Binding of quinidine radically increases the stability and decreases the flexibility of the cytochrome P450 2D6 active site
by Karel Berka
Berka K, Anzenbacherova E, Hendrychova T, Lange R, Masek V, Anzenbacher P, Otyepka M: Binding of Quinidine Radically Increases the Stability and Decreases the Flexibility of the Cytochrome P450 2D6 Active Site. J. Inorg. Biochem., 110, 46-50, 2012
Human cytochrome P450 2D6 (CYP2D6) is an enzyme of the CYP superfamily responsible for biotransformation of about 20%... more Human cytochrome P450 2D6 (CYP2D6) is an enzyme of the CYP superfamily responsible for biotransformation of about 20% of drugs of known metabolism containing a basic nitrogen and a planar aromatic ring. Here, we present a combined experimental and computational study on the compressibility and flexibility of unliganded and quinidine-bound CYP2D6. Experimentally, high-pressure induced Soret band shifts of the enzyme were measured by UV/VIS spectroscopy, while 100 ns all atomic molecular dynamics (MD) simulations in explicit water were used in the computational analysis. We identified sharp differences between ligand-free and quinidine-bound CYP2D6 forms in compressibility, flexibility parameters and active site solvation. While the unliganded CYP2D6 is compressible, quinidine binding significantly rigidifies the CYP2D6 active site. In addition, MD simulations show that quinidine binding results in pronounced reductions in active site flexibility and solvation.
Download (.pdf) Giant amyloid spherulites reveal their true colours
by Mike Smith
The prevalence of degenerative conditions such as Alzheimer's and Parkinson's disease amongst an increasingly elderly... more The prevalence of degenerative conditions such as Alzheimer's and Parkinson's disease amongst an increasingly elderly population has led to substantial research efforts into understanding the properties and structures of amyloid protein aggregates. One such aggregate, the amyloid spherulite, consists of a central core surrounded by long fibres of aggregated protein (fibrils) which grow radially outwards. Spherulites (5–50 μm in diameter) exhibit four white lobes when observed using crossed polarised microscopy, due to their birefringence. Here we report the growth of giant amyloid spherulites (GAS, diameters 0.4–1 mm) that produce colourful patterns when placed between crossed polarisers. A ray tracing model was developed that accounts for these patterns by calculating the effects of birefringence on light passing through the GAS. This new model links for the first time the optical properties of spherulites to the density and orientation of the fibrils, providing a route to understanding the formation of these important protein aggregates.
Novel proteins of the phosphotransferase system encoded within the …
Powell, BS, Court DL, Inada T, Nakamura Y, Michotey V, Cui X, Reizer A, Saier MH, Jr, Reizer J. J Biol Chem. 1995 Mar 3;270(9):4822-39.
Two rpoN-linked delta Tn10-kan insertions suppress the conditionally lethal erats allele. One truncates rpoN while the... more Two rpoN-linked delta Tn10-kan insertions suppress the conditionally lethal erats allele. One truncates rpoN while the second disrupts another gene (ptsN) in the rpoN operon and does not affect classical nitrogen regulation. Neither alter expression of era indicating that suppression is post-translational. Plasmid clones of ptsN prevent suppression by either disruption mutation indicating that this gene is important for lethality caused by erats. rpoN and six neighboring genes were sequenced and compared with sequences in the database. Two of these genes encode proteins homologous to Enzyme IIAFru and HPr of the phosphoenolpyruvate:sugar phosphotransferase system. We designate these proteins IIANtr (ptsN) and NPr (npr). Purified IIANtr and NPr exchange phosphate appropriately with Enzyme I, HPr, and Enzyme IIA proteins of the phosphoenolpyruvate: sugar phosphotransferase system. Several sugars and tricarboxylic acid cycle intermediates inhibited growth of the ptsN disruption mutant on medium containing an amino acid or nucleoside base as a combined source of nitrogen, carbon, and energy. This growth inhibition was relieved by supplying the ptsN gene or ammonium salts but was not aleviated by altering levels of exogenously supplied cAMP. These results support our previous proposal of a novel mechanism linking carbon and nitrogen assimilation and relates IIANtr to the unknown process regulated by the essential GTPase Era.
