Theoretical conformational analysis of phospholipids II. Role of hydration in the gel to liquid crystal transition of phospholipids

To obtain a satisfactory agreement between computed transition temperatures and those determined experimentally, we introduce explicitly water molecules which hydrate the polar headgroup of dipalmitoylphosphatidylethanolamine molecules. The calculated free energy curves as a function of the intermolecular interchain distance and the degree of hydration of the polar groups permit the determination of the transition of the phospholipid system from the gel to the liquid crystalline phase. The detailed structure of the hydration shell is defined using the supermolecular approach.     

Hume and the argument for biological design

There seems to be a widespread conviction — evidenced, for example, in the work of Mackie, Dawkins and Sober — that it is Darwinian rather than Humean considerations which deal the fatal logical blow to arguments for intelligent design. I argue that this conviction cannot be well-founded. If there are current logically decisive objections to design arguments, they must be Humean — for Darwinian considerations count not at all against design arguments based upon apparent cosmological fine-tuning. I argue, further, that there are good Humean reasons for atheists and agnostics to resist the suggestion that apparent design — apparent biological design and/or apparent cosmological fine-tuning — establishes (or even strongly supports) the hypothesis of intelligent design.     

Do NOE distances contain enough information to assess the relative populations of multi-conformer structures?

Summary The feasibility of determining the relative populations of multi-conformer structures from NOE-derived distances alone is assessed. Without cross-validation of the NOE restraints, any population ratio can be refined to a similar quality of the fit. Complete cross-validation provides a less biased measure of fit and allows the estimation of the correct population ratio when used in conjunction with very tight distance restraints. With the qualitative distance restraints most commonly used in NMR structure determination, cross-validation is unsuccessful in providing the correct answer. Other experimental sources are therefore needed to determine relative populations of multi-conformer structures.To whom correspondence should be addressed.     

Denaturation and unfolding of human anaphylatoxin C3a: an unusually low covalent stability of its native disulfide bonds

The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl]_1/2 at 3.4-5 M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1 mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys³⁶-Cys⁴⁹ and two disulfide bonds formed by two pair of consecutive cysteines, Cys²²-Cys²³ and Cys⁵⁶-Cys⁵⁷, a unique disulfide structure of polypeptide that has not been documented previously.     

Conformational control in structure-based drug design

In structure-based drug design, the basic goal is to design molecules that fit complementarily to a given binding pocket. Since such computationally modeled molecules may not adopt the intended bound conformation outside the binding pocket, one challenge is to ensure that the designed ligands adopt similar low energy conformations both inside and outside of the binding pocket. Computational chemistry methods and conformational preferences of small molecules from PDB and Cambridge Structural Database (CSD) can be used to predict the bound structures of the designed molecules. Herein, we review applications of conformational control in structure-based drug design using selected examples from the recent medicinal chemistry literature. The main purpose is to highlight some intriguing conformational features that can be applied to other drug discovery programs.     

Deciphering the “Fuzzy” Interaction of FG Nucleoporins and Transport Factors Using Small-Angle Neutron Scattering

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Calculation of Vapour-Liquid Equilibria of Lennard-Jones Binary Systems by the Gibbs Ensemble Monte Carlo Simulation

Abstract

The Gibbs ensemble Monte Carlo simulation has been used to calculate vapour-liquid equilibria of a Lennard-Jones (LJ) binary mixture. The mixture studied is the LB-2-1 model which has been used in our previous calculations on PVT relation and density-dependent local composition. The P-x-y relation has been established at two different temperatures and used to determine vapour-liquid coexistence region in the PVTx space.     

Conformational transition state is responsible for assembly of microtubule-binding domain of tau protein

In the brains of Alzheimer's disease patients, the tau protein dissociates from the axonal microtubule and abnormally aggregates to form a paired helical filament (PHF). One of the priorities in Alzheimer research is to clarify the mechanism of PHF formation. Although several reports on the regulation of tau assembly have been published, it is not yet clear whether in vivo PHFs are composed of beta-structures or alpha-helices. Since the four-repeat microtubule-binding domain (4RMBD) of the tau protein has been considered to play an essential role in PHF formation, its heparin-induced assembly propensity was investigated by the thioflavin fluorescence method to clarify what conformation is most preferred for the assembly. We analyzed the assembly propensity of 4RMBD in Tris-HCl buffer with different trifluoroethanol (TFE) contents, because TFE reversibly induces the transition of the random structure to the alpha-helical structure in an aqueous solution. Consequently, it was observed that the 4RMBD assembly is most significantly favored to proceed in the 10-30% TFE solution, the concentration of which corresponds to the activated transition state of 4RMBD from a random structure to an alpha-helical structure, as determined from the circular dichroism (CD) spectral changes. Since such an assembly does not occur in a buffer containing TFE of < 10% or > 40%, the intermediate conformation between the random and alpha-helical structures could be most responsible for the PHF formation of 4RMBD. This is the first report to clarify that the non-native alpha-helical intermediate in transition from random coil is directly associated with filament formation at the start of PHF formation.     

High-throughput screening of bacteriorhodopsin mutants in whole cell pastes

A high-throughput screening method has been developed which enables functional analysis of bacteriorhodpsin in whole cell pastes. Reflectance spectra, from as little as 5 ml of Halobacterium salinarum cells, show close correspondence to that obtained from the purified purple membrane (PM), containing bacteriorhodopsin (BR) as the sole protein component. We demonstrate accurate quantification of BR accumulation by ratiometric analysis of BR (A_max 568) and a membrane-bound cytochrome (A_max 410). In addition, ground-state light- and dark-adapted (LA and DA, respectively) spectral differences were determined with high accuracy and precision. Using cells expressing the BR mutant D85N, we monitored transitions between intermediate-state homologues of the reprotonation phase of the light-activated proton pumping mechanism. We demonstrate that phenotypes of three mutants (D85N/T170C, D85N/D96N, and D85N/R82Q) previously characterized for their effect on photocycle transitions are reproduced in the whole cell samples. D85N/T170C stabilizes accumulation of the N state while D85N/D96N accumulates no N state. D85N/R82Q was found to have perturbed the pK_a of M accumulation. These studies illustrate the correspondence between pH-dependent ground-state transitions accessed by D85N and the transitions accessed by the wild-type protein following photoexcitation. We demonstrate that whole cell reflectance spectroscopy can be used to efficiently characterize the large numbers of mutants generated by engineering strategies that exploit saturation mutagenesis.