The B-Z conformational transition and aggregation of poly[d(G-C)] induced by moderate concentrations of Mg(CIO4)2

Mg(ClO4)2 induces the cooperative B-to-Z transition of poly[d(G-C)]; the salt concentration at the midpoint is 0.26 M. A comparison with previous data for NaCl, MgCl2 and NaClO4 (F.M. Pohl and T.M. Jovin, J. Mol. Biol. 67 (1972) 375) indicates that Mg(ClO4)2 is more effective than would be anticipated from the simple additive effects of the Mg2+ and ClO4- ions (the ionic strengths of the respective transition points are: NaCl, 2.4; MgCl2, 2.1; NaClO4, 1.8 and Mg(ClO4)2, 0.78). These results suggest the importance of specific interactions involving ClO4-, particularly in the presence of Mg2+. The B-Z transition of poly[d(G-C)] can be monitored spectroscopically via the large hyperchromic shift at 295 nm and the inversion in the CD spectrum. The reaction is fully reversible and can be fitted by a monoexponential function with half times varying between 8 and 150 min. The observed relaxation times are strongly dependent on the concentration of Mg(ClO4)2 with a distinct maximum at the transition point, in accordance with a concerted mechanism involving only the B and Z states. As the polymer assumes the Z conformation it progressively aggregates into a gel-like precipitate, which, however, redissolves rapidly upon lowering the salt concentration. The natural DNA from Micrococcus lysodeikticus which has a high GC content of 72% is also precipitated by Mg(ClO4)2 but we do not have direct spectroscopic evidence for the involvement of the Z conformation in this phenomenon. Neither calf thymus DNA (41% GC) nor poly[d(A-T)] (0% GC) aggregates under the same conditions.     

Permeability and morphology of low temperature phases in bilayers of single and of mixtures of phosphatidylcholines

The properties of subtransitions were studied in aqueous dispersions of saturated phosphatidylcholines (PC) by means of permeability measurements, freeze-fracture electron microscopy, and differential scanning calorimetry (DSC). For dispersions of C16PC, a C16PC analog (2,3-dipalmitoyl-cyclopentano-1-phosphocholine with four methylene residues between the nitrogen and the phosphorus atoms) and C17PC, there was good agreement between phase properties (including subtransitions) as observed by DSC and temperature-related permeability. C16PC and C17PC dispersions also displayed a 'crinkled' surface morphology in the subgel state. The phase diagram for mixtures of C14PC and C16PC was consistent with ideal mixing of these two components in the subgel state and also illustrated the relative independence of the subtransition on acyl chain length as compared to the pre- and main transitions. Together, these results indicate that (i) permeability, DSC and freeze-fracture electron microscopy measurements do correlate reasonably well with the existence of a subgel state, (ii) mixtures of lipids with similar acyl chain lengths can be used to investigate subtransitions, (iii) the development of a subtransition appears to be mainly a function of the non-acyl chain moiety of the phospholipid.     

Phosphatidylcholine bilayers: subtransitions in pure and in mixed lipids

Aqueous dispersions of C14, C16, C17 and C18 phosphatidylcholines (PC, where Cn denotes di-acyl of n carbons per chain), and mixtures of C14/C16PC and C16/C17PC were prepared and their thermal properties studied by differential scanning calorimetry (d.s.c.) after sample storage at 2-6 degrees C for up to 22 days. C16PC and C18PC display subtransitions at 22 degrees C and 29 degrees C, respectively, as previously reported by Chen et al. (Proc. Natl. Acad. Sci. U.S.A. 77 (1980) 5060-5063). C17PC shows two subtransitions at 21 degrees C and 26 degrees C, respectively, which are independent of each other. Although C16PC and C17PC individually develop subtransitions, an equimolar mixture does not. However, mixtures of C14/C16PC containing 10 or more mol% of C14PC do display a subtransition. These results underscore the primary dependence of subtransition formation in phosphatidylcholine dispersions on acyl chain structure.     

High resolution thermal denaturation analyses of small sequenced DNA restriction fragments containing Escherichia coli lactose genetic control loci.

