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1.
Int J Mol Sci ; 22(7)2021 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-33810353

RESUMO

Many pairwise additive force fields are in active use for intrinsically disordered proteins (IDPs) and regions (IDRs), some of which modify energetic terms to improve the description of IDPs/IDRs but are largely in disagreement with solution experiments for the disordered states. This work considers a new direction-the connection to configurational entropy-and how it might change the nature of our understanding of protein force field development to equally well encompass globular proteins, IDRs/IDPs, and disorder-to-order transitions. We have evaluated representative pairwise and many-body protein and water force fields against experimental data on representative IDPs and IDRs, a peptide that undergoes a disorder-to-order transition, for seven globular proteins ranging in size from 130 to 266 amino acids. We find that force fields with the largest statistical fluctuations consistent with the radius of gyration and universal Lindemann values for folded states simultaneously better describe IDPs and IDRs and disorder-to-order transitions. Hence, the crux of what a force field should exhibit to well describe IDRs/IDPs is not just the balance between protein and water energetics but the balance between energetic effects and configurational entropy of folded states of globular proteins.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Simulação de Dinâmica Molecular , Engenharia de Proteínas , Teorema de Bayes , Simulação por Computador , Entropia , Espectroscopia de Ressonância Magnética , Peptídeos/química , Polímeros/química , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Solventes , Eletricidade Estática , Temperatura
2.
Data Brief ; 30: 105496, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32368578

RESUMO

The structural conformations of phospholipids and cholesterol in phase-separated lipid domains were determined by surface area, transverse density profile, and lipid acyl chain orientational parameter calculations. Binding kinetics and characterization of membrane-bound states of beta-amyloid fibrils of various sizes (dimer to pentamer), on those lipid domains, were determined using protein residue orientational parameter and fibril-residue-lipid minimum distance analysis methods. The energy of binding and characterization of annular lipid shells surrounding the surface-bound amyloid fibrils were also determined. The calculations described above support the article "Coarse-Grained MD simulations Reveal Diverse Membrane-Bound Conformational States of Beta-Amyloid Fibrils in the Liquid-ordered and Liquid-disordered Regions of Phase-Separated Lipid Rafts Containing Glycolipid, Cholesterol and Oxidized Cholesterol (Cheng et al., 2020 [1])". The reported data is valuable for the future design and analysis of any protein fibrils binding to phase-separated lipid domains in model multi-component lipids membranes using either atomistic or coarse-grained molecular dynamics simulations. Additionally, this data can guide or validate future single-molecule experiments on fibril/membrane interactions in model or cell membranes.

3.
Biophys Chem ; 260: 106355, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32179374

RESUMO

The membrane binding behaviors of beta-amyloid fibrils, dimers to pentamers, from solution to lipid raft surfaces, were investigated using coarse-grained (CG) MD simulations. Our CG rafts contain phospholipid, cholesterol (with or without tail- or headgroup modifications), and with or without asymmetrically distributed monosialotetrahexosylganglioside (GM1). All rafts exhibited liquid-ordered (Lo), liquid-disordered (Ld), and interfacial Lo/Ld (Lod) domains, with domain sizes depending on cholesterol structure. For rafts without GM1, all fibrils bound to the Lod domains. Specifically, dimer fibrils bound exclusively via the C-terminal, while larger fibrils could bind via other protein regions. Interestingly, a membrane-inserted state was detected for a trimer fibril in a raft with tail-group modified cholesterol. For rafts containing GM1, fibrils bound either to the GM1-clusters, with numerous membrane-bound conformations, or to the non-GM1-containing-Lod domains via the C-terminal. Our results indicate beta-amyloid fibrils bind to Lod domains or GM1, with diversified membrane-bound conformations, in structurally heterogeneous lipid membranes.


Assuntos
Peptídeos beta-Amiloides/química , Lipídeos/química , Simulação de Dinâmica Molecular , Sítios de Ligação , Humanos , Conformação Molecular , Tamanho da Partícula
4.
Annu Rev Biophys ; 48: 371-394, 2019 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-30916997

RESUMO

Realistic modeling of biomolecular systems requires an accurate treatment of electrostatics, including electronic polarization. Due to recent advances in physical models, simulation algorithms, and computing hardware, biomolecular simulations with advanced force fields at biologically relevant timescales are becoming increasingly promising. These advancements have not only led to new biophysical insights but also afforded opportunities to advance our understanding of fundamental intermolecular forces. This article describes the recent advances and applications, as well as future directions, of polarizable force fields in biomolecular simulations.