Influence of Glycation of Wheat Albumins and Globulins on Their Immunoreactivity and Physicochemical Properties
published in "Polish Journal of Food and Nutrition Sciences" (2010, 60(4) pp335-340), co-authored (first author)
Glycation (non-enzymatic glycosylation) is a spontaneous reaction that occurs during food processing, storage and... more
Glycation (non-enzymatic glycosylation) is a spontaneous reaction that occurs during food processing, storage and preparation of food, which can have a significant influence on physiochemical and biological properties of food proteins. Glycation has been used mostly for improvement of functional properties of proteins although there is increasing concern of possible changes in biologic properties of glycation products.
The influence of dry-heat glycation with glucose on immunoreactive properties of wheat salt-soluble proteins has been studied. It has been shown that glycation caused differences in electrophoretic patterns (both 1D and 2D), as well as in Western-immunoblotting with rabbit IgG and human IgE. A significant increase in immunoreactivity has been observed, as well as a decrease in free amino group. Thermostable, immunoreactive and susceptible for glycation protein band with molecular weight of approximately 13 kDa (by SDS-PAGE) has been selected for the N-terminal sequence analysis and determined to be an alpha-amylase inhibitor.
Dry heat glycation is a very potent but nondestructive method of protein glycation, that leads to high degree of substitution and changes in both physicochemical and biological (immunological) properties of food proteins.
6 views
Seen by:
Conformation and dynamics of the three-helix bundle UBA domain of p62 from experiment and simulation
Co-authors: Jed Long, Thomas R. A. Gallagher, Jonathan D. Hirst, Mark S. Searle
Publication details: Proteins: Structure, Function, and Bioinformatics, Volume 71, Issue 1, pages 227–240, April 2008
The ubiquitin associated domain of p62 is a small three-helix bundle of ∼50 residues that mediates the recognition of... more The ubiquitin associated domain of p62 is a small three-helix bundle of ∼50 residues that mediates the recognition of polyubiquitin chains and ubiquitylated substrates. The solution structure of a 52 residue construct containing this domain has been characterized using heteronuclear nuclear magnetic resonance (NMR) methods. The resulting ensemble of NMR-derived structures was used in molecular dynamics (MD) simulations to investigate the equilibrium conformation and dynamics of this domain. NOE and 15N relaxation data have been used to validate the structural ensemble produced by the MD simulations and show a good correlation for residues in regions of secondary structure. A similar approach was taken using an ensemble of structures from the MD simulations to calculate electronic circular dichroism (CD) and IR spectra from first principles with an encouraging correlation with the experimental CD and IR data.
Solvent Accessibility and Purifying Selection Within Proteins of Escherichia Coli and Salmonella Enterica
The neutral theory of molecular evolution predicts that variation within species is inversely related to the strength... more The neutral theory of molecular evolution predicts that variation within species is inversely related to the strength of purifying selection, but the strength of purifying selection itself must be related to physical constraints imposed by protein folding and function. In this paper, we analyzed five enzymes for which polymorphic sequence variation within Escherichia coli and/or Salmonella enterica was available, along with a protein structure. Single and multivariate logistic regression models are presented that evaluate amino acid size, physicochemical properties, solvent accessibility, and secondary structure as predictors of polymorphism. A model that contains a positive coefficient of association between polymorphism and solvent accessibility and separate intercepts for each secondary-structure element is sufficient to explain the observed variation in polymorphism between sites. The model predicts an increase in the probability of amino acid polymorphism with increasing solvent accessibility for each protein regardless of physicochemical properties, secondary-structure element, or size of the amino acid. This result, when compared with the distribution of synonymous polymorphism, which shows no association with solvent accessibility, suggests a strong decrease in purifying selection with increasing solvent accessibility.
Expanding the promiscuity of a natural-product glycosyltransferase by directed evolution.
Nat Chem Biol, 2007
Natural products, many of which are decorated with essential sugar residues, continue to serve as a key platform for... more Natural products, many of which are decorated with essential sugar residues, continue to serve as a key platform for drug development. Adding or changing sugars attached to such natural products can improve the parent compound's pharmacological properties, specificity at multiple levels, and/or even the molecular mechanism of action. Though some natural-product glycosyltransferases (GTs) are sufficiently promiscuous for use in altering these glycosylation patterns, the stringent specificity of others remains a limiting factor in natural-product diversification and highlights a need for general GT engineering and evolution platforms. Herein we report the use of a simple high-throughput screen based on a fluorescent surrogate acceptor substrate to expand the promiscuity of a natural-product GT via directed evolution. Cumulatively, this study presents variant GTs for the glycorandomization of a range of therapeutically important acceptors, including aminocoumarins, flavonoids and macrolides, and a potential template for engineering other natural-product GTs.
