Salt induced deprotonation of initially salt-free transfer RNA.

¹H NMR double resonance studies of valinomycin in (CD₃)₂SO were conducted at 90 MHz (FT-mode) and 250 MHz (correlation mode) to determine the mechanism of intramolecular nuclear Overhauser effects (NOE). These studies set specific constraints on any model for the conformation of valinomycin in (CD₃)₂SO and illustrate that NOE experiments of this type hold considerable promise for conformational studies of peptides, proteins and other biomolecules. The NOE's are positive at the lower frequency and negative at the higher frequency. Consideration of the theoretical dependence of the NOE on the proton-proton internuclear correlation time and on the resonance frequency indicates that these results are explained by a predominantly dipolar relaxation mechanism.     

Intensity-fluctuation spectroscopy and tRNA conformation. II. Changes of size and shape of tRNA in the melting process

We have measured the translational (D^T) and rotational (D^R) diffusion coefficients of bulk tRNA from baker's yeast during the thermal unfolding process by means of photon-correlation spectroscopy. It should be noted that our estimate of the rotational diffusion coefficient represented, for the first time, measurements on a small macromolecule in solution by the photoelectron time-of-arrival technique with a delay-time resolution of 1 nsec. The melting curves expressed in terms of δD^T vs temperature were consistent with the literature data in revealing the melting steps and their dependence on NaCl concentration. Additionally, it was possible to prove the existence of an intermediate, more compact structure during the initial steps of the thermal unfolding process. We found that the temperature ranges over which this intermediate structure appears depend strongly on salt concentration. By utilizing both translational and rotational diffusion coefficients and Perrin's equations for ellipsoids of revolution, we have computed the values of the equivalent length and width of tRNA molecules in solution at four different temperatures for NaCl concentrations of 0.2, 0.5, and 1M. The approximate model of ellipsoids of revolution also permits us to obtain an estimate of the radius of gyration, which is in very good agreement with literature data measured by means of small-angle x-ray scattering. Furthermore, we have measured the shape and size changes of tRNA with varying NaCl concentrations at room temperatures (25°C). The molecule becomes smaller and more spherical when NaCl concentration increases. As a result of partial melting at 70°C, the macromolecule is surprisingly elongated with an approximate axial ratio of 8:1 and has dimensions of about 180/22Å. Such information on conformational changes by a simultaneous determination of rotational and translational diffusion coefficients illustrates the potential of this approach, not available by other methods.     

Effect of sodium ion concentration on transfer RNA conformation in solution studied by Rayleigh light scattering

The sodium ion concentration dependent conformational changes of transfer RNA (unfractionated tRNA from baker's yeast) have been studied in unbuffered aqueous solutions by Rayleigh light scattering. Changes of the optical parameters of the molecule indicated the following conformational changes of tRNA with increasing NaCl concentration: in salt-free solution tRNA molecules have an irregular hairpin loop-like structure in which the orientation of base rings is not correlated. Upon addition of a small amount of NaCl (0.005 M) an increasing ordering of this structure is observed. In 0.1 M-NaCl the molecule has an extended structure with ordered regions (arms). Further increase of sodium ion concentration up to 2 M results in folding of the extended structure and formation of a compact and rigid conformation in which most of the bases are nearly perpendicular to the symmetry axis of the molecule.     

The size and conformation of Artemia (brine-shrimp) ribosomal RNA free in solution.

The RNA was isolated from the large ribosomal subunits of the brine shrimp Artemia, and its conformation free in solution was studied by determining its sedimentation and diffusion coefficients. A comparison was made of the hydrodynamic radius of the ribosomal subunit and its isolated RNA in various buffers. The conformation of the rRNA free in solution is more extended than when it is incorporated in the ribosome. This is not only the case when the rRNA solution lacks bivalent and polyvalent cations, but even in the presence of Mg2+ and spermidine, which cause a tightening of RNA. Thus the ribosomal proteins should induce a further tightening of the rRNA during the assembly of the ribosome. In the discussion, the reported data on Escherichia coli rRNA species are presented in such a way that large discrepancies between various studied are revealed, and that they can be compared with the data reported here on the larger rRNA of an eukaryote.     

Stochastic fractal behavior in concentration fluctuation and fluorescence correlation spectroscopy.

Fluctuations in the concentration of Brownian particles in one and two dimensions, or any reasonable measurement of the concentration such as in fluorescence correlation spectroscopy, is shown to be a stochastic fractal with a long tail. Being singular at omega = 0, the power spectrum of the fluctuation S(omega) approximately omega-1/2 for diffusion in one dimension, approximately log omega in two dimensions, but non-singular in three dimensions. This discovery provides one simple physical mechanism for possible long-memory fractal behavior, and its implications to various biological processes are discussed.     

Dehydro-dermorphins. III. Circular dichroism investigation of conformation in solution

Dehydropeptide analogs of dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) and N-terminal fragments containing one or two dehydrophenylalanine residues in the 3rd and/or 5th position, have been investigated by means of CD spectroscopy. The results indicate that the above dehydropeptides can adopt different conformations in alcohol and water solutions. In methanol and trifluoroethanol, the CD spectra are mainly consistent with the presence of folded structures, probably stabilized by intramolecular hydrogen bonds. In water, conversely, CD data indicate disruption of ordered structures and formation of preferentially extended flexible conformations. Models of the involved folded structures are tentatively proposed, taking into account the geometric features of dehydro residues and their tendency to favor hydrogen-bonded 10-membered rings.     