Assuntos
Simulação de Dinâmica Molecular , Eletricidade Estática , Algoritmos , Fenômenos Físicos
5.
J Am Chem Soc ; 139(20): 7110-7116, 2017 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-28463488

RESUMO

High-resolution melting (HRM) analysis of DNA is a closed-tube single-nucleotide polymorphism (SNP) detection method that has shown many advantages in point-of-care diagnostics and personalized medicine. While recently developed melting probes have demonstrated significantly improved discrimination of mismatched (mutant) alleles from matched (wild-type) alleles, no effort has been made to design a simple melting probe that can reliably distinguish all four SNP alleles in a single experiment. Such a new probe could facilitate the discovery of rare genetic mutations at lower cost. Here we demonstrate that a melting probe embedded with a single locked thymidine monomer (tL) can reliably differentiate the four SNP alleles by four distinct melting temperatures (termed the "4Tm probe"). This enhanced discriminatory power comes from the decreased melting temperature of the tL·C mismatched hybrid as compared to that of the t·C mismatched hybrid, while the melting temperatures of the tL-A, tL·G and tL·T hybrids are increased or remain unchanged as compared to those of their canonical counterparts. This phenomenon is observed not only in the HRM experiments but also in the molecular dynamics simulations.


Assuntos
Sondas de DNA/química , Oligonucleotídeos/química , Polimorfismo de Nucleotídeo Único/genética , Timidina/química , Temperatura de Transição , Alelos , Simulação de Dinâmica Molecular
6.
Sci Rep ; 7: 45812, 2017 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-28393861

RESUMO

We introduce a coarse-grained RNA model for molecular dynamics simulations, RACER (RnA CoarsE-gRained). RACER achieves accurate native structure prediction for a number of RNAs (average RMSD of 2.93 Å) and the sequence-specific variation of free energy is in excellent agreement with experimentally measured stabilities (R2 = 0.93). Using RACER, we identified hydrogen-bonding (or base pairing), base stacking, and electrostatic interactions as essential driving forces for RNA folding. Also, we found that separating pairing vs. stacking interactions allowed RACER to distinguish folded vs. unfolded states. In RACER, base pairing and stacking interactions each provide an approximate stability of 3-4 kcal/mol for an A-form helix. RACER was developed based on PDB structural statistics and experimental thermodynamic data. In contrast with previous work, RACER implements a novel effective vdW potential energy function, which led us to re-parameterize hydrogen bond and electrostatic potential energy functions. Further, RACER is validated and optimized using a simulated annealing protocol to generate potential energy vs. RMSD landscapes. Finally, RACER is tested using extensive equilibrium pulling simulations (0.86 ms total) on eleven RNA sequences (hairpins and duplexes).


Assuntos
Simulação de Dinâmica Molecular , Dobramento de RNA , RNA/química , Modelos Moleculares , Termodinâmica
7.
Data Brief ; 7: 658-72, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27054174

RESUMO

This data article supports the research article entitled "Maximally Asymmetric Transbilayer Distribution of Anionic Lipids Alters the Structure and interaction with Lipids of an Amyloidogenic Protein Dimer Bound to the Membrane Surface" [1]. We describe supporting data on the binding kinetics, time evolution of secondary structure, and residue-contact maps of a surface-absorbed beta-amyloid dimer protein on different membrane surfaces. We further demonstrate the sorting of annular and non-annular regions of the protein/lipid bilayer simulation systems, and the correlation of lipid-number mismatch and surface area per lipid mismatch of asymmetric lipid membranes.