Micro/nanopatterning of proteins via contact printing using high aspect ratio PMMA stamps and nanoimprint apparatus
Co-authored with Mateu Pla-Roca, Christopher A. Mills and Josep Samitier, Published in 'Langmuir'
Micro- and nanoscale protein patterns have been produced via a new contact printing method using a nanoimprint... more Micro- and nanoscale protein patterns have been produced via a new contact printing method using a nanoimprint lithography apparatus. The main novelty of the technique is the use of poly(methyl methacrylate) (PMMA) instead of the commonly used poly(dimethylsiloxane) (PDMS) stamps. This avoids printing problems due to roof collapse, which limits the usable aspect ratio in microcontact printing to 10:1. The rigidity of the PMMA allows protein patterning using stamps with very high aspect ratios, up to 300 in this case. Conformal contact between the stamp and the substrate is achieved because of the homogeneous pressure applied via the nanoimprint lithography instrument, and it has allowed us to print lines of protein approximately 150 nm wide, at a 400 nm period. This technique, therefore, provides an excellent method for the direct printing of high-density sub-micrometer scale patterns, or, alternatively, micro-/nanopatterns spaced at large distances. The controlled production of these protein patterns is a key factor in biomedical applications such as cell-surface interaction experiments and tissue engineering
39 views
Seen by:
Microcalorimetric study of thermal unfolding of lysozyme in water/glycerol mixtures: An analysis by solvent exchange model
Folded protein stabilization or destabilization induced by cosolvent in mixed aqueous solutions has been studied by... more
Folded protein stabilization or destabilization induced by cosolvent in mixed aqueous solutions has been studied by differential scanning microcalorimetry and related to difference in preferential solvation of native and denatured states. In particular, the thermal denaturation of a model system formed by lysozyme dissolved in water in the presence of the stabilizing cosolvent glycerol has been considered. Transition temperatures and enthalpies, heat capacity, and standard free energy changes
have been determined when applying a two-state denaturation model to microcalorimetric data. Thermodynamic parameters show an unexpected, not linear, trend as a function of solvent
composition; in particular, the lysozyme thermodynamic stability shows a maximum centered at water molar fraction of about 0.6. Using a thermodynamic hydration model based on the exchange equilibrium between glycerol and water molecules from the protein solvation layer to the bulk, the contribution of protein-solvent interactions to the unfolding free energy and the changes of this contribution with solvent composition have been derived. The preferential solvation data indicate that lysozyme unfolding involves an increase in the solvation surface, with a small reduction of the protein-preferential hydration. Moreover, the derived changes in the excess solvation numbers at
denaturation show that only few solvent molecules are responsible for the variation of lysozyme stability in relation to the solvent composition.
New insights into urea action on proteins: a sans study of the lysozyme case
We present a study on lysozyme dissolved in mixtures of water and urea, which is ubiquitously used as a protein... more
We present a study on lysozyme dissolved in mixtures of water and urea, which is ubiquitously used as a protein denaturant. Despite the wide use of urea, the basic molecular mechanisms inducing protein unfolding are not still clarified. Small-angle neutron scattering (SANS) experiments have been performed using little amounts of denaturant in solutions in order to investigate the urea effect on lysozyme preceding the unfolding
process. A global fit strategy, applied to analyze SANS experiments, provides an estimation of the average composition of the solvent in the close vicinity of the protein surface and the change of the protein-protein interactions due to the presence of urea. In particular, the thermodynamic equilibrium constant responsible for cosolvent balancing between the bulk and solvation layer has been determined. It turns out that urea is
preferentially driven to the protein surface, confirming literature results at infinite dilute conditions. SANS data also reveal a possible variation of the protein net charge as a function of urea concentration, opening new perspectives and questions about the protein surface architecture at the first stages of unfolding processes.
Combining structure and dynamics: non-denaturing high-pressure effect on lysozyme in solution
Small-angle X-ray scattering (SAXS) and elastic and quasi-elastic neutron scattering techniques were used to... more Small-angle X-ray scattering (SAXS) and elastic and quasi-elastic neutron scattering techniques were used to investigate the high-pressure-induced changes on interactions, the low-resolution structure and the dynamics of lysozyme in solution. SAXS data, analysed using a global-fit procedure based on a new approach for hydrated protein form factor description, indicate that lysozyme completely maintains its globular structure up to 1500 bar, but significant modifications in the protein–protein interaction potential occur at approximately 600–1000 bar. Moreover, the mass density of the protein hydration water shows a clear discontinuity within this pressure range. Neutron scattering experiments indicate that the global and the local lysozyme dynamics change at a similar threshold pressure. A clear evolution of the internal protein dynamics from diffusing to more localized motions has also been probed. Protein structure and dynamics results have then been discussed in the context of protein–water interface and hydration water dynamics. According to SAXS results, the new configuration of water in the first hydration layer induced by pressure is suggested to be at the origin of the observed local mobility changes.
