Eberhard Von Kitzing

The structure of the Holliday junction, and its resolution.

The Holliday (four-way) junction is a critical intermediate in homologous genetic recombination. We have studied the structure of a series of four-way junctions, constructed by hybridization of four 80 nucleotide synthetic oligonucleotides. These molecules migrate anomalously slowly in gel electrophoresis. Each arm of any junction could be selectively shortened by cleavage at a unique restriction site, and we have studied the relative gel mobilities of species in which two arms were cleaved. The pattern of fragments observed argues strongly for a structure with two-fold symmetry, based on an X shape, the long arms of which are made from pairwise colinear association of helical arms. The choice of partners is governed by the base sequence at the junction, allowing a potential isomerization between equivalent structural forms. Resolvase enzymes can distinguish between these structures, and the resolution products are determined by the structure adopted, i.e., by the sequence at the junction. In the absence of cations, the helical arms of the junction are fully extended in a square configuration, and unstacking results in junction thymines becoming reactive to osmium tetroxide.

Rings of Anionic Amino Acids as Structural Determinants of Ion Selectivity in the Acetylcholine Receptor Channel

To gain an insight into the molecular basis of the weak but significant selectivity among alkali metal cations of the nicotinic acetylcholine receptor (AChR) channel, we have determined single-channel conductance and permeability ratios for alkali metal cations on specifically mutated Torpedo californica AChR channels expressed in Xenopus oocytes. The mutations involved charged and polar side chains in the three anionic rings (extracellular, interm ediate and cytoplasmic ring) which have previously been found to determine the rate of K + transport through the AChR channel. The results obtained reveal that mutations in the intermediate ring exert much stronger effects on ion selectivity than do mutations in the extracellular and the cytoplasmic ring. The experimental results, together with simulations of the channel’s energy profile, suggest that the amino acid residues forming the intermediate ring come into close contact with permeating cations and possibly represent part of the physical correlate of the postulated selectivity filter in the AChR channel.

IN)VALIDITY OF THE CONSTANT FIELD AND CONSTANT CURRENTS ASSUMPTIONS IN THEORIES OF ION TRANSPORT

Constant electric fields and constant ion currents are often considered in theories of ion transport. Therefore, it is important to understand the validity of these helpful concepts. The constant field assumption requires that the charge density of permeant ions and flexible polar groups is virtually voltage independent. We present analytic relations that indicate the conditions under which the constant field approximation applies. Barrier models are frequently fitted to experimental current-voltage curves to describe ion transport. These models are based on three fundamental characteristics: a constant electric field, negligible concerted motions of ions inside the channel (an ion can enter only an empty site), and concentration-independent energy profiles. An analysis of those fundamental assumptions of barrier models shows that those approximations require large barriers because the electrostatic interaction is strong and has a long range. In the constant currents assumption, the current of each permeating ion species is considered to be constant throughout the channel; thus ion pairing is explicitly ignored. In inhomogeneous steady-state systems, the association rate constant determines the strength of ion pairing. Among permeable ions, however, the ion association rate constants are not small, according to modern diffusion-limited reaction rate theories. A mathematical formulation of a constant currents condition indicates that ion pairing very likely has an effect but does not dominate ion transport.

Modeling DNA structures : molecular mechanics and molecular dynamics

Abstract There are several tools to improve initial structural estimates of a molecule under study. The methods described in this section weight the structural models according to the approximate internal energy as a function of the atomic coordinates provided by a force field. The method of conjugated gradients is a powerful tool to find the way downhill toward a local minimum. Surmounting barriers and escaping local minima require nonlocal optimization procedures. Of the various possible choices the Bremermann method has been described in greatest detail. The central idea for the implementation of this method is its application to a small number of relevant degrees of freedom, allowing the remaining ones to relax to a local energetic minimum. Monte Carlo methods may be used to obtain statistical averaged quantities. Again the convergence of this method may be considerably increased if the randomly chosen directions mainly concentrate on the relevant directions. Molecular dynamics calculations not only provide averaged quantities but also give information about time-dependent processes.

Modeling DNA Structures

Publisher Summary This chapter presents the methods used to build the models of large molecules. They are based on empirical force-fields, which can be considered as data bases for the physicochemical properties of moleculesm, such as DNA, RNA, or proteins, and give the internal energy as a function of atomic coordinates. In polynucleotides, special attention must be paid to the interaction of charged phosphate groups with their counterion clouds. The complexity of the problem of model building requires an intelligent application of various local and global optimization procedures and of Monte Carlo and molecular-dynamics methods. Including experimental restraints, this method helps to design hypothetical atomic models that can be used to plan specific experiments. Understanding the biological function of large molecules is one of the main reasons for studying their physical properties. Without detailed knowledge of the three-dimensional atomic structure of a molecule, it is unlikely that adequate insight into its biological function can be obtained.

A Novel Model for Saturation of Ion Conductivity in Transmembrane Channels

An important experimental finding in measuring ion currents through ion channels like the nicotinic acetylcholine receptor channel is the saturation of the current with increasing ionic strength. Practically all measurements are done at saturating ionic strength. Thus any theory which is supposed to describe the physical behavior of these channels, must predict this saturation, which appears to result from the finite volume of the site to be filled with ions. An Eyring rate theory that takes into account several ions entering the site is presented here. This new model possesses unexpected properties.

Structure Modeling of the Acetylcholine Receptor Channel and Related Ligand Gated Channels

In this model-building study a model for the pore of the acetylcholine receptor channel is proposed. The pore is formed by five α-helices of the M2 segment where three rings of hydrophilic side chains point into the channel lumen. This model is in agreement with most experimental data like photolabeling, drug affinity studies, single channel conductivity measurements and cryo electron microscopy known about this channel.This study predicts a strong coupling of the motion of the ions in the channel to that of the charged and highly hydrophilic amino acid side chains at the channel wall. Due to the negative net-charge in the pore more than a single cation may occupy the pore region. The resulting strong local electric fields make the commonly used constant field approximation obsolete for this type of ion channel.

Configurational space of biological macromolecules as seen by semi-empirical force fields: inherent problems for molecular design and strategies to solve them by means of hierarchical force fields

Abstract Atomic force fields have become valuable tools for studying biophysical properties of biological macromolecules. “Realistic” simulations require increasingly large systems (e.g. to include a sufficient amount of water molecules) and long time scales (e.g. to study protein folding). An important step toward large-scale simulations consists of distinguishing between biologically relevant and irrelevant motions of the molecular system. This paper reviews heuristic as well as rigid approaches to reduce the demands on computer resources. It also presents recent progress in building up hierarchically structured modular molecular force fields. The techniques involved include domain decomposition methods and expansion of energy approximations using radial functions, splines and wavelets.

The Structure of DNA Four-Way Junctions

The stereochemical conformation of the four-way helical junction in DNA (the Holliday junction; the putative central intermediate of genetic recombination) has been analyzed, using molecular mechanical computer modelling. A version of the AMBER program package was employed, that had been modified to include the influence of counterions and a nonlocal optimisation procedure.