Analysis of the conformational properties of κ‐ and ι‐carrageenan by size‐exclusion chromatography combined with low‐angle laser light scattering

The conformational properties of κ‐carrageenan in 0.2M LiI and ι‐carrageenan in 0.2M LiCl were analyzed by size exclusion chromatography combined with low‐angle laser light scattering. Fractionated samples with narrow molecular weight distributions (M_w/M_n ∼ 1.4) were used, and M_w in the disordered states were 35,000 (κ‐35) and 200,000 (κ‐200) for κ‐carrageenan and 65,000 (ι‐65) and 170,000 (ι‐170) for ι‐carrageenan, respectively. The analyses were performed across a temperature range where the conformational transitions occurred, and at extremely low concentrations (2–50 μg/mL) due to low amounts of samples injected and the subsequent dilution occurring during the separation. The results indicate that a twofold increase of the molecular weight (M_w) occurs for κ‐carrageenan upon inducing the ordered conformation. For ι‐carrageenan an additional increase in M_w may take place, which is attributed to the strong tendency for aggregation of ordered chains especially at high molecular weights. The results thus suggest that both κ‐ and ι‐carrageenan are double (or multiple) stranded in their ordered conformations, within the concentration range studied here. © 1999 John Wiley & Sons, Inc. Biopoly 49: 71–80, 1999     

Polyelectrolytic aspects of the thermodynamics of conformational transition: κ‐carrageenan in formamide

A recently published method for the determination of the enthalpy and entropy changes of nonionic origin upon conformational transition of linear biopolyelectrolytes in solution [J. C. Benegas, A. Cesàro, R. Rizzo and S. Paoletti (1998) Biopolymers, Vol. 45, pp. 203–216] has been extended from the case of aqueous salt solutions to that of the organic solvent formamide (FA). The calculation have been applied to the case of the intramolecular transition of the K⁺ salt form of the sulfated polysaccharide κ‐carrageenan. The method proved to be effective in providing the desired data in FA, as it has been previously been successful for the water cases. The comparison between the predicted enthalpy change of transition and the microcalorimetric experimental one turned out to be excellent, thereby ensuring on the validity of the approach. © 1999 John Wiley & Sons, Inc. Biopoly 49: 127–130, 1999     

Characteristics of two-step RNA dimerization in avian sarcoma and leukosis viruses

Dimerization of two genomic RNA copies is essential for the assembly of retrovirus particles. This process has been studied in detail, and a two-step mechanism has been proposed for the human immunodeficiency virus type 1 (HIV-1). A similar model can be assumed for avian sarcoma and leukosis viruses (ASLV), despite the lack of homology between the dimerization initiation site (DIS) of ASLV and that of HIV-1. The structural features of the ASLV DIS were studied with the examples of the avian leukosis virus HPRS-103 and the avian sarcoma virus CT-10. The rate of spontaneous transition from loose to tight dimers at a higher temperature was studied as dependent on the stem length in the DIS hairpin. Dimers of both types were formed by the selected RNA fragments of the two viruses. The conditions of loose dimer formation differed considerably, although the two viruses had identical sequences (5-A-CUGCAG-3) of the hairpin loop. Dimerization of CT-10 RNA fragments required an RNA concentration at least an order of magnitude higher than in the case of HPRS-103. The difference was explained by deletion of an adenine from the hairpin stem of C-10.Translated from Molekulyarnaya Biologiya, Vol. 39, No. 1, 2005, pp. 147–154.Original Russian Text Copyright © 2005 by Beniaminov, Samokhin, Ulyanov, Minyat.     

Identification of dynamical hinge points of the L1 ligase molecular switch

The L1 ligase is an in vitro selected ribozyme that uses a noncanonically base-paired ligation site to catalyze regioselectively and regiospecifically the 5′ to 3′ phosphodiester bond ligation, a reaction relevant to origin of life hypotheses that invoke an RNA world scenario. The L1 ligase crystal structure revealed two different conformational states that were proposed to represent the active and inactive forms. It remains an open question as to what degree these two conformers persist as stable conformational intermediates in solution, and along what pathway are they able to interconvert. To explore these questions, we have performed a series of molecular dynamics simulations in explicit solvent of the inactive–active conformational switch in L1 ligase. Four simulations were performed departing from both conformers in both the reactant and product states, in addition to a simulation where local unfolding in the active state was induced. From these simulations, along with crystallographic data, a set of four virtual torsion angles that span two evolutionarily conserved and restricted regions were identified as dynamical hinge points in the conformational switch transition. The ligation site visits three distinct states characterized by hydrogen bond patterns that are correlated with the formation of specific contacts that may promote catalysis. The insights gained from these simulations contribute to a more detailed understanding of the coupled catalytic/conformational switch mechanism of L1 ligase that may facilitate the design and engineering of new catalytic riboswitches.