Drew C. Tilley

An Integrated Framework Advancing Membrane Protein Modeling and Design

Membrane proteins are critical functional molecules in the human body, constituting more than 30% of open reading frames in the human genome. Unfortunately, a myriad of difficulties in overexpression and reconstitution into membrane mimetics severely limit our ability to determine their structures. Computational tools are therefore instrumental to membrane protein structure prediction, consequently increasing our understanding of membrane protein function and their role in disease. Here, we describe a general framework facilitating membrane protein modeling and design that combines the scientific principles for membrane protein modeling with the flexible software architecture of Rosetta3. This new framework, called RosettaMP, provides a general membrane representation that interfaces with scoring, conformational sampling, and mutation routines that can be easily combined to create new protocols. To demonstrate the capabilities of this implementation, we developed four proof-of-concept applications for (1) prediction of free energy changes upon mutation; (2) high-resolution structural refinement; (3) protein-protein docking; and (4) assembly of symmetric protein complexes, all in the membrane environment. Preliminary data show that these algorithms can produce meaningful scores and structures. The data also suggest needed improvements to both sampling routines and score functions. Importantly, the applications collectively demonstrate the potential of combining the flexible nature of RosettaMP with the power of Rosetta algorithms to facilitate membrane protein modeling and design.

Tarantula toxins use common surfaces for interacting with Kv and ASIC ion channels

Tarantula toxins that bind to voltage-sensing domains of voltage-activated ion channels are thought to partition into the membrane and bind to the channel within the bilayer. While no structures of a voltage-sensor toxin bound to a channel have been solved, a structural homolog, psalmotoxin (PcTx1), was recently crystalized in complex with the extracellular domain of an acid sensing ion channel (ASIC). In the present study we use spectroscopic, biophysical and computational approaches to compare membrane interaction properties and channel binding surfaces of PcTx1 with the voltage-sensor toxin guangxitoxin (GxTx-1E). Our results show that both types of tarantula toxins interact with membranes, but that voltage-sensor toxins partition deeper into the bilayer. In addition, our results suggest that tarantula toxins have evolved a similar concave surface for clamping onto α-helices that is effective in aqueous or lipidic physical environments. DOI: http://dx.doi.org/10.7554/eLife.06774.001

Chemoselective tarantula toxins report voltage activation of wild-type ion channels in live cells

Significance Electrically excitable cells, such as neurons, exhibit tremendous variation in their patterns of electrical signals. These variations arise from the collection of ion channels present in any specific cell, but understanding which ion channels are at the root of particular electrical signals remains a significant challenge. Here, we describe novel probes, derived from a tarantula venom peptide, that are able to report the activity of voltage-gated ion channels in living cells. This technology uses state-selective binding to optically monitor the activation of ion channels during cellular electrical signaling. Activity-reporting probes based on these prototypes could potentially identify when endogenous ion channels contribute to electrical signaling, thus facilitating the identification of ion channel targets for therapeutic drug intervention. Electrically excitable cells, such as neurons, exhibit tremendous diversity in their firing patterns, a consequence of the complex collection of ion channels present in any specific cell. Although numerous methods are capable of measuring cellular electrical signals, understanding which types of ion channels give rise to these signals remains a significant challenge. Here, we describe exogenous probes which use a novel mechanism to report activity of voltage-gated channels. We have synthesized chemoselective derivatives of the tarantula toxin guangxitoxin-1E (GxTX), an inhibitory cystine knot peptide that binds selectively to Kv2-type voltage gated potassium channels. We find that voltage activation of Kv2.1 channels triggers GxTX dissociation, and thus GxTX binding dynamically marks Kv2 activation. We identify GxTX residues that can be replaced by thiol- or alkyne-bearing amino acids, without disrupting toxin folding or activity, and chemoselectively ligate fluorophores or affinity probes to these sites. We find that GxTX–fluorophore conjugates colocalize with Kv2.1 clusters in live cells and are released from channels activated by voltage stimuli. Kv2.1 activation can be detected with concentrations of probe that have a trivial impact on cellular currents. Chemoselective GxTX mutants conjugated to dendrimeric beads likewise bind live cells expressing Kv2.1, and the beads are released by channel activation. These optical sensors of conformational change are prototype probes that can indicate when ion channels contribute to electrical signaling.

What keeps Kv channels small? The molecular physiology of modesty

All known K+ channels use the same molecular architecture to achieve K+ selectivity. Selectivity filters, even from distantly related K+ channels, appear nearly identical in atomic resolution crystal structures; remarkably, the deviation in the coordinates of K+-coordinating backbone carbonyls is

Azide–Alkyne Click Conjugation on Quantum Dots by Selective Copper Coordination

Functionalization of nanocrystals is essential for their practical application, but synthesis on nanocrystal surfaces is limited by incompatibilities with certain key reagents. The copper-catalyzed azide-alkyne cycloaddition is among the most useful methods for ligating molecules to surfaces, but has been largely useless for semiconductor quantum dots (QDs) because Cu+ ions quickly and irreversibly quench QD fluorescence. To discover nonquenching synthetic conditions for Cu-catalyzed click reactions on QD surfaces, we developed a combinatorial fluorescence assay to screen >2000 reaction conditions to maximize cycloaddition efficiency while minimizing QD quenching. We identify conditions for complete coupling without significant quenching, which are compatible with common QD polymer surfaces and various azide/alkyne pairs. Based on insight from the combinatorial screen and mechanistic studies of Cu coordination and quenching, we find that superstoichiometric concentrations of Cu can promote full coupling if accompanied by ligands that selectively compete with the Cu from the QD surface but allow it to remain catalytically active. Applied to the conjugation of a K+ channel-specific peptidyl toxin to CdSe/ZnS QDs, we synthesize unquenched QD conjugates and image their specific and voltage-dependent affinity for K+ channels in live cells.

Molecular Dynamics Models of Two Proposed Protein Structures of Salmon Protamine

Protamine is an arginine rich 32 residue DNA binding protein. It is hypothesized that protamine allows DNA to be densely packed in the later stages of spermogenesis, although no experimental structures of DNA-bound protamine have been solved. We present models of two possible bound structures for salmon protamine to DNA. The first structure has an extended conformation within the major groove (s1) and a second structure modelled with an alpha-helix between the 19th and 23rd residues (s2). These protamine structures were modeled into a 40mer of double stranded B-form DNA and simulated by molecular dynamics with explicit water using the Amber99SB force field. The structure of protamine bound DNA did not significantly differ from a control simulation of DNA. The positively charged protamine displaced sodium counter ions from the DNA backbone decreasing the density of sodium around the nucleotide. The arginines additionally displaced the water molecules within 20 A from the phosphates on the DNA backbone. Calculated binding energies for the s1 and s2 protamine:DNA complexes were −680.6 kJ/mol and −692.7 kJ/mol, respectively. The protamine displacing the water and counter ions could allow DNA packing with greater density, while retaining a near-B-form DNA structure.