Bulged adenosine influence on the RNA duplex conformation in solution

The RNA single bulge motif is an unpaired residue within a strand of several complementary base pairs. To gain insight into structural changes induced by the presence of the adenosine bulge on RNA duplex, the solution structures of RNA duplex containing a single adenine bulge (5'-GCAGAAGAGCG-3'/5'-CGCUCUCUGC-3') and a reference duplex with all Watson-Crick base pairs (5'-GCAGAGAGCG-3'/5'-CGCUCUCUGC-3') have been determined by NMR spectroscopy. The reference duplex structure is a regular right-handed helix with all of the attributes of an A-type helix. In the bulged duplex, single adenine bulge stacks into the helix, and the bulge region forms a well-defined structure. Both structures were analyzed by the use of calculated helical parameters. Distortions induced by the accommodation of unpaired residue into the helical structure propagate over the entire structure and are manifested as the reduced base pairs inclination and x-displacement. Intrahelical position of bulged adenine A5 is stabilized by efficient stacking with 5'-neighboring residues G4.     

On the conformation of serine-specific transfer RNA. Studies by small-angle X-ray scattering and ultraviolet absorption of the molecule in solution.

The scattered X-ray intensities from dilute solutions of tRNASer (yeast) in 0.1 M Soerensen buffer at pH 7.0 were measured at 25 degrees C. The radius of gyration, molecular weight and volume were determined. A model equivalent in scattering is given. The change of the conformation of tRNASer by heating was followed by small-angle X-ray measurements and ultraviolet absorption in a temperature range 20-70 degrees C. The molecule begins to unfold at about 40 degrees C and 70 degrees C has a random coil conformation. Addition of magnesium stabilizes the tRNASer molecule. The reversibility of the melting process was also studied by both methods. An interesting effect was found by ultraviolet absorption: by heating the tRNASer solutions to 55 degrees C and 60 degrees C and subsequently slowly cooling, the melting curves lie at higher absorption values than the corresponding cooling curves. The small-angle data and optical properties of tRNASer are compared with those of tRNAPhe which has already been thoroughly investigated.     

Role of modifications in tyrosine transfer RNA. II. Ribothymidylate-deficient tRNA

The ability of suppressor tyrosine transfer RNA to participate in protein synthesis was studied in an E. coli mutant unable to synthesize ribothymidylate. Sequence analysis has shown that uracil replaces thymine in the common TψCG sequence. This transfer RNA is able to support protein synthesis at a rate equivalent to that observed with normally methylated transfer RNA.     

The size and shape of human and bovine antithrombin III.

Human and bovine antithrombin, purified by affinity chromatography on heparin-agarose, have been characterized with regard to chemical composition, size, shape and conformation. Both preparations were found to contain several active components of identical or similar size but different electrical charge. Amino acids and carbohydrate analyses revealed striking similarities between human and bovine antithrombin, while immunological analyses failed to demonstrate any cross-reactivity. The molecular weights were determined by sedimentation equilibrium to be 58 000 for human and 56 000 for bovine antithrombin. The small molecular weight difference suggested by these values was verified by several empirical methods of molecular weight estimation. Hydrodynamic measurements indicated that the two proteins have similar molecular shapes, both of which are slightly more extended that that of typical globular proteins. The internal folding of the two polypeptide chains is also similar, as evidenced by the identity of the far-ultraviolet circular dichroism spectra. Specifically, these analyses suggested a low alpha-helix content of both proteins. In conclusion, the marked structural similarity of human and bovine antithrombin indicates that the two proteins may also exhibit extensive functional similarities in the binding of heparin and the inhibition of various coagulation factors.     

Influence of the shape of phospholipid vesicles on the measurement of their size by photon correlation spectroscopy

The influence of shape transformation of large unilamellar vesicles (LUV) on their size measurement by photon correlation spectroscopy (PCS) has been investigated. The experimental size of vesicles after hyperosmotic contractions of increasing intensities have been compared to the theoretical volume decrease determined by applying Boyle Van't Hoff's law. The main observation is that PCS size measurement gives overestimated values when LUV have been subjected to a volume decrease of more than 20% of their initial volume. The PCS size overestimation is related to the influence of the shape transformation of the vesicles on their diffusion coefficient (D) as shown by modelling the evolution of D of a sphere which is transformed into an ellipsoid by internal volume reduction under constant area. Received: 4 December 1997 / Revised version: 2 March 1998 / Accepted: 15 April 1998     

The conformation of double-stranded DNA inside bacteriophages depends on capsid size and shape

The packaging of double-stranded DNA into bacteriophages leads to the arrangement of the genetic material into highly-packed and ordered structures. Although modern experimental techniques reveal the most probable location of DNA inside viral capsids, the individual conformations of DNA are yet to be determined. In the current study we present the results of molecular dynamics simulations of the DNA packaging into several bacteriophages performed within the framework of a coarse-grained model. The final DNA conformations depend on the size and shape of the capsid, as well as the size of the protein portal, if any. In particular, isometric capsids with small or absent portals tend to form concentric spools, whereas the presence of a large portal favors coaxial spooling; slightly and highly elongated capsids result in folded and twisted toroidal conformations, respectively. The results of the simulations also suggest that the predominant factor in defining the global DNA arrangement inside bacteriophages is the minimization of the bending stress upon packaging.