Differential melting curves are reported for four DNA restriction fragments (789, 301, 203, and 95 base pairs in length) spanning the lactose control region. All but the smallest melt with two or more subtransitions. Maps are proposed which identify the positions of regions of different thermal stability in the sequences. The sizes of regions comprising subtransitions range from 60 to 200 base pairs. An analysis is made of the cooperativity exhibited between regions in the sequence. The effect on the shape of the differential melting curves of Na+ between 10 mM and 0.5 M as well as that of Mg2+ and glycerol has been determined. An 81-bp-long sequence of unusual thermal stability occurs at the lactose promoter. Its TM change, resulting from the above change in salt concentration, is out of step by 1.5 degree C with the neighboring DNA sequence. The potential biological significance of this behavior is discussed.     

Point mutations change the thermal denaturation profile of a short DNA fragment containing the lactose control elements. Comparison between experiment and theory.

To understand the denaturation process of short DNA segments we have chosen a 203-base pair (bp) restriction fragment containing the lactose control region. A steady decrease in GC content exists between its i proximal and z proximal ends. We confirm that this fragment melts at low salt in two subtransitions. A GC to AT mutation in the AT-rich region (mutation UV5) increases the number of denatured base pairs in the first subtransition and decreases the cooperativity of the melting process. A GC to AT mutation in the GC-rich region (mutation L8) decreases the number of denatured base pairs in the first subtransition and increases the cooperativity. These mutations induce the same shift in the temperature of half denaturation. The effects of both mutations are additive. A short deletion at the z end of the fragment affects only the first subtransition. When four GC pairs are added to both end, the fragment melts in one transition. Comparison with the results obtained with a larger 789-bp lac fragment reveals strong end effects on base pair stability and suggests that denaturation of the 203-bp fragment proceeds unidirectionally from the z end. Good agreement is shown with the predictions made with the "z ipper model" of Crothers et al. (1965).     

A statistical mechanical model of the pre- and subtransitions of lecithin membranes

A statistical mechanical model with experimentally proved facts as starting points is presented. This model explains on molecular level, the pre- and subtransitions appearing in lipid membranes. The model describes the main features of the transitions, the hysteresis of the subtransition and the mobility changes of the heads and chains at these transitions. The model was expanded for phosphatidylcholine homologues with arbitrary chain lengths, and a qualitative agreement in the case of pretransition as far as a quantitative one for the subtransition were found.     

The essential role of Ca2+ in the activity of bovine pancreatic deoxyribonuclease.

DNase requires Ca2+ for activity against DNA with Mg2+. The Ca2+ selective chelating agent, ethylene glycol bis(beta-aminoethyl ether)-N, N'-tetraacetic acid, (EGTA) inhibits DNase completely at pH 7 or 8, and subsequent addition of excess Ca2+ reverses inhibition in less than one second. DNase action can be stopped at any point by the addition of excess EGTA over Ca2+. Ca2+ is required for DNase to bind substrate. Gel filtration experiments fail to show DNase binding to 0.2 mg per ml of DNA at 5 mm Mg2+ and 10-4 M EGTA. The concentration of Ca2+ needed for half of maximum DNase activity decreases with increases DNA concentration, from 1.2 times 10-5 M Ca2+ at 2.3 times 10-5 M DNA-P to about 4 times 10-7 M Ca2+ at 2.3 DNA-P. Kinetic analysis by the titrametic assay of protons releases shows that V max is independent of Ca2+ concentration while Km increases from 7.7 times 10-5 M DNA-P at 5 times 10-4 M Ca2+ to 3.4 times 10-4 M DNA-P at 5 times 10-6 M Ca2+. Both of these results are predicted by a rate equation which is derived from the assumption that DNase must bind Ca2+ before it can bind DNA. The essential Ca2+ atom probably binds to the one of two high affinity Ca2+ binding sites on DNase which cannont bind Mg2+ or Mn2+. The only other divalent metal ions which can bind to this site, Sr2+ and Ba2+, are also the only metal ions which can substitute for Ca2+ in DNase action against DNA with Mg2+. Some DNase activity is obtained in the absence of added Ca2+ with Mg2+ at pH 6 or below and with Mn2+ or Co2+ at pH 8. These assay solutions are contaminated by 1 to 3 muM Ca2+, which may be sufficient to account for the observed activity.     

