Kinetics and Thermodynamics of Membrane Protein Folding

Understanding protein folding has been one of the great challenges in biochemistry and molecular biophysics. Over the past 50 years, many thermodynamic and kinetic studies have been performed addressing the stability of globular proteins. In comparison, advances in the membrane protein folding field lag far behind. Although membrane proteins constitute about a third of the proteins encoded in known genomes, stability studies on membrane proteins have been impaired due to experimental limitations. Furthermore, no systematic experimental strategies are available for folding these biomolecules in vitro. Common denaturing agents such as chaotropes usually do not work on helical membrane proteins, and ionic detergents have been successful denaturants only in few cases. Refolding a membrane protein seems to be a craftsman work, which is relatively straightforward for transmembrane {\beta}-barrel proteins but challenging for {\alpha}-helical membrane proteins. Additional complexities emerge in multidomain membrane proteins, data interpretation being one of the most critical. In this review, we will describe some recent efforts in understanding the folding mechanism of membrane proteins that have been reversibly refolded allowing both thermodynamic and kinetic analysis. This information will be discussed in the context of current paradigms in the protein folding field

Steady state kinetic analysis of Legionella pneumophila Cu+ transport ATPase: The activation by Cu+ and ATP )

P-type ATPases are a family of membrane proteins which couple ATPhydrolysis to the transport of substrates across biological membranes. Withinthem, Cu + -ATPases are the most widespread and conserved heavy metal iontransporting ATPases (PIB-ATPases). Its reaction cycle is assumed to bedescribed by the so-called Albers-Post model postulated for the most studied P-ATPases such as the Na + ,K + -ATPase or the Ca 2+ -ATPases. However, as somestructural and functional particularities arise for Cu + -ATPases, several authorsposit some doubts about their reaction cycle mechanism. The aim of our work isto perform a functional characterization of Legionella pneumophila Cu⁺-ATPase(LpCopA) by measuring steady state ATPase activity. Cu + -ATPase activity of theenzyme presents a maximum at ∼37ºC and pH 6.6-6.8. Phospholipids enhanceLpCopA Cu + -ATPase activity in a non-essential mode where optimal activity isachieved at an asolectin mole fraction of 0.15 and an amphiphile-protein ratio of~30000. As described for other P-ATPases, Mg 2+ acts as an essential activator.When evaluating the role of ATP and Cu + in the reaction cycle of LpCopA weobserved that ATPase activity increases as Cu + concentration increases with afunctional dependence that can be described by a sum of two hyperboles. Onthe other hand, the increment on ATP concentration in the reaction mediaproduces an increment of ATPase activity that can be described by a hyperbolaplus a constant value. Based on that, and the [Cu + ] and [ATP] dependencies ofthe best fitting parameters of the functions pointed above, we propose aminimal reaction scheme for LpCopA catalytic mechanism that contemplatestwo enzyme conformations with different affinities for ATP, enzymephosphorylation and binding of at least two Cu + ions with different affinities. Thismodel is compatible with the structural information available and the maincharacteristics of the reaction cycle models for the most characterized P-TypeATPases.Fil: Placenti, Maria Agueda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Roman, Ernesto Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Gonzalez Flecha, Francisco Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Gonzalez-Lebrero, Rodolfo Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina20th Internaitional Congress of the INternational Union for Pure Applied Biophysics; 50th Annual Meeting of the Brazilian Society for Biochemistry and Molecular Biology; 45th Congress of Brazilian Biophysics Society anda 13th Brazilian Society on Nuclear Biosciences CongressBrasilSociedade Brasileira de Bioquímica e Biología Molecula

Cooperativity in binding processes: new insights from phenomenological modeling

Cooperative binding is one of the most interesting and not fully understood phenomena involved in control and regulation of biological processes. Here we analyze the simplest phenomenological model that can account for cooperativity (i.e. ligand binding to a macromolecule with two binding sites) by generating equilibrium binding isotherms from deterministically simulated binding time courses. We show that the Hill coefficients determined for cooperative binding, provide a good measure of the Gibbs free energy of interaction among binding sites, and that their values are independent of the free energy of association for empty sites. We also conclude that although negative cooperativity and different classes of binding sites cannot be distinguished at equilibrium, they can be kinetically differentiated. This feature highlights the usefulness of pre-equilibrium time-resolved strategies to explore binding models as a key complement of equilibrium experiments. Furthermore, our analysis shows that under conditions of strong negative cooperativity, the existence of some binding sites can be overlooked, and experiments at very high ligand concentrations can be a valuable tool to unmask such sites.Fil: Cattoni, Diego Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universite Montpellier II; FranciaFil: Chara, Osvaldo. Universite Montpellier II; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Física de Líquidos y Sistemas Biológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Física de Líquidos y Sistemas Biológicos; ArgentinaFil: Kaufman, Sergio Benjamín. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Gonzalez Flecha, Francisco Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin

Conserved Glu-47 and Lys-50 residues are critical for UDP-N-acetylglucosamine/UMP antiport activity of the mouse Golgi-associated transporter Slc35a3

Nucleotide sugar transporters (NSTs) regulate the flux of activated sugars from the cytosol into the lumen of the Golgi apparatus where glycosyltransferases use them for the modification of proteins, lipids, and proteoglycans. It has been well established that NSTs are antiporters that exchange nucleotide sugars with the respective nucleoside monophosphate. Nevertheless, information about the molecular basis of ligand recognition and transport is scarce. Here, using topology predictors, cysteine-scanning mutagenesis, expression of GFP-tagged protein variants, and phenotypic complementation of the yeast strain Kl3, we identified residues involved in the activity of a mouse UDP-GlcNAc transporter, murine solute carrier family 35 member A3 (mSLC35A3). We specifically focused on the putative transmembrane helix 2 (TMH2) and observed that cells expressing E47C or K50C SLC35A3 variants had lower levels of GlcNAc-containing glycoconjugates than WT cells, indicating impaired UDP-GlcNAc transport activity of these two variants. A conservative substitution analysis revealed that single or double substitutions of Glu-47 and Lys-50 does not restore GlcNAc glycoconjugates. Analysis of mSLC35A3 and its genetic variants reconstituted into proteoliposomes disclosed that i) all variants act as UDP-GlcNAc/UMP antiporters; ii) conservative substitutions (E47D, E47Q, K50R or K50H) impair UDP-GlcNAc uptake; and iii) substitutions of Glu-47 and Lys-50 dramatically alter kinetic parameters, consistent with a critical role of these two residues in mSLC35A3 function. A bioinformatics analysis revealed that a EXXK motif in TMH2 is highly conserved across SLC35 A subfamily members, and a 3D-homology model predicted that Glu-47 and Lys-50 are facing the central cavity of the proteinFil: Toscanini, María Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Favarolo, Maria Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Gonzalez Flecha, Francisco Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Ebert, Berit. University of Melbourne; AustraliaFil: Rautengarten, Carsten. University of Melbourne; AustraliaFil: Bredeston, Luis María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin

Modulation of plasma membrane Ca2-ATPase by neutral Phospholipids: effect of the micelle-vesicle transition and the bilayer thickness

Background: Membrane proteins require phospholipids to be biologically active. Results: An increase of phosphatidylcholine/detergent molar ratio leads to a biphasic behavior of the PMCA Ca2-ATPase activity, whose maximum depends on phosphatidylcholine characteristics. Conclusion: The optimum hydrophobic thickness for PMCA structure and Ca2-ATPase activity is about 24 Å. Significance: Differential modulation by neutral phospholipids could be a general mechanism for regulating membrane protein function.Fil: Pignataro, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Dodes Traian, Martín Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Gonzalez Flecha, Francisco Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Sica, Mauricio Pablo. Comision Nacional de Energia Atomica. Gerencia del área de Seguridad Nuclear y Ambiente. Instituto de Energía y Desarrollo Sustentable. Instituto de Energía y Desarrollo Sustentable - Sede Bariloche; ArgentinaFil: Mangialavori, Irene Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Rossi, Juan Pablo Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin

Pilot study of the characteristics of acute stroke events in patients discharged from the Carolina University Hospital, Puerto Rico in 2007

BACKGROUND: Stroke is the third leading cause of death in Puerto Rico. We examined the pre-hospital phase, management and case-fatality-rates (CFR) of patients discharged with acute stroke from the Carolina University of Puerto Rico Hospital during 2007. METHODS: Trained personnel collected information on demographics, delay-time, mode-of-transportation, management, and mortality from all medical records. STATAa was utilized to conduct univariate comparison of demographics, mode-of-transportation, therapeutics and diagnostic characteristics. Logistic regression analysis assessed cohort effect and controlled for confounders. RESULTS: The average age was 69.1 years, and 53% were males. The average delay between onset of symptoms suggestive of stroke and arrival at the emergency department was 4.5 hours. Only 62% of patients utilized Emergency Medical Services (EMS). Intravenous thrombolysis was not administered. Stroke mortality increased with age. Ischemic vs. hemorrhagic CFR was significantly higher (63.9% vs. 36.10%; p = 0.034). CONCLUSIONS: These findings highlight the potential benefit of evidence-based therapeutics and EMS use among stroke patients

Outcomes of abdominal wall reconstruction in patients with the combination of complex midline and lateral incisional hernias.

