Application of Funnel Metadynamics to the Platelet Integrin αIIbß3 in Complex with an RGD Peptide.

Integrin αIIbß3 mediates platelet aggregation by binding the Arginyl-Glycyl-Aspartic acid (RGD) sequence of fibrinogen. RGD binding occurs at a site topographically proximal to the αIIb and ß3 subunits, promoting the conformational activation of the receptor from bent to extended states. While several experimental approaches have characterized RGD binding to αIIbß3 integrin, applying computational methods has been significantly more challenging due to limited sampling and the need for a priori information regarding the interactions between the RGD peptide and integrin. In this study, we employed all-atom simulations using funnel metadynamics (FM) to evaluate the interactions of an RGD peptide with the αIIb and ß3 subunits of integrin. FM incorporates an external history-dependent potential on selected degrees of freedom while applying a funnel-shaped restraint potential to limit RGD exploration of the unbound state. Furthermore, it does not require a priori information about the interactions, enhancing the sampling at a low computational cost. Our FM simulations reveal significant molecular changes in the ß3 subunit of integrin upon RGD binding and provide a free-energy landscape with a low-energy binding mode surrounded by higher-energy prebinding states. The strong agreement between previous experimental and computational data and our results highlights the reliability of FM as a method for studying dynamic interactions of complex systems such as integrin.

Drunken lipid membranes, not drunken SNARE proteins, promote fusion in a model of neurotransmitter release.

Alcohol affects many neuronal proteins that are upstream or down-stream of synaptic vesicle fusion and neurotransmitter release. Less well studied is alcohol's effect on the fusion machinery including SNARE proteins and lipid membranes. Using a SNARE-driven fusion assay we show that fusion probability is significantly increased at 0.4% v/v (68 mM) ethanol; but not with methanol up to 10%. Ethanol appears to act directly on membrane lipids since experiments focused on protein properties [circular dichroism spectrometry, site-directed fluorescence interference contrast (sdFLIC) microscopy, and vesicle docking results] showed no significant changes up to 5% ethanol, but a protein-free fusion assay also showed increased lipid membrane fusion rates with 0.4% ethanol. These data show that the effects of high physiological doses of ethanol on SNARE-driven fusion are mediated through ethanol's interaction with the lipid bilayer of membranes and not SNARE proteins, and that methanol affects lipid membranes and SNARE proteins only at high doses.

A review of TNP-ATP in protein binding studies: benefits and pitfalls.

We review 50 years of use of 2',3'-O-trinitrophenyl (TNP)-ATP, a fluorescently tagged ATP analog. It has been extensively used to detect binding interactions of ATP to proteins and to measure parameters of those interactions such as the dissociation constant, Kd, or inhibitor dissociation constant, Ki. TNP-ATP has also found use in other applications, for example, as a fluorescence marker in microscopy, as a FRET pair, or as an antagonist (e.g., of P2X receptors). However, its use in protein binding studies has limitations because the TNP moiety often enhances binding affinity, and the fluorescence changes that occur with binding can be masked or mimicked in unexpected ways. The goal of this review is to provide a clear perspective of the pros and cons of using TNP-ATP to allow for better experimental design and less ambiguous data in future experiments using TNP-ATP and other TNP nucleotides.