John C Voss - Academia.edu (original) (raw)

Papers by John C Voss

ACS Applied Materials & Interfaces, Apr 20, 2015

The changes in orientation and conformation of three different membrane scaffold proteins (MSPs) ... more The changes in orientation and conformation of three different membrane scaffold proteins (MSPs) upon entrapment in sol-gel derived mesoporous silica monoliths were investigated. MSPs were examined in either a lipid-free conformation or a lipid-bound conformation, where the proteins were associated with lipids to form nanolipoprotein particles (NLPs). NLPs are water soluble, disk-shaped patches of lipid bilayer that have amphiphilic MSPs shielding the hydrophobic lipid tails. The NLPs in this work had an average thickness of 5 nm and diameters of 9.2 nm, 9.7 nm, and 14.8 nm. We have previously demonstrated that NLPs are more suitable lipidbased structures for silica gel entrapment than liposomes due to their size compatibility with the mesoporous network (2-50 nm) and minimally altered structure after encapsulation. Here we further elaborate on that work by using a variety of spectroscopic techniques to elucidate whether or not different MSPs maintain their protein-lipid interactions after encapsulation. Fluorescence spectroscopy and quenching of the tryptophan residues with acrylamide, 5-DOXYL-stearic acid, and 16-DOXYL-stearic acid was used to determine MSP orientation. We also utilized fluorescence anisotropy of tryptophans to measure the relative size of NLPs and MSP aggregates after entrapment. Finally, circular dichroism spectroscopy was used to examine the secondary structure of the MSPs. Our results showed that after entrapment, all of the lipid-bound MSPs maintained orientations that were minimally changed and indicative of association with lipids in NLPs. The tryptophan residues appeared to remain buried within the hydrophobic core of the lipid tails in the NLPs and appropriately spaced from the bilayer center. Also after entrapment, lipid-bound MSPs maintained a high degree of α-helical content -a secondary structure associated with protein-lipid interactions. These findings demonstrate that NLPs are capable of serving as viable hosts for functional integral membrane proteins in the synthesis of sol-gel derived bio-inorganic hybrid nanomaterials.

Biochemistry, Sep 20, 2008

Phosphorylation drives the disassembly of the vimentin Intermediate Filament (IF1) cytoskeleton a... more Phosphorylation drives the disassembly of the vimentin Intermediate Filament (IF1) cytoskeleton at mitosis. Chromatographic analysis has suggested that phosphorylation produces a soluble vimentin tetramer, but little has been determined about the structural changes that are caused by phosphorylation, or the structure of the resulting tetramer. In this study, Site Directed Spin Labeling and Electron Paramagnetic Resonance (SDSL-EPR) were used to examine the structural changes resulting from Protein Kinase A phosphorylation of vimentin IFs in vitro. EPR spectra suggest that the tetrameric species resulting from phosphorylation is the A11 configuration. EPR spectra also establish that the greatest degree of structural change was found in the linker 2, and the C-terminal half of the rod domain, despite the fact that most phosphorylation occurs in the N-terminal head domain. The phosphorylation-induced changes notably affected the proposed "trigger sequences" located in the linker 2 region, which have been hypothesized to mediate the induction of coiled-coil formation. These data are the first to document specific changes in IF structure resulting from a physiologic regulatory mechanism, and provide further evidence, also generated by SDSL EPR, that the linker regions play a key role in IF structure, and regulation of assembly/disassembly. The intermediate filament (IF) protein family is the largest of the three major classes of cytoskeleton proteins, and consists of more than 65 human IF genes (1). IF proteins exhibit considerable primary sequence variation, but share a well-conserved predicted secondary structure that consists of an alpha-helical central rod domain flanked by head and tail domains that are highly variable in both size and sequence (2-4). The rod domain is approximately 310 amino acids in length and is often described as consisting of four alpha helical "coil" regions (1A, 1B, 2A and 2B), separated by three, non-helical "linker" regions L1, L12, and L2 (5,6) (see schematic in figure ). IF protein expression varies by cell type as well as the state of development/differentiation of the cell (reviewed (7)). The best known functions of IF proteins include protection against mechanical and metabolic stresses, but IF proteins have been implicated in a wide range of †

