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Dive into the research topics where David Punihaole is active.

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Featured researches published by David Punihaole.


Analytical Chemistry | 2014

2D Photonic Crystal Protein Hydrogel Coulometer for Sensing Serum Albumin Ligand Binding

Zhongyu Cai; Jian Tao Zhang; Fei Xue; Zhenmin Hong; David Punihaole; Sanford A. Asher

Bovine and human serum albumin (BSA and HSA) are globular proteins that function as bloodstream carriers of hydrophobes such as fatty acids and drugs. We fabricated novel photonic crystal protein hydrogels by attaching 2D colloidal arrays onto pure BSA and HSA hydrogels. The wavelengths of the diffracted light sensitively report on the protein hydrogel surface area. The binding of charged species to the protein hydrogel gives rise to Donnan potentials that change the hydrogel volume causing shifts in the diffraction. These photonic crystal protein hydrogels act as sensitive Coulometers that monitor the hydrogel charge state. We find multiple high-affinity BSA and HSA binding sites for salicylate, ibuprofen and picosulfate by using these sensors to monitor binding of charged drugs. We demonstrate proof-of-concept for utilizing protein hydrogel sensors to monitor protein-ionic species binding.


Angewandte Chemie | 2015

A Photonic Crystal Protein Hydrogel Sensor for Candida albicans

Zhongyu Cai; Daniel H. Kwak; David Punihaole; Zhenmin Hong; Sachin S. Velankar; Xinyu Liu; Sanford A. Asher

We report two-dimensional (2D) photonic crystal (PC) sensing materials that selectively detect Candida albicans (C. albicans). These sensors utilize Concanavalin A (Con A) protein hydrogels with a 2D PC embedded on the Con A protein hydrogel surface, that multivalently and selectively bind to mannan on the C. albicans cell surface to form crosslinks. The resulting crosslinks shrink the Con A protein hydrogel, reduce the 2D PC particle spacing, and blue-shift the light diffracted from the PC. The diffraction shifts can be visually monitored, measured with a spectrometer, or determined from the Debye diffraction ring diameter. Our unoptimized hydrogel sensor has a detection limit of around 32 CFU/mL for C. albicans. This sensor distinguishes between C. albicans and those microbes devoid of cell-surface mannan such as the gram-negative bacterium E. coli. This sensor provides a proof-of-concept for utilizing recognition between lectins and microbial cell surface carbohydrates to detect microorganisms in aqueous environments.


The Cardiology | 2012

Characterization of the Role of Nitric Oxide and Its Clinical Applications

Arlene B. Levine; David Punihaole; T. Barry Levine

Nitric oxide (NO) has long been known as endothelium-derived relaxing factor. It is a vasodilator, modulating vascular tone, blood pressure and hemodynamics, a role exploited by nitrate donor therapy for angina, heart failure, pulmonary hypertension and erectile dysfunction. In addition, its powerful antioxidant, anti-inflammatory and antithrombotic actions are antiatherogenic with antiatherothrombotic impact. NO signaling modulates skeletal muscle and myocardial contractility and metabolism and is intimately linked with insulin signaling. Vascular and muscle NO signaling coordinate skeletal muscle and myocardial energy demand with supply and are critical for both carbohydrate and fatty acid total-body homeostasis. NO signaling in mitochondria underlies much of NO’s metabolic effect, which, at low physiologic levels, links cellular energy demand with mitochondrial energy supply, while beneficially affecting mitochondrial oxidative stress and calcium handling. Mitochondria are also the site for the life-threatening deleterious effects arising from inflammation-related excessive NO levels. NO-deficient states are characterized by cell senescence, oxidative stress, inflammation, endothelial dysfunction, vascular disease, insulin resistance and type 2 diabetes mellitus. NO-enriching therapy would be expected to be of benefit not only for its hemodynamic but also for its metabolic impact. In contrast, strategies are needed to curtail excessive NO in states such as septic shock.


