Daniel K. Weber

Selective mitochondrial accumulation of cytotoxic dinuclear polypyridyl ruthenium(II) complexes

The lipophilic ligand-bridged dinuclear cation Rubb₁₆ is significantly cytotoxic and preferentially accumulates in the mitochondria of the L1210 murine leukemia cancer cell line.

Bacteria May Cope Differently from Similar Membrane Damage Caused by the Australian Tree Frog Antimicrobial Peptide Maculatin 1.1

Background: Mac1 specificity and bactericidal concentrations may be due to a distinctive membrane disrupting mechanism. Results: S. aureus growth is inhibited at 16-fold lower Mac1 concentration than E. coli, but both lipid membranes are compromised at similar concentrations. Conclusion: Bacteria may cope differently with membrane damage. Significance: Antimicrobial peptide mode of action needs to be better understood to develop new drug candidate. Maculatin 1.1 (Mac1) is an antimicrobial peptide from the skin of Australian tree frogs and is known to possess selectivity toward Gram-positive bacteria. Although Mac1 has membrane disrupting activity, it is not known how Mac1 selectively targets Gram-positive over Gram-negative bacteria. The interaction of Mac1 with Escherichia coli, Staphylococcus aureus, and human red blood cells (hRBC) and with their mimetic model membranes is here reported. The peptide showed a 16-fold greater growth inhibition activity against S. aureus (4 μm) than against E. coli (64 μm) and an intermediate cytotoxicity against hRBC (30 μm). Surprisingly, Sytox Green uptake monitored by flow cytometry showed that Mac1 compromised both bacterial membranes with similar efficiency at ∼20-fold lower concentration than the reported minimum inhibition concentration against S. aureus. Mac1 also reduced the negative potential of S. aureus and E. coli membrane with similar efficacy. Furthermore, liposomes mimicking the cell membrane of S. aureus (POPG/TOCL) and E. coli (POPE/POPG) were lysed at similar concentrations, whereas hRBC-like vesicles (POPC/SM/Chol) remained mostly intact in the presence of Mac1. Remarkably, when POPG/TOCL and POPE/POPG liposomes were co-incubated, Mac1 did not induce leakage from POPE/POPG liposomes, suggesting a preference toward POPG/TOCL membranes that was supported by surface plasma resonance assays. Interestingly, circular dichroism spectroscopy showed a similar helical conformation in the presence of the anionic liposomes but not the hRBC mimics. Overall, the study showed that Mac1 disrupts bacterial membranes in a similar fashion before cell death events and would preferentially target S. aureus over E. coli or hRBC membranes.

Measuring translational diffusion coefficients of peptides and proteins by PFG‑NMR using band‑selective RF pulses

Abstract Molecular translational self-diffusion, a measure of diffusive motion, provides information on the effective molecular hydrodynamic radius, as well as information on the properties of media or solution through which the molecule diffuses. Protein translational diffusion measured by pulsed-field gradient nuclear magnetic resonance (PFG-NMR) has seen increased application in structure and interaction studies, as structural changes or protein–protein interactions are often accompanied by alteration of their effective hydrodynamic radii. Unlike the analysis of complex mixtures by PFG-NMR, for monitoring changes of protein translational diffusion under various conditions, such as different stages of folding/unfolding, a partial region of the spectrum or even a single resonance is sufficient. We report translational diffusion coefficients measured by PFG-NMR with a modified stimulated echo (STE) sequence where band-selective pulses are employed for all three 1H RF pulses. Compared with conventional non-selective sequence, e.g. the BPP-LED sequence, the advantage of this modified band-selective excitation short transient (BEST) version of STE (BEST-STE) sequence is multi-fold, namely: (1) potential sensitivity gain as in generalized BEST-based sequences, (2) water suppression is no longer required as the magnetization of solvent water is not perturbed during the measurement, and (3) dynamic range problems due to the presence of intense resonances from molecules other than the protein or peptide of interest, such as non-deuterated detergent micelles, are avoided.

A practical implementation of de-Pake-ing via weighted Fourier transformation

We provide an NMRPipe macro to meet an increasing need in membrane biophysics for facile de-Pake-ing of axially symmetric deuterium, and to an extent phosphorous, static lineshapes. The macro implements the development of McCabe & Wassall (1997), and is run as a simple replacement for the usual Fourier transform step in an NMRPipe processing procedure.