Biochimica Et Biophysica Acta - Biomembranes, Oct 1, 2020

Membrane active peptides (MAPs) have gained wide interest due to their far reaching applications ... more Membrane active peptides (MAPs) have gained wide interest due to their far reaching applications in drug discovery and drug delivery. The search for new MAPs, however, has been largely skewed with bias selecting for physicochemical parameters believed to be important for membrane activity, such as alpha helicity, cationicity and hydrophobicity. Here we carry out a search-and-find strategy to screen a 100,000-membered one-bead-one-compound (OBOC) combinatorial peptide library for lead compounds, agnostic of those physicochemical constraints. Such a synthetic strategy also permits expansion of our peptide repertoire to include unnatural amino acids. Using this approach, we discovered a structurally unique lead peptide LBF14, a linear 14-mer peptide, that induces gross morphological disruption of membranes, irrespective of membrane composition. Further, we demonstrate that the unique insertion mechanism of the peptide, visualized by spinning disc confocal microscopy and further analyzed by electron paramagnetic resonance measurements, may be the cause of this large scale membrane deformation. We also demonstrate the robustness, reproducibility, and potential application of this technique to discover and characterize new membrane active peptides that display activity by local insertion and subsequent allosteric effects leading to global membrane disruption.

Neurobiology of Aging, 2010

Alzheimer's disease (AD) is characterized by depositions of β-amyloid (Aβ) aggregates as amyloid ... more Alzheimer's disease (AD) is characterized by depositions of β-amyloid (Aβ) aggregates as amyloid in the brain. To facilitate diagnosis of AD by radioligand imaging, several highly specific small-molecule amyloid ligands have been developed. Because amyloid ligands display excellent pharmacokinetics properties and brain bioavailability, and because we have previously shown that some amyloid ligands bind the highly neurotoxic Aβ oligomers (AβO) with high affinities, they may also be valuable candidates for anti-Aβ therapies. Here we identified two fluorene compounds from libraries of amyloid ligands, initially based on their ability to block cell death secondary to intracellular AβO. We found that the lead fluorenes were able to reduce the amyloid burden including the levels of AβO in cultured neurons and in 5xFAD mice. To explain these in vitro and in vivo effects, we found that the lead fluorenes bind and destabilize AβO as shown by electron paramagnetic resonance spectroscopy studies, and block the harmful AβO-synapse interaction. These fluorenes and future derivatives, therefore, have a potential use in AD therapy and research.

Frontiers in Chemistry

Several neurodegenerative diseases are driven by misfolded proteins that assemble into soluble ag... more Several neurodegenerative diseases are driven by misfolded proteins that assemble into soluble aggregates. These “toxic oligomers” have been associated with a plethora of cellular dysfunction and dysregulation, however the structural features underlying their toxicity are poorly understood. A major impediment to answering this question relates to the heterogeneous nature of the oligomers, both in terms of structural disorder and oligomer size. This not only complicates elucidating the molecular etiology of these disorders, but also the druggability of these targets as well. We have synthesized a class of bifunctional stilbenes to modulate both the conformational toxicity within amyloid beta oligomers (AβO) and the oxidative stress elicited by AβO. Using a neuronal culture model, we demonstrate this bifunctional approach has the potential to counter the molecular pathogenesis of Alzheimer’s disease in a powerful, synergistic manner. Examination of AβO structure by various biophysical...

