We examined the permeabilization of lipid bilayers by the β-sheet, cyclic antimicrobial decapeptide gramicidin S (GS) in phospholipid bilayers formed either by mixtures of zwitterionic diphytanoylphosphatidylcholine and anionic diphytanoylphosphatidylglycerol or by single zwitterionic unsaturated phosphatidylcholines having various hydrocarbon chain lengths, with and without cholesterol. In the zwitterionic bilayers formed by the phosphatidylcholines, without or with cholesterol, the peptide concentrations and membrane potentials required to initiate membrane permeabilization vary little as function of bilayer thickness and cholesterol content. In all the systems tested, the GS-induced transient ion conductance events exhibit a broad range of conductances, which are little affected by the bilayer composition or thickness. In the zwitterionic phosphatidylcholine bilayers, the effect of GS does not depend on the polarity of the transmembrane potential; however, in bilayers formed from mixtures of phosphatidylcholines and anionic phospholipids, the polarity of the transmembrane potential becomes important, with the GS-induced conductance events being much more frequent when the GS-containing solution is positive relative to the GS-free solution. Overall, these results suggest that GS does not form discrete, well-defined, channel-like structures in phospholipid bilayers, but rather induces a wide variety of transient, differently sized defects which serve to compromise the bilayer barrier properties for small electrolytes.

Aptamers are short RNA ⁄ DNA sequences that are identified through the process of systematic evolution of ligands by exponential enrichment and that bind to diverse biomolecular targets. Aptamers have strong and specific binding through molecular recognition and are promising tools in studying molecular biology. They are recognized as having potential therapeutic and diagnostic clinical applications. The success of the systematic evolution of ligands by exponential enrichment process requires that the RNA ⁄ DNA pools used in the process have a sufficient level of sequence diversity and structural complexity. While the systematic evolution of ligands by exponential enrichment technology is well developed, it remains a challenge in the efficient identification of correct aptamers. In this article, we propose a novel information-driven approach to a theoretical design of aptamer templates based solely on the knowledge regarding the biomolecular target structures. We have investigated both theoretically and experimentally the applicability of the proposed approach by considering two specific targets: the serum protein thrombin and the cell membrane phospholipid phosphatidylserine. Both of these case studies support our method and indicate a promising advancement in theoretical aptamer design. In unfavorable cases where the designed sequences show weak binding affinity, these template sequences can be still modified to enhance their affinities without going through the systematic evolution of ligands by exponential enrichment process.Key words: bioinformatics, mechanism-based drug design, molecular recognition, structure-based drug design Received 18 October 2010, revised 24 February 2011 and accepted for publication 1 April 2011 Aptamers, short RNA ⁄ DNA sequences, are selected through an experimental technique known as systematic evolution of ligands by exponential enrichment (SELEX) to bind to specific biomolecular targets including small molecules, proteins, nucleic acids, phospholipids as well as complex structures such as cells, tissues, bacteria, and other organisms. Aptamers have strong and specific binding through molecular recognition and are promising tools in studying molecular biology with recognized therapeutic and diagnostic clinical applications (1-5). SELEX consists of a number of rounds of in vitro selection in which the RNA ⁄ DNA pool is incubated with the binding target. The non-binding or loosely binding sequences are discarded, while the binding sequences are expanded using the polymerase chain reaction method to provide a pool of sequences for the next round of testing. In practice, multiple rounds of selection and expansion are required before unique tightly binding sequences can be identified. Additionally, isolated aptamers will often need to be reengineered to reduce their sequence length and impart additional favorable biological properties. These issues pose a challenge for the efficient identification of correct aptamers.Over the past several years, various research groups have attempted...

Linear rate-equilibrium (RE) relations, also known as linear free energy relations, are widely observed in chemical reactions, including protein folding, enzymatic catalysis, and channel gating. Despite the widespread occurrence of linear RE relations, the principles underlying the linear relation between changes in activation and equilibrium energy in macromolecular reactions remain enigmatic. When examining amphiphile regulation of gramicidin channel gating in lipid bilayers, we noted that the gating process could be described by a linear RE relation with a simple geometric interpretation. This description is possible because the gating process provides a well-understood reaction, in which structural changes in a bilayer-embedded model protein can be studied at the singlemolecule level. It is thus possible to obtain quantitative information about the energetics of the reaction transition state and its position on a spatial coordinate. It turns out that the linear RE relation for the gramicidin monomer-dimer reaction can be understood, and the quantitative relation between changes in activation energy and equilibrium energy can be interpreted, by considering the effects of amphiphiles on the changes in bilayer elastic energy associated with channel gating. We are not aware that a similar simple mechanistic explanation of a linear RE relation has been provided for a chemical reaction in a macromolecule. RE relations generally should be useful for examining how amphiphile-induced changes in bilayer properties modulate membrane protein folding and function, and for distinguishing between direct (e.g., due to binding) and indirect (bilayer-mediated) effects.Brønsted | linear free energy relationships | phi value | bilayer elasticity I t is a common observation that manipulations that alter the energetics of a reaction:alter the rate constants (k 1 and k −1 ) and equilibrium constant (K eq ¼ k 1 ∕k −1 ), such that lnfk 1 g is a linear function of lnfK eq g. This linearity is interpreted to mean that, for a series of manipulations of the reaction, the activation free energy (ΔG ‡ ) varies as a linear function of the equilibrium free energy (ΔG 0 ):where a is a slope factor and b a constant (1-3). Such linear relations, known as linear rate-equilibrium (RE) relations (4), linear free energy relations (5), or linear rate-equilibrium free energy relations (6), have been observed and studied since the early 1900s. First observed in acid-based reactions (7-9), they have been identified in a wide range of reactions (1-3). Leffler proposed, based on studies of covalent reactions, that the slope factor a (Eq. 2) identifies the position of the transition state on a reaction coordinate, where S and P are positioned at 0 and 1, respectively (5). Marcus and coworkers deduced the principles underlying linear RE relations in electron transfer processes using a model in which the transfer is described as a one-step transition between two harmonic energy wells (10, 11). Fersht and coworkers used linear RE relations associated with p...

We demonstrate the effects on membrane of the tubulin-binding chemotherapy drugs: thiocolchicoside and taxol. Electrophysiology recordings across lipid membranes in aqueous phases containing drugs were used to investigate the drug effects on membrane conductance. Molecular dynamics simulation of the chemotherapy drug-lipid complexes was used to elucidate the mechanism at an atomistic level. Both drugs are observed to induce stable ion-flowing pores across membranes. Discrete pore current-time plots exhibit triangular conductance events in contrast to rectangular ones found for ion channels. Molecular dynamics simulations indicate that drugs and lipids experience electrostatic and van der Waals interactions for short periods of time when found within each other's proximity. The energies from these two interactions are found to be similar to the energies derived theoretically using the screened Coulomb and the van der Waals interactions between peptides and lipids due to mainly their charge properties while forming peptide-induced ion channels in lipid bilayers. Experimental and in silico studies together suggest that the chemotherapy drugs induce ion pores inside lipid membranes due to drug-lipid physical interactions. The findings reveal cytotoxic effects of drugs on the cell membrane, which may aid in novel drug development for treatment of cancer and other diseases.