracts X-rays to 0.85 angstrom resolution. The crystal structure has been solved and refined to a residual factor of 9.5%. The two dinucleoside phosphate molecules form an antiparallel double helix with the acridine orange intercalated between them. The two base pairs of the double helical fragment have a twist angle of 10 degrees and it is found to have a C3' endo-(3', 5')-C2' endo mixed sugar puckering along the nucleotide backbone as has been observed for other simple intercalator complexes. Twenty-five water molecules have been located in the lattice together with a sodium ion. The intercalator double helical fragments form sheets which are held together by van der Waals interactions in one direction and hydrogen bonding interactions in the other. The crystal lattice contains aqueous channels in which sixteen water molecules are hydrogen bonded to the nucleotide, none to the intercalator, five water molecules are coordinated about the sodium ion and four water molecules bind solely to other water molecules. The bases in the base pairs have a dihedral angle of 7 to 8 degrees between them.     

Molecular structure of short-chain (SC) cartilage collagen by electron microscopy

We have recently observed that aged and/or hypertrophying chondrocytes in culture synthesize a small collagen molecule termed short-chain (SC) collagen. Our previous biochemical studies have suggested that this molecule is slightly less than half the length of "typical" interstitial collagens and should have both a helical, collagenous domain and a nonhelical, globular one. In the present study we have examined the structure of this molecule by electron microscopy of rotary-shadowed preparations and segment-long-spacing crystallites. Rotary-shadowed SC collagen molecules appear as rods with a length of 132 nm and a knob at one end. Preparations of native molecules that have been treated by limited pepsin digestion show only the rod-like domain. These results are consistent with the rod-like domain having the molecular structure of a collagen helix, which is refractory to pepsin digestion, and the knob representing a globular, nonhelical domain. Segment-long-spacing crystallites of pepsin-digested molecules confirm the length of the helical domain to be 132 nm. Positively stained crystallites show a banding pattern different from other collagens.     

Three-dimensional reconstruction of tubular crystals of catalase

On the basis of electron microscope data the structure of tubular crystals of catalase has been determined with resolution of approximately 25 A. The symmetry of the helical packing of molecules is 142/17. The three-dimensional reconstruction has been carried out in real space. The catalase molecule consists of four subunits whose centers from a fairly flattened tetrahedron. The molecule has dimensions of 69X87X92 A.     

Molecular modelling study of changes induced by netropsin binding to nucleosome core particles.

It is well known that certain sequence-dependent modulators in structure appear to determine the rotational positioning of DNA on the nucleosome core particle. That preference is rather weak and could be modified by some ligands as netropsin, a minor-groove binding antibiotic. We have undertaken a molecular modelling approach to calculate the relative energy of interaction between a DNA molecule and the protein core particle. The histones particle is considered as a distribution of positive charges on the protein surface that interacts with the DNA molecule. The molecular electrostatic potentials for the DNA, simulated as a discontinuous cylinder, were calculated using the values for all the base pairs. Computing these parameters, we calculated the relative energy of interaction and the more stable rotational setting of DNA. The binding of four molecules of netropsin to this model showed that a new minimum of energy is obtained when the DNA turns toward the protein surface by about 180 degrees, so a new energetically favoured structure appears where netropsin binding sites are located facing toward the histones surface. The effect of netropsin could be explained in terms of an induced change in the phasing of DNA on the core particle. The induced rotation is considered to optimize non-bonded contacts between the netropsin molecules and the DNA backbone.     

Model of biologically active apolipoprotein E bound to dipalmitoylphosphatidylcholine

Apolipoprotein (apo)E plays a critical role in cholesterol transport, through high affinity binding to the low density lipoprotein receptor. This interaction requires apoE to be associated with a lipoprotein particle. To determine the structure of biologically active apoE on a lipoprotein particle, we crystallized dipalmitoylphosphatidylcholine particles containing two apoE molecules and determined the molecular envelope of apoE at 10 Angstroms resolution. On the basis of the molecular envelope and supporting biochemical evidence, we propose a model in which each apoE molecule is folded into a helical hairpin with the binding region for the low density lipoprotein receptor at its apex.     

Structural changes that occur in scallop myosin filaments upon activation

Myosin filaments isolated from scallop striated muscle have been activated by calcium-containing solutions, and their structure has been examined by electron microscopy after negative staining. The orderly helical arrangement of myosin projections characteristic of the relaxed state is largely lost upon activation. The oblique striping that arises from alignment of elongated projections along the long-pitched helical tracks is greatly weakened, although a 145 A axial periodicity is sometimes partially retained. The edges of the filaments become rough, and the myosin heads move outwards as their helical arrangement becomes disordered. Crossbridges at various angles appear to link thick and thin filaments after activation. The transition from order to disorder is reversible and occurs over a narrow range of free calcium concentration near pCa 5.7. Removal of nucleotide, as well as dissociation of regulatory light chains, also disrupts the ordered helical arrangement of projections. We suggest that the relaxed arrangement of the projections is probably maintained by intermolecular interactions between myosin molecules, which depend on the regulatory light chains. Calcium binding changes the interactions between light chains and the rest of the head, activating the myosin molecule. Intermolecular contacts between molecules may thus be altered and may propagate activation cooperatively throughout the thick filament.     