<p>Small oligomers used in this study are formed within 60 minutes (scale bar 10 nm). The d... more <p>Small oligomers used in this study are formed within 60 minutes (scale bar 10 nm). The direct visualization of oligomers by using AFM revealed the formation of AβO aggregates. (A) 50 µM of Aβ after 24 hours of incubation at room temperature (diameter 5–10 nm). B) 50 µM of Aβ and 50 µM of SLF after 24 hours of incubation at room temperature. (C) Staining for beta-rich assemblies by the amyloid dye thioflavin T for incubations of Aβ with and without SLF HO-4160 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035443#s3" target="_blank">Methods</a>. Data are the averages from 3 separate experiments with the error bars representing the SEM.</p

Biochemical and Biophysical Research Communications, Jul 1, 2013

In Saccharomyces cerevisiae, Pho89 mediates a cation-dependent transport of Pi across the plasma ... more In Saccharomyces cerevisiae, Pho89 mediates a cation-dependent transport of Pi across the plasma membrane. This integral membrane protein belongs to the Inorganic Phosphate Transporter (PiT) family, a group that includes the mammalian Na + /Pi cotransporters Pit1 and Pit2. Here we report that the Pichia pastoris expressed recombinant Pho89 was purified in the presence of Foscholine-12 and functionally reconstituted into proteoliposomes with a similar substrate specificity as observed in an intact cell system. The alpha-helical content of the Pho89 protein was estimated to 44%. EPR analysis showed that purified Pho89 protein undergoes conformational change upon addition of substrate.

Nanoscale Advances, 2021

Extracellular vesicles can carry Aβ associated with Alzheimer's disease. In this article we i... more Extracellular vesicles can carry Aβ associated with Alzheimer's disease. In this article we identify specific Raman spectroscopic features that can be associated with Aβ present in the molecular cargo of small extracellular vesicles.

Journal of Materials Chemistry B, 2020

Using a regular CMOS sensor as a template, we are able to fabricate a simple but highly effective... more Using a regular CMOS sensor as a template, we are able to fabricate a simple but highly effective superhydrophobic SERS substrate.

Frontiers in Chemistry, 2021

Apolipoprotein E (ApoE), an important mediator of lipid transportation in plasma and the nervous ... more Apolipoprotein E (ApoE), an important mediator of lipid transportation in plasma and the nervous system, plays a large role in diseases such as atherosclerosis and Alzheimer's. The major allele variants ApoE3 and ApoE4 differ only by one amino acid. However, this difference has major consequences for the physiological behaviour of each variant. In this paper, we follow (i) the initial interaction of lipid-free ApoE variants with model membranes as a function of lipid saturation, (ii) the formation of reconstituted High-Density Lipoprotein-like particles (rHDL) and their structural characterisation, and (iii) the rHDL ability to exchange lipids with model membranes made of saturated lipids in the presence and absence of cholesterol [1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) with and without 20 mol% cholesterol]. Our neutron reflection results demonstrate that the protein variants interact differently with the model ...

The Journal of Physical Chemistry C, 2017

The soluble oligomeric form of the amyloid beta (Aβ) peptide is the major causative agent in the ... more The soluble oligomeric form of the amyloid beta (Aβ) peptide is the major causative agent in the molecular pathogenesis of Alzheimer's disease (AD). We have previously developed a pyrrolinenitroxyl fluorene compound (SLF) that blocks the toxicity of Aβ. Here we introduce the multiparametric surface plasmon resonance (MP-SPR) approach to quantify SLF binding and effect on the self-association of the peptide via a label-free, real-time approach. Kinetic analysis of SLF binding to Aβ and measurements of layer thickness alterations inform on the mechanism underlying the ability of SLF to inhibit Aβ toxicity and its progression towards larger oligomeric assemblies. Depending on the oligomeric state of Aβ, distinct binding affinities for SLF are revealed. The Aβ monomer and dimer uniquely possess sub-nanomolar affinity for SLF via a nonspecific mode of binding. SLF binding is weaker in oligomeric Aβ, which displays an affinity for SLF on the order of 100 μM. To complement these experiments we carried out molecular docking and molecular dynamics simulations to explore how SLF interacts with the Aβ peptide. The MP-SPR results together with in silico modeling provide affinity data for the SLF-Aβ interaction and allow us to develop a new general method for examining protein aggregation.

