Protein Structure and Function Continued
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Nucleosome-Mediated Cooperativity Between Transcription Factors
Nucleosome-mediated cooperativity between transcription factors Leonid A. Mirny1 Harvard–MIT Division of Health Sciences and Technology, and Department of Physics, Massachusetts Institute of Technology, Cambridge, MA 02139 Edited* by José N. Onuchic, University of California San Diego, La Jolla, CA, and approved October 12, 2010 (received for review December 3, 2009) Cooperative binding of transcription factors (TFs) to promoters and A B other regulatory regions is essential for precise gene expression. The classical model of cooperativity requires direct interactions between TFs, thus constraining the arrangement of TF sites in reg- ulatory regions. Recent genomic and functional studies, however, demonstrate a great deal of flexibility in such arrangements with variable distances, numbers of sites, and identities of TF sites lo- cated in cis-regulatory regions. Such flexibility is inconsistent with L C N O D T R cooperativity by direct interactions between TFs. Here, we demon- P 0 0 0 0 P O O strate that strong cooperativity among noninteracting TFs can be K K 2 2 NN O O T R achieved by their competition with nucleosomes. We find that the P 1 1 P 1 1 K O O mechanism of nucleosome-mediated cooperativity is analogous to c = O 10 1..10 3 2 2 K cooperativity in another multimolecular complex: hemoglobin. This N N2 O2 T R [P] P 2 2 = 15 P surprising analogy provides deep insights, with parallels between O2 O2 KO … … the heterotropic regulation of hemoglobin (e.g., the Bohr effect) [N ] L = 0 100 1000 N O and the roles of nucleosome-positioning sequences and chromatin [O0] n n T4 R4 modifications in gene expression. -
Pi-Pi Stacking Mediated Cooperative Mechanism for Human Cytochrome P450 3A4
Molecules 2015, 20, 7558-7573; doi:10.3390/molecules20057558 OPEN ACCESS molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Article Pi-pi Stacking Mediated Cooperative Mechanism for Human Cytochrome P450 3A4 Botao Fa 1, Shan Cong 2 and Jingfang Wang 1,* 1 Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China; E-Mail: [email protected] 2 Department of Bioinformatics and Biostatistics, College of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China; E-Mail: [email protected] * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +86-21-3420-7344 (ext. 123); Fax: +86-21-3420-4348. Academic Editor: Antonio Frontera Received: 13 January 2015 / Accepted: 19 March 2015 / Published: 24 April 2015 Abstract: Human Cytochrome P450 3A4 (CYP3A4) is an important member of the cytochrome P450 superfamily with responsibility for metabolizing ~50% of clinical drugs. Experimental evidence showed that CYP3A4 can adopt multiple substrates in its active site to form a cooperative binding model, accelerating substrate metabolism efficiency. In the current study, we constructed both normal and cooperative binding models of human CYP3A4 with antifungal drug ketoconazoles (KLN). Molecular dynamics simulation and free energy calculation were then carried out to study the cooperative binding mechanism. Our simulation showed that the second KLN in the cooperative binding model had a positive impact on the first one binding in the active site by two significant pi-pi stacking interactions. The first one was formed by Phe215, functioning to position the first KLN in a favorable orientation in the active site for further metabolism reactions. -
The Oxyhaemoglobin Dissociation Curve in Critical Illness
Basic sciences review The Oxyhaemoglobin Dissociation Curve in Critical Illness T. J. MORGAN Intensive Care Facility, Division of Anaesthesiology and Intensive Care, Royal Brisbane Hospital, Brisbane, QUEENSLAND ABSTRACT Objective: To review the status of haemoglobin-oxygen affinity in critical illness and investigate the potential to improve gas exchange, tissue oxygenation and outcome by manipulations of the oxyhaemoglobin dissociation curve. Data sources: Articles and published peer-review abstracts. Summary of review: The P50 of a species is determined by natural selection according to animal size, tissue metabolic requirements and ambient oxygen tension. In right to left shunting