Ionic effects on the structure of nucleoprotein cores from adenovirus

Nucleoprotein cores, prepared from adenovirus type 5 with a deoxycholate/heat treatment, consist of the viral DNA and two major internal proteins. The core particles exhibit structural characteristics that are highly reproducible and dependent on their ionic environment. In low-ionic-strength buffer, the cores had a sedimentation coefficient of 180 S and appeared in the electron microscope as homogeneous particles with distinct centers from which numerous arms and loops radiated. Condensation of the cores was induced by Mg2+ or Ca2+ over the range 0 to 1 mM. The sedimentation coefficient increased monotonically with divalent cation concentration, reaching a maximum of 405 S in 1 mM Mg2+. A corresponding condensation in the core structure was observed by electron microscopy. Increasing concentrations of NaCl also produced a conformational change in the cores, with an almost linear increase in sedimentation velocity up to 274 S in 0.04 M NaCl. Between 0.05 and 1.0 M NaCl, the cores were insoluble. In 2.0 M NaCl, the cores were again soluble with an s20,w of 228 S. Under all ionic strength conditions in which the cores were soluble, both core proteins remained bound to the DNA.     

The DNA melting transition in aqueous magnesium salt solutions.

The melting transition of the magnesium salt of DNA has been systematically examined in the presence of various types of anions. The addition of ClO4- to a concentration of 3.0 N results in the biphasic optical transition, with the first phase exhibiting rapid reversibility and independence of the DNA concentration. This subtransition, which is interpreted as an intramolecular condensation to a collapsed form of DNA, is followed by a DNA concentration-dependent aggregation reaction. The aggregation can be reversed by increasing the ClO4- concentration to 6.0 N while elevating the temperature to post-transition levels. Alternatively, both the collapse and the aggregation can be prevented by melting in the presence of trichloroacetate, the most strongly chaotropic solvent for DNA which has been reported (K. Hamaguchi and E. P. Geiduschek (1962), J. Am. Chem. Soc. 84, 1329). The forces responsible for mediating both the collapse and the aggregation are superficially similar to those involved in maintaining duplex stability. The collapsed form, in particular, possibly possesses features in common with the condensed structures which can be produced in aqueous solution of certain polymers, such as polyethylene glycol (Lerman, L.S. (1971), Proc. Natl. Acad. Sci. U.S.A. 68, 1886).     

A study of phi X-174 DNA torus and lambda DNA torus tertiary structure and the implications for DNA self-assembly

Hydrated torus shaped complexes were examined by transmission electron microscopy in both spermidine-condensed linear and nicked circular phi X-174 DNA and lambda DNA preparations. Freeze-etch replicas of both these torus samples, produced with very low Pt metal deposition levels (9APt/C), were found to have circumferentially wound single DNA double helix size surface fibers in the range of 30A width. Measurements of torus inner and outer circumference as well as ring thickness were performed. Observed differences in the torus dimension distributions from circular phi X-174 DNA and linear phi X-174 DNA may be related to the different topological constraints on DNA folding in these two samples (1). On the basis of annulus thickness measurements phi X-174 DNA toruses, in contrast to lambda DNA toruses, were observed to fall into two classes identified as being formed from monomer DNA condensation and multimer DNA condensation. All of the torus substructure and population dimensions observed here are consistent with the continuous circumferential DNA winding model of torus organization proposed by Marx and Reynolds (1) to explain the micrococcal nuclease cleavage properties of the toruses. End-on view measurements of the torus thickness were made from micrographs obtained by extensive tilting of the object replica. These direct measurements confirmed quaternary structure interpretations made from simple strand packing models. We compared the measured torus properties in this linear DNA size series (5386-48000 bp). With increasing DNA length the pattern of DNA strand self-assembly was found to be more varied producing lambda DNA toruses of varying shape. The relevance of our study to the problem of lambda bacteriophage DNA head packaging was discussed.     