The nature of the helix-random coil transition of biological macromolecules attached to a surface

The effect of the presence of a surface on the helix–random coil transition is investigated. It is found that a grand canonical ensemble formation used previously to solve exactly the problem of a polymer between two plates can be used to solve approximately the problem of DNA near a plane surface. The formalism is applied to the homogeneous perfect-matching model of infinite molecular weight. A crucial part of the calculation for double-stranded molecules involves the evaluation of the entropy of the loops connecting the helical portions of the double-stranded chains. One obtains as a measure of the configurational freedom of a loop Asⁱ/j^c where c has the following values: for a loop connecting two helical regions both off the surface c = 3/2; for a loop connecting a helical region on the surface to a helical region off the surface c = 5/2; for a loop connecting helixes both on the surface c = 4. Corresponding values for an n-stranded molecule are c = (3n ? 3)/2, c = (4n ? 3)/2, c = (5n ? 2)/2. In all cases, the effect of the surface is to sharpen the transition. In the case of double-stranded molecules, the transition becomes first order. We take the view that self-assembly of biological macromolecules can be considered as a sharp thermodynamic phase transition. Thus, the above systems become models of self-assembling systems. They are also relevant to the problem of surface-induced enzymatic activity.     

Thermodynamics of Forming a Parallel DNA Crossover

The process of genetic recombination involves the formation of branched four-stranded DNA structures known as Holliday junctions. The Holliday junction is known to have an antiparallel orientation of its helices, i.e., the crossover occurs between strands of opposite polarity. Some intermediates in this process are known to involve two crossover sites, and these may involve crossovers between strands of identical polarity. Surprisingly, if a crossover occurs at every possible juxtaposition of backbones between parallel DNA double helices, the molecules form a paranemic structure with two helical domains, known as PX-DNA. Model PX-DNA molecules can be constructed from a variety of DNA molecules with five nucleotide pairs in the minor groove and six, seven or eight nucleotide pairs in the major groove. A topoisomer of the PX motif is the juxtaposed JX₁ molecule, wherein one crossover is missing between the two helical domains. The JX₁ molecule offers an outstanding baseline molecule with which to compare the PX molecule, so as to measure the thermodynamic cost of forming a crossover in a parallel molecule. We have made these measurements using calorimetric and ultraviolet hypochromicity methods, as well as denaturing gradient gel electrophoretic methods. The results suggest that in relaxed conditions, a system that meets the pairing requirements for PX-DNA would prefer to form the PX motif relative to juxtaposed molecules, particularly for the 6:5 structure.     

Mg2+ Dependence of the Structure and Thermodynamics of Wheat Germ and Lupin Seeds 5S rRNA

Abstract

The formation and stability of structural elements in two 5S rRNA molecules from wheat germ (WG) and lupin seeds (LS) as a function of Mg²⁺ concentration in solution was determined using the adiabatic differential scanning microcalorimetry (DSC). The experimentally determined thermodynamic parameters are compared with calculations using thermodynamic databases used for prediction of RNA structure. The 5S rRNA molecules which show minor differences in the nucleotide sequence display very different thermal unfolding profiles (DSC profiles). Numerical deconvolution of DSC profiles provided information about structural transformations that take place in both 5S rRNA molecules. A comparative analysis of DSC data and the theoretical thermodynamic models of the structure was used to establish a relationship between the constituting transitions found in the melting profiles and the unfolding of structural domains of the 5S rRNA and stability of its particular helical elements.

Increased concentration of Mg²⁺ ions induces additional internal interactions stabilising 5S rRNA structures found at low Na⁺ concentrations. Observed conformational transitions suggest a structural model in which the extension of helical region E dominates over the postulated tertiary interaction between hairpin loops. We propose that helix E is stabilised by a sequence of non-standard pairings extending this helix by the formation of tetra loop e and an almost total reduction of loop d between helices E and D. Two hairpin structures in both 5S rRNA molecules: the extended C-C' and the extended E-E'-E” hairpins appear as the most stable elements of the structure. The cooperativity of the unfolding of helixes in these 5S rRNA molecules changes already at 2 mM Mg²⁺.     