mathematical modeling indicates that an increased P50 defends capillary oxygenation, the one exception being sustained hypercapnia. Increasing the P50 should also be protective in tissue ischaemia, and this is supported by modeling and experimental evidence. Most studies of critically ill patients have indicated reduced 2,3-DPG concentrations. This is probably due to acidaemia, and the in vivo P50 is likely to be normal despite low 2,3-DPG levels. It may soon be possible to achieve significant P50 elevations without potentially harmful manipulations of acid-base balance or hazardous drug therapy. Conclusions: Despite encouraging theoretical and experimental data, it is not known whether manipulations of the P50 in critical illness can improve gas exchange and tissue oxygenation or improve outcome. The status of the P50 may warrant more routine quantification and consideration along with the traditional determinants of tissue oxygen availability. (Critical Care and Resuscitation 1999; 1: 93-100) Key words: Critical illness, haemoglobin-oxygen affinity, ischaemia, P50, tissue oxygenation, shunt In intensive care practice, manipulations to improve in the tissues. -
Allosteric Effects and Cooperative Binding
Biochemistry I, Fall Term Lecture 13 Sept 28, 2005 Lecture 13: Allosteric Effects and Cooperative Binding Assigned reading in Campbell: Chapter 4.7, 7.2 Key Terms: • Homotropic Allosteric effects • Heterotropic Allosteric effects • T and R states of cooperative systems • Role of proximal His residue in cooperativity of O2 binding by Hb • Regulation of O2 binding to Hb by bis-phosphoglycerate (BPG) Oxygen Binding to Myoglobin and Hemolobin: Myoglobin binds one O2: Hemoglobin binds four O2: Allosteric Effects and Cooperativity: Allosteric effects occur when the binding properties of a macromolecule change as a consequence of a second ligand binding to the macromolecule and altering its affinity towards the first, or primary, ligand. There need not be a direct connection between the two ligands (i.e. they may bind to opposite sides of the protein, or even to different subunits) • If the two ligands are the same (e.g. oxygen) then this is called a homo-tropic allosteric effect. • If the two ligands are different (e.g. oxygen and BPG), then this is called a hetero-tropic allosteric effect. In the case of macromolecules that have multiple ligand binding sites (e.g. Hb), allosteric effects can generate cooperative behavior. Allosteric effects are important in the regulation of enzymatic reactions. Both allosteric activators (which enhance activity) and allosteric inhibitors (which reduce activity) are utilized to control enzyme reactions. Allosteric effects require the presence of two forms of the macromolecule. One form, usually called the T or tense state, binds the primary ligand (e.g. oxygen) with low affinity. The other form, usually called the R or relaxed state, binds ligand with high affinity. -
Mathematical Toolkit for Quantitative Analysis of Cooperative Binding of Two Or More Ligands to a Substrate Jacob Peacock and James B
Mathematical toolkit for quantitative analysis of cooperative binding of two or more ligands to a substrate Jacob Peacock and James B. Jaynes Dept. of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia PA 19107 United States of America [email protected] Abstract We derive mathematical expressions for quantitative analysis of cooperative binding covering the following cases: 1) a single ligand binds to either two non-equivalent sites, or an arbitrary number of equivalent sites, on a substrate (Fig. 1), and 2) two different ligands bind distinct sites on a substrate (Figs. 2, 3). We show how to analyze "competition experiments" using non-linear regression, where a ligand binds to a single site on a labeled substrate in the presence of increasing amounts of identical but unlabeled competitor substrate, to simultaneously determine the Kd and active ligand concentration (Fig. 4A). We compare the performance of this competition method with the commonly used saturation binding method (Fig. 4B). We also provide methods to analyze such experiments that include a second competitor substrate with non-specific binding sites. We show how to build on results from single-ligand competition experiments to fully characterize cooperative binding in systems with two distinct ligands and binding sites (Fig. 4C and Section 5). We generalize the methodology to more than two cooperating ligands, such as an array of DNA binding proteins (Fig. 6). See Peacock and Jaynes [1] for discussion of the various ways these tools can be used, and results using them. • Visualize characteristics and limitations of Hill plots applied to more realistic binding models than those described by the Hill equation, which implies multiple simultaneous ligand binding. -