Copper Modulation of Amyloid Beta 42 Interactions with Model Membranes

Growing evidence supports that interactions of the amyloid-β peptide Aβ(1–42) with neuronal cell membranes and copper are involved in Alzheimer’s disease pathogenesis. We report using solid-state NMR that the peptide significantly perturbed the phosphate and upper acyl chain region of bilayers comprising brain total lipid extract to cause domain segregation. Deep headgroup perturbations were also realized for palmitoyloleoylphospatidylcholine–cholesterol model systems; however, incorporating 10 % palmitoyloleoylphosphatidylserine or the ganglioside GM1 resulted in a more peripheral interaction. Cu2+ at a 1 : 7 molar ratio to peptide caused deeper penetration into model systems, but partially attenuated interactions with brain total lipid extract. Thioflavin T assay showed that bilayers affected amyloid formation in a mode dependant on lipid content, and was further modulated by addition of Cu2+. Our data support that ternary interactions between Cu2+, lipids and Aβ(1–42) may have significance in Alzheimer’s disease, and challenge the validity of model bilayers as substitutes for natural systems.

Dynamic Modelling Reveals 'Hotspots' on the Pathway to Enzyme-Substrate Complex Formation.

Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step in the diaminopimelate pathway of bacteria, yielding amino acids required for cell wall and protein biosyntheses. The essentiality of the enzyme to bacteria, coupled with its absence in humans, validates DHDPS as an antibacterial drug target. Conventional drug design efforts have thus far been unsuccessful in identifying potent DHDPS inhibitors. Here, we make use of contemporary molecular dynamics simulation and Markov state models to explore the interactions between DHDPS from the human pathogen Staphylococcus aureus and its cognate substrate, pyruvate. Our simulations recover the crystallographic DHDPS-pyruvate complex without a priori knowledge of the final bound structure. The highly conserved residue Arg140 was found to have a pivotal role in coordinating the entry of pyruvate into the active site from bulk solvent, consistent with previous kinetic reports, indicating an indirect role for the residue in DHDPS catalysis. A metastable binding intermediate characterized by multiple points of intermolecular interaction between pyruvate and key DHDPS residue Arg140 was found to be a highly conserved feature of the binding trajectory when comparing alternative binding pathways. By means of umbrella sampling we show that these binding intermediates are thermodynamically metastable, consistent with both the available experimental data and the substrate binding model presented in this study. Our results provide insight into an important enzyme-substrate interaction in atomistic detail that offers the potential to be exploited for the discovery of more effective DHDPS inhibitors and, in a broader sense, dynamic protein-drug interactions.

Aggregation kinetics in the presence of brain lipids of Aβ(1–40) cleaved from a soluble fusion protein

The cleavage of the amyloid precursor protein by β- and γ-secretases is a key event in Alzheimers disease. A fusion protein was constructed to investigate the cleavage rate and aggregation kinetics of amyloid-beta (1-40) (Aβ(1-40)) peptides. The peptide was expressed with a Small Ubiquitin-Like Modifier (SUMO) on the N-terminus and cleaved by a SUMO protease Ulp1. The time course of the cleavage reaction was monitored by SDS-PAGE gel with 100:1 or 1000:1 SUMO-Aβ(1-40) to Ulp1 molar ratio and in the presence of brain total lipid extract unilamellar vesicles. Similarly, the aggregation of Aβ(1-40) peptides upon cleavage was monitored by thioflavin T fluorescence assays and by circular dichroism. The cleavage reaction was modulated by the concentration of Ulp1, with fast release of Aβ(1-40) peptides producing shorter lag time before fibril formation, but with similar elongation rate. The presence of lipids significantly reduced the cleavage completion at 1000:1, but reduced the lag time before fibril formation, while at 100:1 similar cleavage and aggregation kinetics were observed compared to the lipid-free condition. Overall, the results showed that the fusion protein SUMO-Aβ(1-40) is a means to study the cleavage and aggregation of amyloid peptides and that the presence of lipids and the fast release rate accelerated the aggregation of Aβ(1-40) peptides.