8.
Chem Phys Lipids ; 196: 33-51, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26827904

RESUMO

We used molecular dynamics simulations to explore the effects of asymmetric transbilayer distribution of anionic phosphatidylserine (PS) lipids on the structure of a protein on the membrane surface and subsequent protein-lipid interactions. Our simulation systems consisted of an amyloidogenic, beta-sheet rich dimeric protein (D42) absorbed to the phosphatidylcholine (PC) leaflet, or protein-contact PC leaflet, of two membrane systems: a single-component PC bilayer and double PC/PS bilayers. The latter comprised of a stable but asymmetric transbilayer distribution of PS in the presence of counterions, with a 1-component PC leaflet coupled to a 1-component PS leaflet in each bilayer. The maximally asymmetric PC/PS bilayer had a non-zero transmembrane potential (TMP) difference and higher lipid order packing, whereas the symmetric PC bilayer had a zero TMP difference and lower lipid order packing under physiologically relevant conditions. Analysis of the adsorbed protein structures revealed weaker protein binding, more folding in the N-terminal domain, more aggregation of the N- and C-terminal domains and larger tilt angle of D42 on the PC leaflet surface of the PC/PS bilayer versus the PC bilayer. Also, analysis of protein-induced membrane structural disruption revealed more localized bilayer thinning in the PC/PS versus PC bilayer. Although the electric field profile in the non-protein-contact PS leaflet of the PC/PS bilayer differed significantly from that in the non-protein-contact PC leaflet of the PC bilayer, no significant difference in the electric field profile in the protein-contact PC leaflet of either bilayer was evident. We speculate that lipid packing has a larger effect on the surface adsorbed protein structure than the electric field for a maximally asymmetric PC/PS bilayer. Our results support the mechanism that the higher lipid packing in a lipid leaflet promotes stronger protein-protein but weaker protein-lipid interactions for a dimeric protein on membrane surfaces.


Assuntos
Peptídeos beta-Amiloides/química , Bicamadas Lipídicas/química , Ânions , Dimerização , Simulação de Dinâmica Molecular , Estrutura Molecular , Conformação Proteica , Eletricidade Estática
9.
Biophys Chem ; 206: 22-39, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26164502

RESUMO

We have used coarse-grained (CG) and united atom (UA) molecular dynamics simulations to explore the mechanisms of protein orientational transition of a model peptide (Aß42) in a phosphatidylcholine/cholesterol (PC/CHO) lipid bilayer. We started with an inserted state of Aß42 containing a folded (I) or unfolded (II) K28-A42 lipid insertion domain (LID), which was stabilized by the K28-snorkeling and A42-anchoring to the PC polar groups in the lipid bilayer. After a UA-to-CG transformation and a 1000ns-CG simulation for enhancing the sampling of protein orientations, we discovered two transitions: I-to-"deep inserted" state with disrupted K28-snorkeling and II-to-"deep surface" state with disrupted A42-anchoring. The new states remained stable after a CG-to-UA transformation and a 200ns-UA simulation relaxation. Significant changes in the cholesterol-binding domain of Aß42 and protein-induced membrane disruptions were evident after the transitions. We propose that the conformation of the LID regulates protein orientational transitions in the lipid membrane.


Assuntos
Peptídeos beta-Amiloides/química , Colesterol/química , Bicamadas Lipídicas/química , Fragmentos de Peptídeos/química , Fosfatidilcolinas/química , Desdobramento de Proteína , Sequência de Aminoácidos , Humanos , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Estrutura Terciária de Proteína
10.
Biophys Chem ; 198: 22-35, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25637891

RESUMO

Quantifying protein-induced lipid disruptions at the atomistic level is a challenging problem in membrane biophysics. Here we propose a novel 3D Voronoi tessellation nearest-atom-neighbor shell method to classify and characterize lipid domains into discrete concentric lipid shells surrounding membrane proteins in structurally heterogeneous lipid membranes. This method needs only the coordinates of the system and is independent of force fields and simulation conditions. As a proof-of-principle, we use this multiple lipid shell method to analyze the lipid disruption profiles of three simulated membrane systems: phosphatidylcholine, phosphatidylcholine/cholesterol, and beta-amyloid/phosphatidylcholine/cholesterol. We observed different atomic volume disruption mechanisms due to cholesterol and beta-amyloid. Additionally, several lipid fractional groups and lipid-interfacial water did not converge to their control values with increasing distance or shell order from the protein. This volume divergent behavior was confirmed by bilayer thickness and chain orientational order calculations. Our method can also be used to analyze high-resolution structural experimental data.


Assuntos
Peptídeos beta-Amiloides/química , Fosfolipídeos/química , Sequência de Aminoácidos , Bicamadas Lipídicas , Simulação de Dinâmica Molecular , Dados de Sequência Molecular
11.
J Chem Theory Comput ; 10(7): 2792-2801, 2014 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-25018674

RESUMO

The orthogonal space random walk (OSRW) method has shown enhanced sampling efficiency in free energy calculations from previous studies. In this study, the implementation of OSRW in accordance with the polarizable AMOEBA force field in TINKER molecular modeling software package is discussed and subsequently applied to the hydration free energy calculation of 20 small organic molecules, among which 15 are positively charged and five are neutral. The calculated hydration free energies of these molecules are compared with the results obtained from the Bennett acceptance ratio method using the same force field, and overall an excellent agreement is obtained. The convergence and the efficiency of the OSRW are also discussed and compared with BAR. Combining enhanced sampling techniques such as OSRW with polarizable force fields is very promising for achieving both accuracy and efficiency in general free energy calculations.