Thermodynamic Versus Kinetic Discrimination of Cooperativity of Enzymatic Ligand Binding
Open Access Austin Biochemistry Special Article - Enzyme Kinetics Thermodynamic versus Kinetic Discrimination of Cooperativity of Enzymatic Ligand Binding Das B1, Banerjee K2 and Gangopadhyay G1* 1Department of Chemical Physics, SN Bose National Abstract Centre for Basic Sciences, India In this work, we have introduced thermodynamic measure to characterize the 2Department of Chemistry, A.J.C. Bose College, India nature of cooperativity in terms of the variation of standard free energy change *Corresponding author: Gangopadhyay G, SN Bose with the fraction of ligand-bound sub-units of a protein in equilibrium, treating National Centre for Basic Sciences, Block-JD, Sector-III, the protein-ligand attachment as a stochastic process. The fraction of ligand- Salt Lake, Kolkata-700106, India bound sub-units of cooperative ligand-binding processes are calculated by the formulation of stochastic master equation for both the KNF and MWC Allosteric Received: December 26, 2018; Accepted: May 23, cooperative model. The proposed criteria of this cooperative measurement is 2019; Published: May 30, 2019 valid for all ligand concentrations unlike the traditional kinetic measurement of Hill coefficient at half-saturation point. A Kullback-Leibler distance is also introduced which indicates how much average standard free energy is involved if a non-cooperative system changes to a cooperative one, giving a quantitative synergistic measure of cooperativity as a function of ligand concentration which utilizes the full distribution function beyond the mean and variance. For the validation of our theory to provide a systematic approach to cooperativity, we have considered the experimental result of the cooperative binding of aspartate to the dimeric receptor of Salmonella typhimurium. -
Pranayama Redefined/ Breathing Less to Live More
Pranayama Redefined: Breathing Less to Live More by Robin Rothenberg, C-IAYT Illustrations by Roy DeLeon ©Essential Yoga Therapy - 2017 ©Essential Yoga Therapy - 2017 REMEMBERING OUR ROOTS “When Prana moves, chitta moves. When prana is without movement, chitta is without movement. By this steadiness of prana, the yogi attains steadiness and should thus restrain the vayu (air).” Hatha Yoga Pradipika Swami Muktabodhananda Chapter 2, Verse 2, pg. 150 “As long as the vayu (air and prana) remains in the body, that is called life. Death is when it leaves the body. Therefore, retain vayu.” Hatha Yoga Pradipika Swami Muktabodhananda Chapter 2, Verse 3, pg. 153 ©Essential Yoga Therapy - 2017 “Pranayama is usually considered to be the practice of controlled inhalation and exhalation combined with retention. However, technically speaking, it is only retention. Inhalation/exhalation are methods of inducing retention. Retention is most important because is allows a longer period for assimilation of prana, just as it allows more time for the exchange of gases in the cells, i.e. oxygen and carbon dioxide.” Hatha Yoga Pradipika Swami Muktabodhananda Chapter 2, Verse 2, pg. 151 ©Essential Yoga Therapy - 2017 Yoga Breathing in the Modern Era • Focus tends to be on lengthening the inhale/exhale • Exhale to induce relaxation (PSNS activation) • Inhale to increase energy (SNS) • Big ujjayi - audible • Nose breathing is emphasized at least with inhale. Some traditions teach mouth breathing on exhale. • Focus on muscular action of chest, ribs, diaphragm, intercostals and abdominal muscles all used actively on inhale and exhale to create the ‘yoga breath.’ • Retention after inhale and exhale used cautiously and to amplify the effect of inhale and exhale • Emphasis with pranayama is on slowing the rate however no discussion on lowering volume. -