Mechanism and kinetics of the subtransition in hydrated L-dipalmitoylphosphatidylcholine

The mechanism of the subtransitions (Lc to L beta') in L-dipalmitoylphosphatidylcholine bilayers in excess water has been investigated by time-resolved X-ray diffraction using synchrotron radiation. The temperature dependence of the diffraction patterns closely correlate with the asymmetric excess specific heat variation recorded by differential scanning calorimetry. During the subtransition two prominent wide-angle reflections, characteristic of the low-temperature crystalline phase, Lc, gradually change such that a sharp peak at a spacing of 0.430 nm decreases in intensity and ultimately disappears while a broader peak initially located at 0.375 nm progressively shifts to an eventual spacing of 0.410 nm. This behaviour is interpreted as a lateral deformation of the acyl chain packing subcell as the chains begin to rotate until a state is reached where the chains pack on a regular hexagonal array characteristic of the L beta phase. An increase in lamellar repeat distance from 6.0 to 6.4 nm takes place simultaneously with the acyl chain rearrangement at relatively low (5 K/min) as well as high (6 K/s) heating rates. As judged from the shape of the wide-angle peak, transformation to L beta' phase occurs some minutes after transition to the L beta phase. The X-ray data characterise the subtransition as a continuous (second order) phase transition in which a presumably orthorhombic subcell is transformed into a hexagonal subcell in a gradual process. In temperature jump experiments at 6 K/s between 0 degree C and 80 degrees C the relaxation time of the subtransition was found to be about 5 s while the relaxation time of the main gel to liquid-crystalline transition was about 2 s.     

Cloning of beta-isopropylmalate dehydrogenase gene from Bacillus coagulans in Escherichia coli and purification and properties of the enzyme

The beta-isopropylmalate dehydrogenase (2-hydroxy-4-methyl-3-carboxyvalerate: NAD+ oxidoreductase, EC 1.1.1.85) gene from Baccilus coagulans was cloned and expressed in Escherichia coli C600, using pBR322 as a vector plasmid. The B. coagulans enzyme was purified to a homogeneous state from the E. coli carrying a pBR322 - the B. coaglulans enzyme gene hybrid plasmid. The enzyme consists of two subunits of equal molecular weight (4.4 X 10(4) ). The enzyme activity was stimulated by 0.5 mM Mn2+, Mg2+ and Co2+. The enzyme was strongly inhibited by 0.2 mM p-chloromercuribenzoate and the inhibition was completely recovered by 1 mM dithiothreitol. The B. coagulans enzyme was thermostabilized by 1.5 M NaCl. The B. coagulans enzyme is a composite of alpha-helix, beta-sheet and remainder. The secondary structure of the enzyme was appreciably altered by 0.5 mM MgCl2 and 1.5 M NaCl.     

Cobalt complexes of terpyridine ligand: crystal structure and photocleavage of DNA

Two new cobalt complexes, [Co(pytpy)(2)](ClO(4))(2), 1, and [Co(pytpy)(2)](ClO(4))(3), 2 where pytpy=pyridine terpyridine, have been synthesized and characterized. Single-crystal X-ray structure of both the complexes has been resolved. The structure shows the complexes to be a monomeric cobalt(II) and cobalt(III) species with two pytpy ligands coordinated to the metal ion to give a six coordinate complex. Both cobalt(II) and cobalt(III) complexes crystallize in meridional configuration. The interaction of these complexes with calf thymus DNA has been explored by using absorption, emission spectral, electrochemical studies and viscosity measurements. From the experimental results the DNA binding constants of 1 and 2 are found to be (1.97+/-0.15)x10(4)M(-1) and (2.7+/-0.20)x10(4)M(-1) respectively. The ratio of DNA binding constants of 1 and 2 have been estimated to be 0.82 from electrochemical studies, which is in close agreement with the value of 0.73 obtained from spectral studies. The observed changes in viscosity of DNA in the presence of increasing amount of complexes 1 and 2 suggest intercalating binding of these complexes to DNA. Results of DNA cleaving experiments reveal that complex 2 efficiently cleaves DNA under photolytic conditions while complex 1 does not cleave DNA under similar conditions.     