Biochemistry | 2012

UV resonance raman spectroscopy monitors polyglutamine backbone and side chain hydrogen bonding and fibrillization

Kan Xiong; David Punihaole; Sanford A. Asher

We utilize 198 and 204 nm excited UV resonance Raman spectroscopy (UVRR) and circular dichroism spectroscopy (CD) to monitor the backbone conformation and the Gln side chain hydrogen bonding (HB) of a short, mainly polyGln peptide with a D(2)Q(10)K(2) sequence (Q10). We measured the UVRR spectra of valeramide to determine the dependence of the primary amide vibrations on amide HB. We observe that a nondisaggregated Q10 (NDQ10) solution (prepared by directly dissolving the original synthesized peptide in pure water) exists in a β-sheet conformation, where the Gln side chains form hydrogen bonds to either the backbone or other Gln side chains. At 60 °C, these solutions readily form amyloid fibrils. We used the polyGln disaggregation protocol of Wetzel et al. [Wetzel, R., et al. (2006) Methods Enzymol.413, 34-74] to dissolve the Q10 β-sheet aggregates. We observe that the disaggregated Q10 (DQ10) solutions adopt PPII-like and 2.5(1)-helix conformations where the Gln side chains form hydrogen bonds with water. In contrast, these samples do not form fibrils. The NDQ10 β-sheet solution structure is essentially identical to that found in the NDQ10 solid formed upon evaporation of the solution. The DQ10 PPII and 2.5(1)-helix solution structure is essentially identical to that in the DQ10 solid. Although the NDQ10 solution readily forms fibrils when heated, the DQ10 solution does not form fibrils unless seeded with the NDQ10 solution. This result demonstrates very high activation barriers between these solution conformations. The NDQ10 fibril secondary structure is essentially identical to that of the NDQ10 solution, except that the NDQ10 fibril backbone conformational distribution is narrower than in the dissolved species. The NDQ10 fibril Gln side chain geometry is more constrained than when NDQ10 is in solution. The NDQ10 fibril structure is identical to that of the DQ10 fibril seeded by the NDQ10 solution.


Journal of Physical Chemistry B | 2016

Polyglutamine Fibrils: New Insights into Antiparallel β-Sheet Conformational Preference and Side Chain Structure

David Punihaole; Riley J. Workman; Zhenmin Hong; Jeffry D. Madura; Sanford A. Asher

Understanding the structure of polyglutamine (polyQ) amyloid-like fibril aggregates is crucial to gaining insights into the etiology of at least ten neurodegenerative disorders, including Huntingtons disease. Here, we determine the structure of D2Q10K2 (Q10) fibrils using ultraviolet resonance Raman (UVRR) spectroscopy and molecular dynamics (MD). Using UVRR, we determine the fibril peptide backbone Ψ and glutamine (Gln) side chain χ3 dihedral angles. We find that most of the fibril peptide bonds adopt antiparallel β-sheet conformations; however, a small population of peptide bonds exist in parallel β-sheet structures. Using MD, we simulate three different potential fibril structural models that consist of either β-strands or β-hairpins. Comparing the experimentally measured Ψ and χ3 angle distributions to those obtained from the MD simulated models, we conclude that the basic structural motif of Q10 fibrils is an extended β-strand structure. Importantly, we determine from our MD simulations that Q10 fibril antiparallel β-sheets are thermodynamically more stable than parallel β-sheets. This accounts for why polyQ fibrils preferentially adopt antiparallel β-sheet conformations instead of in-register parallel β-sheets like most amyloidogenic peptides. In addition, we directly determine, for the first time, the structures of Gln side chains. Our structural data give new insights into the role that the Gln side chains play in the stabilization of polyQ fibrils. Finally, our work demonstrates the synergistic power and utility of combining UVRR measurements and MD modeling to determine the structure of amyloid-like fibrils.