Biophysical Journal, 2016

Structures of over 30 different G protein-coupled receptors (GPCRs) have advanced our understandi... more Structures of over 30 different G protein-coupled receptors (GPCRs) have advanced our understanding of cell signaling and provided a foundation for structure-guided drug design. This exciting progress required the development of three complimentary methods to facilitate GPCR crystallization, one of which is the thermostabilization of receptors by systematic mutagenesis. However, the reason why a particular mutation, or combination of mutations, stabilizes the receptor is not always evident from a static crystal structure. We have used extensive Molecular Dynamics (MD) simulations to identify the structural dynamics features that lead to stability. We have also calculated the energetic (the enthalpic and entropic) contributions that affect thermostability through comparing the dynamics of the thermostabilized receptors with structurebased models of the wild-type receptor. The data indicate that receptors are stabilized through a combination of factors, including an increase in receptor rigidity, a decrease in collective motion, reduced stress at specific residues, and the presence of ordered water molecules. Predicting thermostabilizing mutations computationally represents a major challenge for the field. We will elaborate on how the MD simulations provide insight into the structural features that can be used in making predictions of thermostable mutations ahead of experiments.

Protein Expression and Purification, 2008

Saporin, a ribosome inactivating protein is widely used for immunotoxin construction. Here we des... more Saporin, a ribosome inactivating protein is widely used for immunotoxin construction. Here we describe a mutation of saporin (sap)-3 DNA by introducing a cysteine residue, followed by protein expression and purification by ion exchange chromatography. The purified Cys255sap-3, sap-3 isomer and commercially purchased saporin, were tested for toxicity using assays measuring inhibition for protein synthesis. The IC 50 values showed that the toxicity of the Cys255sap-3 is equivalent to the sap-3 isomer and commercial saporin. Reactivity of Cys255sap-3 was confirmed by labeling with a thio-specific fluorescent probe as well as conjugation with a nonspecific mouse IgG. We have found that a single cysteine within saporin provides a method for antibody conjugation that ensures a uniform and reproducible modification of a saporin variant retaining high activity.

Nature Communications, 2013

An outstanding unresolved question is how does the mitotic spindle utilize microtubules and mitot... more An outstanding unresolved question is how does the mitotic spindle utilize microtubules and mitotic motors to coordinate accurate chromosome segregation during mitosis? This process depends upon the mitotic motor, kinesin-5, whose unique bipolar architecture, with pairs of motor domains lying at opposite ends of a central rod, allows it to crosslink microtubules within the mitotic spindle and to coordinate their relative sliding during spindle assembly, maintenance and elongation. The structural basis of kinesin-5's bipolarity is, however, unknown, as protein asymmetry has so far precluded its crystallization. Here we use electron microscopy of single molecules of kinesin-5 and its subfragments, combined with hydrodynamic analysis plus mass spectrometry, circular dichroism and site-directed spin label electron paramagnetic resonance spectroscopy, to show how a staggered antiparallel coiledcoil 'BASS' (bipolar assembly) domain directs the assembly of four kinesin-5 polypeptides into bipolar minifilaments.

Biophysical Journal, 2010

Cells derive their mechanical properties largely from the cytoskeleton: a network of stiff biopol... more Cells derive their mechanical properties largely from the cytoskeleton: a network of stiff biopolymers and associated proteins, capable of cross-linking and force generation. The short-timescale mechanical response of biopolymer gels has been studied extensively, while little is known about the biologically more relevant long-time behavior. It is on these longer timescales that the cytoskeleton remodels in response to internal and external cues. Here we present predictions for the viscoelastic behaviour of semiflexible polymer networks cross-linked with physiological transient linkers. Our model allows us to elucidate the mechanisms by which the network can relax as a result of the constant breakage and formation of links in the network.