Ultrastructural and anti-proteinase activity modifications of human alpha 2-macroglobulin induced by zinc and other divalent cations

Native tetrameric alpha 2-macroglobulin molecules (alpha 2M) can be converted into a population of dimers by incubation with various divalent cations such as Zn, Cd, Mg, Cu, Ni, Co. This dissociation is completed within 30 min at 37 degrees C. These dimers have a characteristic shape and a size of about 16 X 8 nm, and appear to be the half of the native alpha 2M molecule which has a clear tetrameric structure as seen in the electron microscope. At room temperature or below, dimers obtained with 5 to 100 mM Zn++ can reassociate in long linear polymers which display a regular chain-like arrangement and a helical periodicity. The structural characteristics of this polymer are described. The trypsin inhibitory capacity of Zn++-treated alpha 2M has been studied in an attempt to correlate its Zn++-induced conformational changes with its functional modifications.     

The effect of sequence patterns and local conformational preferences on the structure of collapsed polypeptide

We studied the structure of a polypeptide model by means of the Monte Carlo method. The model chain consisting of two kinds of residues (hydrophobic and hydrophilic) was confined on the (310) hybrid lattice. The residues interacted with the long-range contact potential. The short-range potential was also used by introducing the preferences of conformations corresponding to the helical structure. Simulations of the coil-to-globule collapse were done by an annealing process starting from high-temperature structures and then gradual cooling of the system. The parameters describing the behavior of the system were monitored. It has been found that in a case of a helical pattern -HHPPHPP- the collapsed chains consisted of helical fragments. The resulting structures were formed in such way that the polar residues were located in the outer shell of the molecule since the hydrophobic residues filled the inner part of molecule. The results showed that the proper balance between the magnitude of the potentials used in a model is important and its influence on final structures of molecules was discussed.     

The molecular structure of adrenal medulla kinesin

The molecular structure of bovine adrenal kinesin was studied by electron microscopy using the low-angle rotary shadowing technique. Adrenal kinesin exhibited either a folded or an extended configuration; the ratio of the two is dependent on the salt concentration. Almost all adrenal kinesin molecules were folded in a low-ionic solution, and the ratio of extended molecules increased to 40-50% in a solution containing 1 M ammonium acetate. Kinesin in the extended configuration displayed a rod-shaped structure with a mean length of about 80 nm. The morphologies of the ends were different; one end was composed of two globular particles, similar to the two-headed structure of myosin, while the other end had a more ill-defined structure, appearing either as a globular particle, an aggregate of two to four small granules, or a frayed, fan-like structure. The folded kinesin molecule possessed a hinge region in the middle of the rod, at about 32 nm from the neck of the two heads. In our preparations, the majority of adrenal kinesin molecules were folded at physiological salt concentrations. Adrenal kinesin bound to microtubules in the presence of adenylyl imidodiphosphate (AMP-PNP) also displayed a folded morphology.     

A novel mutation causes a perinatal lethal form of osteogenesis imperfecta. An insertion in one alpha 1(I) collagen allele (COL1A1)

We have identified an infant with the perinatal lethal form of osteogenesis imperfecta (type II) whose cells synthesize in equal amounts two different pro alpha 1(I) chains of type I procollagen: one chain is normal in length, the other contains an insertion of approximately 50-70 amino acid residues within the triple helical domain defined by amino acids 123-220. The structure of the insertion is consistent with duplication of an approximately 600-base pair segment in one allele of the alpha 1(I) gene (COL1A1). These cells synthesize normal type I procollagen molecules as well as molecules that contain one or two mutant chains. Unlike type I procollagen molecules synthesized by cells from most other infants with osteogenesis imperfecta type II which contain increased lysyl hydroxylation and hydroxylysyl glycosylation along the triple helical domain, the abnormal molecules synthesized by these cells are not overmodified. The lethal effect of this mutation may result from secretion of about one-quarter the normal amount of normal type I procollagen and secretion of a large amount of a molecule which has a lowered melting temperature, is extended asymmetrically, and which has altered structure in domains important for cross-link formation and bone mineralization.     

A comparison of optimal and suboptimal RNA secondary structures predicted by free energy minimization with structures determined by phylogenetic comparison.

This article describes the latest version of an RNA folding algorithm that predicts both optimal and suboptimal solutions based on free energy minimization. A number of RNA's with known structures deduced from comparative sequence analysis are folded to test program performance. The group of solutions obtained for each molecule is analysed to determine how many of the known helixes occur in the optimal solution and in the best suboptimal solution. In most cases, a structure about 80% correct is found with a free energy within 2% of the predicted lowest free energy structure.     