Background The best treatment for the combined defects of midline and lateral incisional hernia is not known. The aim of our multicenter study was to evaluate the operative and patient-reported outcomes using a modified posterior component separation in patients who present with the combination of midline and lateral incisional hernia. Methods We identified patients from a prospective, multicenter database who underwent operative repairs of a midline and lateral incisional hernia at 4 centers with minimum 2-year follow-up. Hernias were divided into a main hernia based on the larger size and associated abdominal wall hernias. Outcomes reported were short- and long-term complications, including recurrence, pain, and bulging. Quality of life was assessed with the European Registry for Abdominal Wall Hernias Quality of Life score. Results Fifty-eight patients were identified. Almost 70% of patients presented with a midline defect as the main incisional hernia. The operative technique was a transversus abdominis release in 26 patients (45%), a modification of transversus abdominis release 27 (47%), a reverse transversus abdominis release in 3 (5%), and a primary, lateral retromuscular preperitoneal approach in 2 (3%). Surgical site occurrences occurred in 22 patients (38%), with only 8 patients (14%) requiring procedural intervention. During a mean follow-up of 30.1 ± 14.4 months, 2 (3%) cases of recurrence were diagnosed and required reoperation. There were also 4 (7%) patients with asymptomatic but visible bulging. The European Registry for Abdominal Wall Hernias Quality of Life score showed a statistically significant decrease in the 3 domains (pain, restriction, and cosmetic) in the postoperative score compared with the preoperative score. Conclusion The different techniques of posterior component separation in the treatment of combined midline and lateral incisional hernia show acceptable results, despite the associated high complexity. Patient-reported outcomes after measurement of the European Registry for Abdominal Wall Hernias Quality of Life score demonstrated a clinically important improvement in quality of life and pain.post-print2.323 K

Filaggrin and cytokines in respiratory samples of preterm infants at risk for respiratory viral infection

Respiratory viral infections (RVIs) are frequent in preterm infants possibly inducing long-term impact on respiratory morbidity. Immune response and respiratory barriers are key defense elements against viral insults in premature infants admitted to Neonatal Intensive Care Units (NICUs). Our main goals were to describe the local immune response in respiratory secretions of preterm infants with RVIs during NICU admission and to evaluate the expression and synthesis of lung barrier regulators, both in respiratory samples and in vitro models. Samples from preterm infants that went on to develop RVIs had lower filaggrin gene and protein levels at a cellular level were compared to never-infected neonates (controls). Filaggrin, MIP-1α/CCL3 and MCP-1 levels were higher in pre-infection supernatants compared to controls. Filaggrin, HIF-1α, VEGF, RANTES/CCL5, IL-17A, IL-1β, MIP-1α and MIP-1β/CCL5 levels were higher during and after infection. ROC curve and logistic regression analysis shows that these molecules could be used as infection risk biomarkers. Small airway epithelial cells stimulated by poly:IC presented reduced filaggrin gene expression and increased levels in supernatant. We conclude that filaggrin gene and protein dysregulation is a risk factor of RVI in newborns admitted at the NICU.This study has been partially supported by ISCIII – Instituto de Salud Carlos III, FIS (Fondo de Investigación Sanitaria—Spanish Health Research Fund) grants FI19/00067, PI18/00167, PI21/00896, PI18CIII/00009 and FEDER funds (Fondo Europeo de Desarrollo Regional); Sociedad Española de Alergología e Inmunología Clínica (SEAIC)Beca19A04_Valverde; Alfonso X El Sabio University Grant: VIII Convocatoria Santander-UAX; CIBER de Enfermedades Respiratorias (CIBERES), a Carlos III Institute of Health Initiative.S

Measurement of H₂O₂ within living drosophila during aging using a ratiometric mass spectrometry probe targeted to the mitochondrial matrix

Hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) is central to mitochondrial oxidative damage and redox signaling, but its roles are poorly understood due to the difficulty of measuring mitochondrial H<sub>2</sub>O<sub>2</sub> in vivo. Here we report a ratiometric mass spectrometry probe approach to assess mitochondrial matrix H<sub>2</sub>O<sub>2</sub> levels in vivo. The probe, MitoB, comprises a triphenylphosphonium (TPP) cation driving its accumulation within mitochondria, conjugated to an arylboronic acid that reacts with H<sub>2</sub>O<sub>2</sub> to form a phenol, MitoP. Quantifying the MitoP/MitoB ratio by liquid chromatography-tandem mass spectrometry enabled measurement of a weighted average of mitochondrial H<sub>2</sub>O<sub>2</sub> that predominantly reports on thoracic muscle mitochondria within living flies. There was an increase in mitochondrial H<sub>2</sub>O<sub>2</sub> with age in flies, which was not coordinately altered by interventions that modulated life span. Our findings provide approaches to investigate mitochondrial ROS in vivo and suggest that while an increase in overall mitochondrial H<sub>2</sub>O<sub>2</sub> correlates with aging, it may not be causative