Biochemistry, 1995

We have previously shown that the basic, amphipathic peptide melittin inhibits the Ca-ATPase of t... more We have previously shown that the basic, amphipathic peptide melittin inhibits the Ca-ATPase of the sarcoplasmic reticulum membrane by inducing large-scale aggregation of the enzyme via electrostatic cross-linking. To better understand the physical mechanism by which melittin-induced Ca-ATPase aggregation inhibits the enzyme, we have performed time-resolved phosphorescence anisotropy (TPA) and steady-state fluorescence experiments in combination with enzyme kinetic assays, utilizing (1) native and charge-modified melittin in order to characterize the peptide charge dependence of the melittin-SR interaction, and (2) various calcium levels in order to define the effect of melittin on the enzyme's E l and E2 conformational equilibrium. TPA results showed that decreasing melittin's positive charge dramatically decreases the ability of the peptide to aggregate the enzyme, which correlates with a reduced potency of the modified peptide to inhibit enzymatic activity. Steady-state fluorescence of fluorescein isothiocyanatelabeled Ca-ATPase showed that melittin reduces Ca-ATPase affinity for calcium by shifting the enzyme's E l-E2 conformational equilibrium toward E2, but increasing calcium progressively reverses this shift. Kinetic experiments showed that melittin does not prevent ATP-dependent enzyme phosphorylation, but it completely inhibits Pi-dependent EP formation and substantially slows Pi release during steady-state cycling. We conclude that melittin-induced aggregation of the Ca-ATPase depends on the electrostatic interaction of the peptide with cytoplasmic Ca2+-dependent sites on the enzyme, and that enforced Ca-ATPase protein-protein interactions inhibit the conformational transitions that facilitate phosphoenzyme hydrolysis. The Ca-ATPase in fast-twitch skeletal sarcoplasmic reticulum (SR)' is a transmembrane protein of approximately 110 kD, which couples the transport of 2 mol of Ca2+ across the SR membrane per mole of ATP hydrolyzed (Inesi, 1985). A thoroughly tested and widely accepted model for the Ca-ATPase enzymatic cycle is shown in Scheme 1. In this model, the enzyme cycles between two fundamental conformations, E l and E2, which couple ATP hydrolysis to calcium transport via differences in their affinities and vectorial specificities for ATP and Ca2+. In the absence of substrates and/or ligands, the enzyme is in equilibrium between E l and E2 (step 8), but micromolar calcium shifts this conformational equilibrium [& x 1 x 10l2 M-2 (Alonso

Biophysical Journal, 1994

The Ca-ATPase in the cardiac sarcoplasmic reticulum membrane is regulated by an amphipathic trans... more The Ca-ATPase in the cardiac sarcoplasmic reticulum membrane is regulated by an amphipathic transmembrane protein, phospholamban. We have used time-resolved phosphorescence anisotropy to detect the microsecond rotational dynamics, and thereby the self-association, of the Ca-ATPase as a function of phospholamban phosphorylation and physiologically relevant calcium levels. The phosphorylation of phospholamban increases the rotational mobility of the Ca-ATPase in the sarcoplasmic reticulum bilayer, due to a decrease in large-scale protein association, with a [Ca2+] dependence parallel to that of enzyme activation. These results support a model in which phospholamban phosphorylation or calcium free the enzyme from a kinetically unfavorable associated state.

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Hepatology (Baltimore, Md.), May 18, 2017

The identification of environmental factors that lead to loss of tolerance has been coined the Ho... more The identification of environmental factors that lead to loss of tolerance has been coined the Holy Grail of autoimmunity. Our work has focused on the reactivity of antimitochondrial autoantibodies (AMA) to chemical xenobiotics and has hypothesized that a modified peptide within PDC-E2, the major mitochondrial autoantigen, will have been immunologically recognized at the time of loss of tolerance. Herein we successfully applied intein technology to construct a PDC-E2 protein fragment containing amino acid residues 177-314 of PDC-E2 by joining a recombinant peptide spanning residues 177 to 252 (PDC-228) with a 62 residue synthetic peptide from 253 to 314 (PP), which encompasses PDC-E2 ILD. We named this intein-constructed fragment PPL. Importantly, PPL, as well as lipoic acid conjugated PPL (LA-PPL) and xenobiotic 2-octynoic acid conjugated PPL (2OA-PPL), are recognized by AMA. Of great importance, AMA has specificity for the 2OA modified PDC-E2 ILD peptide backbone distinct from ant...