Comparison of the structures of the native form of rat liver 5S rRNA and yeast tRNAphe: small-angle and wide-angle X-ray scattering study

The small-angle and wide-angle X-ray scattering of tRNA^phe (yeast) and ribosomal 5S RNA (rat liver) in solution have been analysed and compared. tRNA^phe in solution is folded into a compact L-shaped structure similar to its structure in crystals. The geometry of the secondary structure of the double helical regions is also equivalent to the A-form in the crystalline state. Despite differences between the molar mosses of 5S rRNA (40 000 g mol^?1) and tRNA^phe (25 000 g mol^?1), and the fact that the 5S rRNA molecule is more anisometric than the tRNA^phe molecule, there are many structural similarities. The geometrical parameters of the secondary structure of double helical regions in both RNA molecules are almost identical; the mean rise per base pair is about 0.253–0.28 nm and the mean turn angle is about 32.5–33.5. Identical cross-sectional radii of gyration, R_sq,1 ≈ 1.16 nm and R_sq,2 = 0.92 nm, identical molar mass per unit length, , and a mean thickness of the molecules D ≈ 1.65 nm suggest a similar, nearly coplanar organization of isolated, double helical arms. Furthermore, there are compact regions in the central parts of both molecules, which are the sites of tertiary interactions in the tRNA^phe molecule and are a potential site of tertiary interactions in the SS rRNA molecule for stabilization of the complicated L-shape of the two molecules. Both molecules have a pseudo-twofold axis,w hich may play a role in recognition for binding of specific proteins.     

Crystal structure analysis of the tetragonal crystal form are preliminary molecular model of pig-heart citrate synthase.

The crystal structure of pig heart citrate synthase was analyzed at 0.35-nm resolution. Chain tracing was possible and an initial molecular model constructed. The dimensions of the dimer molecule (located on a crystallographic diad) are 7.5 x 6.0 x 9.0 nm. The chain folding is characterized by the predominance of helices and the absence of sheet structure. The electron density accounts for 355 residues per monomer, so that about 80 residues must be disordered in the crystal. The disordered segment in probably N-terminal. The ordered part consists of two closely associated domains, a large domain with 300 residues and a C-terminal domain of 55 residues consisting of 3(anti)parallel helices. The large domain is built from 12 helical segments, some of which are buried in the interior of the molecule. Inhibitor binding studies with citrate and CoA revealed citrate binding sites but showed no electron density for CoA. It is suggested that CoA binds to the disordered, flexible N-terminal domain. Experiments of limited proteolysis with trypsin showed that under conditions a segment of Mr 9000 is cleaved off selectively. The remaining 35 000-Mr part is dimeric.     

Stabilization of acetylcholine receptor secondary structure by cholesterol and negatively charged phospholipids in membranes

Fourier-transform infrared (FTIR) spectroscopy was used to study the secondary structure of purified Torpedo californica nicotinic acetylcholine receptor (AChR) in reconstituted membranes. Functional studies have previously demonstrated that the ion channel activity requires the presence of both sterol and negatively charged phospholipids in membranes. The present studies are designed to test the hypothesis that the alpha-helical structure of AChR may be stabilized by specific lipid molecules (sterol and/or negatively charged phospholipids) and that these alpha-helices may be responsible for the formation of a potential ion channel. FTIR data show statistically significant (p less than 0.005) spectral changes due to cholesterol and negatively charged phospholipids, respectively. On the basis of standard curves describing the relationship between the spectral properties and the secondary structural contents of water-soluble proteins, the observed spectral change at 931 cm-1 can be interpreted as an apparent change in the alpha-helix content from about 17% in the absence of sterols to about 20% in the presence of sterols, suggesting that protein-sterol interactions increase the helical structure of the AChR molecule. Similarly, the spectral change at 988 cm-1 can be interpreted as an apparent increase of beta-sheet content in the AChR molecule from about 20% to about 24% due to the presence of negatively charged phospholipids. Functional AChR in membranes thus appears to be correlated with higher alpha-helical and beta-sheet contents. It is concluded that one role of specific interactions between membrane protein and lipid molecules may be to maintain specific secondary structures necessary to support the ion channel function of AChR.     

Evidence for an extended 7SL RNA structure in the signal recognition particle.

The signal recognition particle (SRP) functions in conjunction with the SRP receptor to target nascent ectoplasmic proteins to the protein translocation machinery of the endoplasmic reticulum membrane. SRP is a ribonucleoprotein consisting of six distinct polypeptides and one molecule of 7SL RNA 300 nucleotides long. SRP has previously been visualized by a variety of electron microscopic techniques as a rod-shaped particle 24 nm long and 6 nm wide. We report here microanalysis by electron spectroscopic imaging which localizes the RNA molecule in SRP to primarily the two ends of the particle. These results suggest that the single 7SL RNA molecule spans the length of the particle. Micrographs from a scanning transmission electron microscope permit visualization of unstained SRP with low electron exposure, as well as the direct measurement of the mol. wt of the particle. These micrographs confirm our earlier suggestion that SRP is divided into three structural domains and allow discrimination of the two ends of the structure. The results of both techniques have been combined in a model for the structure of SRP in which we propose the basic orientation of the 7SL RNA. The structure proposed is consistent with the secondary structure predicted for the RNA and with biochemical data.