Structural Basis for Regiospecific Midazolam Oxidation by Human Cytochrome P450 3A4
Structural basis for regiospecific midazolam oxidation by human cytochrome P450 3A4 Irina F. Sevrioukovaa,1 and Thomas L. Poulosa,b,c aDepartment of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900; bDepartment of Chemistry, University of California, Irvine, CA 92697-3900; and cDepartment of Pharmaceutical Sciences, University of California, Irvine, CA 92697-3900 Edited by Michael A. Marletta, University of California, Berkeley, CA, and approved November 30, 2016 (received for review September 28, 2016) Human cytochrome P450 3A4 (CYP3A4) is a major hepatic and Here we describe the cocrystal structure of CYP3A4 with the intestinal enzyme that oxidizes more than 60% of administered drug midazolam (MDZ) bound in a productive mode. MDZ therapeutics. Knowledge of how CYP3A4 adjusts and reshapes the (Fig. 1) is the benzodiazepine most frequently used for pro- active site to regioselectively oxidize chemically diverse com- cedural sedation (9) and is a marker substrate for the CYP3A pounds is critical for better understanding structure–function rela- family of enzymes that includes CYP3A4 (10). MDZ is hydrox- tions in this important enzyme, improving the outcomes for drug ylated by CYP3A4 predominantly at the C1 position, whereas metabolism predictions, and developing pharmaceuticals that the 4-OH product accumulates at high substrate concentrations have a decreased ability to undergo metabolism and cause detri- (up to 50% of total product) and inhibits the 1-OH metabolic mental drug–drug interactions. However, there is very limited pathway (11–14). The reaction rate and product distribution structural information on CYP3A4–substrate interactions available strongly depend on the assay conditions and can be modulated by to date. -
Breathing Issues: in Depth Tutorial Hypo=Low Hyper= High Or More
Breathing Issues: In Depth Tutorial Hypo=low Hyper= high or more In the normal state, when we breathe in, we take in oxygen, the oxygen goes to the lungs and gets absorbed into the circulation, in exchange for carbon dioxide. Then, we exhale the carbon dioxide. One way to think of this is that you take in oxygen and you breathe out carbon dioxide. There are two issues that can occur with breathing problems. One is a low oxygen problem (hyoxemia). The other is retention of carbon dioxide (hypercarbia). A low oxygen and a high carbon dioxide level (in lungs and blood) can sometimes be related. Pulmonologist talk about these two issues using the following terms: 1. you can have a problem with oxygenation AND/OR 2. you can have a problem with ventilation (ventilation means the process by which your lungs expand and by taking in the air you exchange the oxygen for carbon dioxide). Ventilation is the problem for people with neuromuscular disease. Ventilation problems lead to carbon dioxide build up. Ventilation requires you to have the muscle support to expand your lungs during inhalation (your chest wall muscles assist in this effort as does the diaphragm. Scoliosis can impair chest expansion). Depending on how weak you are, you may or not be able to take an effective large enough breath. Because you are not taking a deep enough breath in, when you exhale you have a smaller amount of air to breathe out and it is not enough volume to get rid of the carbon dioxide. The carbon dioxide slowly starts to build up over time. -
Binding Modes and Metabolism of Caffeine
This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. Article Cite This: Chem. Res. Toxicol. 