Journal of Physical Chemistry B | 2017

Monomeric Polyglutamine Structures That Evolve into Fibrils

David Punihaole; Ryan S. Jakubek; Riley J. Workman; Lauren E. Marbella; Patricia Campbell; Jeffry D. Madura; Sanford A. Asher

We investigate the solution and fibril conformations and structural transitions of the polyglutamine (polyQ) peptide, D2Q10K2 (Q10), by synergistically using UV resonance Raman (UVRR) spectroscopy and molecular dynamics (MD) simulations. We show that Q10 adopts two distinct, monomeric solution conformational states: a collapsed β-strand and a PPII-like structure that do not readily interconvert. This clearly indicates a high activation barrier in solution that prevents equilibration between these structures. Using metadynamics, we explore the conformational energy landscape of Q10 to investigate the physical origins of this high activation barrier. We develop new insights into the conformations and hydrogen bonding environments of the glutamine side chains in the PPII and β-strand-like conformations in solution. We also use the secondary structure-inducing cosolvent, acetonitrile, to investigate the conformations present in low dielectric constant solutions with decreased solvent-peptide hydrogen bonding. As the mole fraction of acetonitrile increases, Q10 converts from PPII-like structures into α-helix-like structures and β-sheet aggregates. Electron microscopy indicates that the aggregates prepared from these acetonitrile-rich solutions show morphologies similar to our previously observed polyQ fibrils. These aggregates redissolve upon the addition of water! These are the first examples of reversible fibril formation. Our monomeric Q10 peptides clearly sample broad regions of their available conformational energy landscape. The work here develops molecular-level insight into monomeric Q10 conformations and investigates the activation barriers between different monomer states and their evolution into fibrils.


Journal of Physical Chemistry B | 2015

UV Resonance Raman Investigation of the Aqueous Solvation Dependence of Primary Amide Vibrations

David Punihaole; Ryan S. Jakubek; Elizabeth M. Dahlburg; Zhenmin Hong; Nataliya S. Myshakina; Steven J. Geib; Sanford A. Asher

We investigated the normal mode composition and the aqueous solvation dependence of the primary amide vibrations of propanamide. Infrared, normal Raman, and UV resonance Raman (UVRR) spectroscopy were applied in conjunction with density functional theory (DFT) to assign the vibrations of crystalline propanamide. We examined the aqueous solvation dependence of the primary amide UVRR bands by measuring spectra in different acetonitrile/water mixtures. As previously observed in the UVRR spectra of N-methylacetamide, all of the resonance enhanced primary amide bands, except for the Amide I (AmI), show increased UVRR cross sections as the solvent becomes water-rich. These spectral trends are rationalized by a model wherein the hydrogen bonding and the high dielectric constant of water stabilizes the ground state dipolar (-)O-C═NH2(+) resonance structure over the neutral O═C-NH2 resonance structure. Thus, vibrations with large C-N stretching show increased UVRR cross sections because the C-N displacement between the electronic ground and excited state increases along the C-N bond. In contrast, vibrations dominated by C═O stretching, such as the AmI, show a decreased displacement between the electronic ground and excited state, which result in a decreased UVRR cross section upon aqueous solvation. The UVRR primary amide vibrations can be used as sensitive spectroscopic markers to study the local dielectric constant and hydrogen bonding environments of the primary amide side chains of glutamine (Gln) and asparagine (Asn).


Journal of Physical Chemistry B | 2015

Glutamine and Asparagine Side Chain Hyperconjugation-Induced Structurally Sensitive Vibrations

David Punihaole; Zhenmin Hong; Ryan S. Jakubek; Elizabeth M. Dahlburg; Steven J. Geib; Sanford A. Asher

We identified vibrational spectral marker bands that sensitively report on the side chain structures of glutamine (Gln) and asparagine (Asn). Density functional theory (DFT) calculations indicate that the Amide III(P) (AmIII(P)) vibrations of Gln and Asn depend cosinusoidally on their side chain OCCC dihedral angles (the χ3 and χ2 angles of Gln and Asn, respectively). We use UV resonance Raman (UVRR) and visible Raman spectroscopy to experimentally correlate the AmIII(P) Raman band frequency to the primary amide OCCC dihedral angle. The AmIII(P) structural sensitivity derives from the Gln (Asn) Cβ-Cγ (Cα-Cβ) stretching component of the vibration. The Cβ-Cγ (Cα-Cβ) bond length inversely correlates with the AmIII(P) band frequency. As the Cβ-Cγ (Cα-Cβ) bond length decreases, its stretching force constant increases, which results in an upshift in the AmIII(P) frequency. The Cβ-Cγ (Cα-Cβ) bond length dependence on the χ3 (χ2) dihedral angle results from hyperconjugation between the Cδ═Oϵ (Cγ═Oδ) π* and Cβ-Cγ (Cα-Cβ) σ orbitals. Using a Protein Data Bank library, we show that the χ3 and χ2 dihedral angles of Gln and Asn depend on the peptide backbone Ramachandran angles. We demonstrate that the inhomogeneously broadened AmIII(P) band line shapes can be used to calculate the χ3 and χ2 angle distributions of peptides. The spectral correlations determined in this study enable important new insights into protein structure in solution, and in Gln- and Asn-rich amyloid-like fibrils and prions.