Modification of Hydroaffinity of Silicone/Hydrophobic Acrylic Surfaces of Medical Implant Devices and laparoscopic lenses to control condensation mechanisms during and after surgery using Visco-Elastic Colloids and Blood Proteins
This manuscript for this abstract is in preparation and is
to be submitted 12/11/11. It was successfully presented as an oral contribution by Ross B. Bennett-Kennett, ASU '14, at the 58th American Vacuum Society (AVS) Fall International Symposium, Oct 31st-Nov 4th, Nashville, in the Bio-Materials Division.
BIOMATERIAL INTERFACES DIVISION
ROOM: 108 - SESSION BI-THM - Thursday, November 3rd, 2011, 11:20 am
BIOMEDICAL MATERIALS MODERATOR:
S.L. MCARTHUR, SWINBURNE UNIVERSITY OF TECHNOLOGY, AUSTRALIA
N. Herbots, ASU / SiO2 NanoTech, R.J. Culbertson, Q.Xing, D.A. Sell, A.M. Murphy, R.B. Bennett-Kennett*, S.D. Whaley,... more
N. Herbots, ASU / SiO2 NanoTech, R.J. Culbertson, Q.Xing, D.A. Sell, A.M. Murphy, R.B. Bennett-Kennett*, S.D. Whaley, ASU, Drs. C.H. Sell, MD & H.M. Kwong, Arizona Vitro-Retinal Consultants , T. Kutz, A.S. Benitez, B.J. Wilkens, ASU.
Silicone inter-occular lenses and laparoscopic lenses can fog during surgery. This work solves the problem by modifying water affinity of silicone and acrylic, as well as silica lenses via a bio-identical visco-elastic colloidal emulsion, VitreOx™ [1-5] with a 100% success rate in the lab. Ten surgical trials yielded success rate of 80% with failure inferred to be due to blood proteins.
The protein that prevents coagulation, heparin, during surgery, is investigated. Heparin behaves identically to H2O on hydrophobic surfaces. It does not prevent fogging nor interfere with our anti-fogging emulsion.
Fibrinogen is also investigated because it causes coagulation. Fibrinogen applied to IOL's in various dilutions does prevent fogging. Our research shows that the blood proteins studied as well as whole do not modify the hydro-affinity of the surfaces we treated nor their condensation behavior.
We presently combine the bio-identical visco-elastic colloidal emulsion we have developed with blood proteins to modify tissue growth and cell accumulation on implant surfaces.
[1] U. S. Patent Pending, Filed 11/9/10
[2] PhD Dissertation, Q. Xing, ASU (2011).
[3] N. Herbots, Q. Xing, et al. Nucl. Instr. & Meth. B, IBMM 17 (2010), www.sciencedirect.com/science/article/pii/S0168583X11001170
* Presenter at the AVS
Proteome-wide post-translational modification statistics: frequency analysis and curation of the swiss-prot database
Published in Nature Scientific Reports, 2011
Post-translational modifications (PTMs) broadly contribute to the recent explosion of proteomic data and possess a... more Post-translational modifications (PTMs) broadly contribute to the recent explosion of proteomic data and possess a complexity surpassing that of protein design. PTMs are the chemical modification of a protein after its translation, and have wide effects broadening its range of functionality. Based on previous estimates, it is widely believed that more than half of proteins are glycoproteins. Whereas mutations can only occur once per position, different forms of post-translational modifications may occur in tandem. With the number and abundances of modifications constantly being discovered, there is no method to readily assess their relative levels. Here we report the relative abundances of each PTM found experimentally and putatively, from high-quality, manually curated, proteome-wide data, and show that at best, less than one-fifth of proteins are glycosylated. We make available to the academic community a continuously updated resource (http://selene.princeton.edu/PTMCuration) containing the statistics so scientists can assess “how many” of each PTM exists.
Self-assembly of Streptavidin/BisBiotin Monolayers and Multilayers.
Thin Film & Solids. 1992, 210-211, 773-775.