On the internal structure of bacteriophage lambda

The structure of bacteriophage lambda has been studied by electron microscopy of negatively stained particles. The phage particles will eject their DNA if they are heated or dialyzed against a chelating agent. The ghost particles, so formed, have a channel running down their tails. Since the channel is not visible in normal particles, the channel may be filled with part of the DNA molecule. Up to 30% of the ghosts contain round objects about half the internal diameter of the head. The round objects, called "cores," have the same buoyant density as the coat protein. The core may be a protein spool about which the phage DNA is wound.     

A Study of øX-174 DNA Torus and Lambda DNA Torus Tertiary Structure and the Implications For DNA Self-Assembly

Abstract

Hydrated torus shaped complexes were examined by transmission electron microscopy in both spermidine-condensed linear and nicked circular øX-174 DNA and lambda DNA preparations. Freeze-etch replicas of both these torus samples, produced with very low Pt metal deposition levels (9APt/C), were found to have circumferentially wound single DNA double helix size surface fibers in the range of 30A width. Measurements of torus inner and outer circumference as well as ring thickness were performed. Observed differences in the torus dimension distributions from circular øX-174 DNA and linear øX ?174 DNA may be related to the different topological constraints on DNA folding in these two samples (1). On the basis of annulus thickness measurements øX ?174 DNA toruses, in contrast to lambda DNA toruses, were observed to fall into two classes identified as being formed from monomer DNA condensation and multimer DNA condensation. All of the torus substructure and population dimensions observed here are consistent with the continuous circumferential DNA winding model of torus organization proposed by Marx and Reynolds (1) to explain the micrococcal nuclease cleavage properties of the toruses. End-on view measurements of the torus thickness were made from micrographs obtained by extensive tilting of the object replica. These direct measurements confirmed quaternary structure interpretations made from simple strand packing models. We compared the measured torus properties in this linear DNA size series (5386–48000 bp). With increasing DNA length the pattern of DNA strand self- assembly was found to be more varied producing lambda DNA toruses of varying shape. The relevance of our study to the problem of lambda bacteriophage DNA head packaging was discussed.     

Temperature-induced changes in copper centers and protein conformation of two fungal laccases from Coriolus hirsutus and Coriolus zonatus.

The paper reports on two fungal laccases from Coriolus hirsutus and Coriolus zonatus and their type-2 copper-depleted derivatives. Temperature-induced changes of the copper centers were characterized by optical and electron paramagnetic resonance (EPR) spectroscopy, and the overall protein stability by differential scanning microcalorimetry. The intact enzymes showed highly cooperative thermal unfolding transitions at about 90 degrees C. Type-2 copper depletion led to uncoupling of the domains characterized by a different melting pattern which resolved three subtransitions. Melting curves monitored optically at 290, 340 and 610 nm showed additional transitions below thermal unfolding temperature. EPR spectra of the intact laccases showed the disintegration of the trinuclear copper cluster accompanied by loss of one of the copper ions and disappearance of the strong antiferromagnetic coupling in the type-3 site at 70 degrees C and above 70 degrees C. The copper centers of type-2 copper-depleted laccase showed reduced thermotolerance.     

The NMR structure of the gpU tail-terminator protein from bacteriophage lambda: identification of sites contributing to Mg(II)-mediated oligomerization and biological function

During the late stages of lambda bacteriophage assembly, the protein gpU terminates tail polymerization and participates at the interface between the mature capsid and tail components. When it engages the lambda tail, gpU undergoes a monomer-hexamer transition to achieve its biologically active form. Towards understanding how gpU participates in multiple protein-protein interactions, we have solved the structure of gpU in its monomeric state using NMR methods. The structure reveals a mixed alpha/beta motif with several dynamic loops at the periphery. Addition of 20 mM MgCl(2) is known to oligomerize gpU in the absence of its protein partners. Multiple image analysis of electron micrographs revealed ring-like structures of magnesium ion saturated gpU with a 30 A pore, consistent with its function as a portal for the passage of viral DNA into the host bacterium. The ability of magnesium ions to promote oligomerization was lost when substitutions were made at a cluster of acidic amino acids in the vicinity of helix alpha2 and the beta1-beta2 loop. Furthermore, substitutions at these sites abolished the biological activity of gpU.