Journal of Physical Chemistry Letters | 2018

Interaction Enthalpy of Side Chain and Backbone Amides in Polyglutamine Solution Monomers and Fibrils

David Punihaole; Ryan S. Jakubek; Riley J. Workman; Sanford A. Asher

We determined an empirical correlation that relates the amide I vibrational band frequencies of the glutamine (Q) side chain to the strength of hydrogen bonding, van der Waals, and Lewis acid-base interactions of its primary amide carbonyl. We used this correlation to determine the Q side chain carbonyl interaction enthalpy (Δ Hint) in monomeric and amyloid-like fibril conformations of D2Q10K2 (Q10). We independently verified these Δ Hint values through molecular dynamics simulations that showed excellent agreement with experiments. We found that side chain-side chain and side chain-peptide backbone interactions in fibrils and monomers are more enthalpically favorable than are Q side chain-water interactions. Q10 fibrils also showed a more favorable Δ Hint for side chain-side chain interactions compared to backbone-backbone interactions. This work experimentally demonstrates that interamide side chain interactions are important in the formation and stabilization of polyQ fibrils.


Journal of Physical Chemistry B | 2018

New Insights into Quinine–DNA Binding Using Raman Spectroscopy and Molecular Dynamics Simulations

David Punihaole; Riley J. Workman; Shiv Upadhyay; Craig Van Bruggen; Andrew J. Schmitz; Theresa M. Reineke; Renee R. Frontiera

Quinines ability to bind DNA and potentially inhibit transcription and translation has been examined as a mode of action for its antimalarial activity. UV absorption and fluorescence-based studies have lacked the chemical specificity to develop an unambiguous molecular-level picture of the binding interaction. To address this, we use Raman spectroscopy and molecular dynamics (MD) to investigate quinine-DNA interactions. We demonstrate that quinines strongest Raman band in the fingerprint region, which derives from a symmetric stretching mode of the quinoline ring, is highly sensitive to the local chemical environment and pH. The frequency shifts observed for this mode in solvents of varying polarity can be explained in terms of the Stark effect using a simple Onsager solvation model, indicating that the vibration reports on the local electrostatic environment. However, specific chemical interactions between the quinoline ring and its environment, such as hydrogen bonding and π-stacking, perturb the frequency of this mode in a more complicated but predictable manner. We use this vibration as a spectroscopic probe to investigate the binding interaction between quinine and DNA. We find that, when the quinoline ring is protonated, quinine weakly intercalates into DNA by forming π-stacking interactions with the base pairs. The Raman spectra indicate that quinine can intercalate into DNA with a ratio reaching up to roughly one molecule per 25 base pairs. Our results are confirmed by MD simulations, which also show that the quinoline ring adopts a t-shaped π-stacking geometry with the DNA base pairs, whereas the quinuclidine head group weakly interacts with the phosphate backbone in the minor groove. We expect that the spectral correlations determined here will enable future studies to probe quinines antimalarial activities, such as disrupting hemozoin biocrystallization, which is hypothesized to be, among other things, one of its primary modes of action against Plasmodium parasites.

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Zhenmin Hong

University of Pittsburgh

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Zhongyu Cai

University of Pittsburgh

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Daniel H. Kwak

University of Pittsburgh

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Steven J. Geib

University of Pittsburgh

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