12.
Nat Med ; 18(8): 1217-23, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22820644

RESUMO

Sepsis, a systemic inflammatory response to infection, commonly progresses to acute lung injury (ALI), an inflammatory lung disease with high morbidity. We postulated that sepsis-associated ALI is initiated by degradation of the pulmonary endothelial glycocalyx, leading to neutrophil adherence and inflammation. Using intravital microscopy, we found that endotoxemia in mice rapidly induced pulmonary microvascular glycocalyx degradation via tumor necrosis factor-α (TNF-α)-dependent mechanisms. Glycocalyx degradation involved the specific loss of heparan sulfate and coincided with activation of endothelial heparanase, a TNF-α-responsive, heparan sulfate-specific glucuronidase. Glycocalyx degradation increased the availability of endothelial surface adhesion molecules to circulating microspheres and contributed to neutrophil adhesion. Heparanase inhibition prevented endotoxemia-associated glycocalyx loss and neutrophil adhesion and, accordingly, attenuated sepsis-induced ALI and mortality in mice. These findings are potentially relevant to human disease, as sepsis-associated respiratory failure in humans was associated with higher plasma heparan sulfate degradation activity; moreover, heparanase content was higher in human lung biopsies showing diffuse alveolar damage than in normal human lung tissue.


Assuntos
Lesão Pulmonar Aguda/fisiopatologia , Endotoxemia/complicações , Glicocálix/fisiologia , Pulmão/fisiopatologia , Neutrófilos/fisiologia , Lesão Pulmonar Aguda/etiologia , Lesão Pulmonar Aguda/prevenção & controle , Transferência Adotiva , Animais , Adesão Celular/fisiologia , Modelos Animais de Doenças , Endotélio/enzimologia , Endotélio/fisiologia , Endotoxemia/fisiopatologia , Ativação Enzimática , Regulação da Expressão Gênica/efeitos dos fármacos , Glucuronidase/análise , Glucuronidase/deficiência , Glucuronidase/fisiologia , Heparitina Sulfato/antagonistas & inibidores , Heparitina Sulfato/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Molécula 1 de Adesão Intercelular/genética , Perfuração Intestinal/complicações , Perfuração Intestinal/microbiologia , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Alvéolos Pulmonares/enzimologia , Alvéolos Pulmonares/patologia , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Receptores Tipo I de Fatores de Necrose Tumoral/fisiologia , Insuficiência Respiratória/enzimologia , Insuficiência Respiratória/patologia , Fator de Necrose Tumoral alfa/fisiologia , Lesão Pulmonar Induzida por Ventilação Mecânica/enzimologia , Lesão Pulmonar Induzida por Ventilação Mecânica/patologia
15.
Intensive Care Med ; 38(4): 642-8, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22231174

RESUMO

PURPOSE: To assess coagulation status and factor Xa inhibition in surgical intensive care unit (ICU) patients administered prophylactic unfractionated heparin for venous thromboembolism (VTE) prophylaxis. METHODS: We conducted a randomized, single-blind study at a tertiary academic medical center. Included were patients 18 years and older admitted to the surgical ICU directly after major abdominal surgery. Exclusion criteria included significant bleeding risk, preoperative anticoagulation, or history of heparin-induced thrombocytopenia. Patients were randomized to two regimens for VTE prophylaxis: standard of care unfractionated heparin, 5,000 units subcutaneously three times daily (SQH) versus unfractionated heparin via intravenous infusion, titrated to an activated partial thromboplastin time of 40-45 s (IVH). Blood samples were taken prior to surgical incision on day 0 and daily for 5 days after surgery. Samples were analyzed for factor Xa inhibition and viscoelastic whole blood clotting parameters (Sonoclot analyzer). RESULTS: A total of 50 patients were randomized to either SQH or IVH. The majority of patients had cancer. Patients in the SQH group had no detectable peak anti-factor Xa (aFXa) activity for 5 days after surgery, while patients in the IVH group had statistically elevated levels compared to the SQH group on days 3-5. SQH patients demonstrated a hypercoagulable profile on Sonoclot, while IVH patients displayed a normal profile. CONCLUSIONS: Standard of care subcutaneous dosing of unfractionated heparin for VTE prophylaxis in surgical ICU patients leads to subtherapeutic levels of factor Xa inhibition.