2019, 32, 1374−1383 pubs.acs.org/crt Binding Modes and Metabolism of Caffeine Zuzana Jandova,† Samuel C. Gill,‡ Nathan M. Lim,§ David L. Mobley,‡ and Chris Oostenbrink*,† † Institute of Molecular Modeling and Simulation, University of Natural Resources and Life Sciences, Vienna, 1180 Vienna, Austria ‡ § Department of Chemistry and Department of Pharmaceutical Sciences, University of California, Irvine, Irvine, California 92697, United States *S Supporting Information ABSTRACT: A correct estimate of ligand binding modes and a ratio of their occupancies is crucial for calculations of binding free energies. The newly developed method BLUES combines molecular dynamics with nonequilibrium candidate Monte Carlo. Nonequilibrium candidate Monte Carlo generates a plethora of possible binding modes and molecular dynamics enables the system to relax. We used BLUES to investigate binding modes of caffeine in the active site of its metabolizing enzyme Cytochrome P450 1A2 with the aim of elucidating metabolite-formation profiles at different concentrations. Because the activation energies of all sites of metabolism do not show a clear preference for one metabolite over the others, the orientations in the active site must play a key role. In simulations with caffeine located in a spacious pocket above the I-helix, it points N3 and N1 to the heme iron, whereas in simulations where caffeine is in close proximity to the heme N7 and C8 are preferably oriented toward the heme iron. -
Improved Oxygen Release: an Adaptation of Mature Red Cells to Hypoxia
Improved oxygen release: an adaptation of mature red cells to hypoxia Miles J. Edwards, … , Carrie-Lou Walters, James Metcalfe J Clin Invest. 1968;47(8):1851-1857. https://doi.org/10.1172/JCI105875. Research Article Blood from patients with erythrocytosis secondary to arterial hypoxemia due either to congenital heart disease or to chronic obstructive pulmonary disease was shown to have a decreased affinity for oxygen; the average oxygen pressure required to produce 50% saturation of hemoglobin with oxygen was 29.8 mm Hg (average normal, 26.3 mm Hg). Such a displacement of the blood oxygen equilibrium curve promotes the release of oxygen from blood to the tissues. Studies were also performed upon blood from a man with complete erythrocyte aplasia who received all of his red cells by transfusion from presumably normal persons. With mild anemia (hematocrit, 28%), the affinity of his blood for oxygen was slightly diminished (an oxygen pressure of 27.0 mm Hg was required to produce 50% saturation of hemoglobin with oxygen). With severe anemia (hematocrit, 13.5%), however, his blood had a markedly decreased oxygen affinity (an oxygen pressure of 29.6 mm Hg was required to produce 50% saturation of hemoglobin with oxygen). We conclude that patients with various conditions characterized by an impairment in the oxygen supply system to tissues respond with a diminished affinity of their blood for oxygen. Although the mechanism which brings about this adaptation is not known, the displacement of the oxygen equilibrium curve is associated with an increase in heme-heme interaction. The decrease in blood oxygen affinity […] Find the latest version: https://jci.me/105875/pdf Improved Oxygen Release: an Adaptation of Mature Red Cells to Hypoxia MUas J. -
Chem 452 - Fall 2012 - Exam II
Name __________________________________Key Chem 452 - Fall 2012 - Exam II Some potentially useful information: pKa values for ionizable groups in proteins: (α-carboxyl, 3.1; α-amino, 8.0; Asp & Glu side chains, 4.1; His side chain, 6.0; Cys side chain, 8.3; Tyr side chain, 10.9; Lys side chain, 10.8; Arg side chain, 12.5) R (ideal gas constant) = 8.314 J/mol•K = 0.08206 L•atm/mol•K Charge on one electron = 1.602 x 10-19 C The answer to one of the questions on this exam is -3.3 kJ/mol 1. In class, we discussed a number of strategies used by enzymes to speed up the rates of the reactions they catalyze. In one or two sentences, describe a specific example for each of the following strategies, drawing from the four systems that we discussed in class. Include each of the four systems as an example for at least one of these strategies. (The systems discussed in class include, chymotrypsin, carbonic anhydrase, a. Covalent catalysis: the EcoRV restriction endonuclease, and the myosin II ATPase.) The ser 195 in chymotrypsin serves as a nucleophile in the proteolysis reactions carried out by serine proteases. It 18/18 catalyzed the hydrolysis of peptide bonds by attacking the the carbonyl carbon of the peptide bond that is being hydrolyzed. This leads to the formation of an intermediate that is covalently bonded as an ester to the ser 195. The ester is later hydrolyzed to return the enzyme to its initial state. b. Catalysis by approximation: Enzymes speed up reactions by placing the various players in a reaction next to one another.