Assuntos
Anticoagulantes/administração & dosagem , Cuidados Críticos , Heparina/administração & dosagem , Tromboembolia Venosa/prevenção & controle , APACHE , Análise de Variância , Testes de Coagulação Sanguínea , Inibidores do Fator Xa , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/prevenção & controle , Estudos Prospectivos , Método Simples-Cego , Estatísticas não Paramétricas , Resultado do Tratamento
16.
Arch Otolaryngol Head Neck Surg ; 132(6): 655-61, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16785412

RESUMO

OBJECTIVE: To identify clinical factors associated with enteral feeding tube placement in a head and neck cancer population. DESIGN: A self-administered survey was given to patients being treated for head and neck cancer while they were waiting to be seen in 1 of 4 otolaryngology clinics. The post hoc analysis presented here combines survey and chart review data to determine clinical and demographic variables associated with feeding tube placement. SETTING: Four otolaryngology clinics. PATIENTS: Otolaryngology clinic patients being treated for head and neck cancer. MAIN OUTCOME MEASURE: Enteral feeding tube placement. RESULTS: Of the 724 patients eligible for this study, 14% (n = 98) required enteral feeding tube placement. Multivariate analysis found the following variables to be independently associated with feeding tube placement: oropharynx/hypopharynx tumor site (odds ratio [OR], 2.4; P = .01), tumor stage III/IV (OR, 2.1; P = .03), flap reconstruction (OR, 2.2; P = .004), current tracheotomy (OR, 8.0; P<.001), chemotherapy (OR, 2.6; P<.001), and increased age (OR, 1.3; P = .02). In addition, there was a curvilinear relationship between time since treatment and feeding tube placement, with about 30% having a feeding tube at 1 month posttreatment, tapering down during the first 3 years to about 8% and leveling off thereafter. CONCLUSIONS: Identification of factors associated with an increased risk of feeding tube placement may allow physicians to better counsel patients regarding the possibility of feeding tube placement during treatment. Since feeding tube placement has been linked to decreased quality of life in head and neck cancer, such counseling is an integral part of the clinical management of these patients.


Assuntos
Nutrição Enteral/métodos , Neoplasias de Cabeça e Pescoço/psicologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos Transversais , Feminino , Gastrostomia , Humanos , Intubação Gastrointestinal , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Qualidade de Vida , Fatores de Risco , Inquéritos e Questionários
17.
J Biol Chem ; 280(32): 28966-72, 2005 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-15958384

RESUMO

Ssq1, a specialized yeast mitochondrial Hsp70, plays a critical role in the biogenesis of proteins containing Fe-S clusters through its interaction with Isu, the scaffold on which clusters are built. Two substitutions within the Ssq1 substrate binding cleft, both of which severely reduced affinity for Isu, had very different effects in vivo. Cells expressing Ssq1(F462S), which had no detectable affinity for Isu, are indistinguishable from Deltassq1 cells, underscoring the importance of the Ssq1-Isu1 interaction in vivo. In contrast, cells expressing Ssq1(V472F), whose affinity for Isu is at least 10-fold lower than that of wild-type Ssq1, had only moderately reduced Fe-S enzyme activities and increased iron levels and grew similarly to wild-type cells. Consistent with the reduced affinity for Isu, the ATPase activity of Ssq1(V472F) was stimulated less well than that of Ssq1 upon addition of Isu and Jac1, the J-protein partner of Ssq1. However, higher concentrations of Jac1 or Isu1, which form a stable complex, could compensate for this defect in stimulation of Ssq1(V472F). Expression of Isu1 was up-regulated 10-fold in ssq1(V472F) compared with wild-type cells, suggesting that formation of a Jac1-Isu1 complex can overcome a lowered affinity of Ssq1 for Isu in vivo as well as in vitro.


Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Proteínas Ferro-Enxofre/química , Mitocôndrias/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/metabolismo , Motivos de Aminoácidos , Anisotropia , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Proteínas Fúngicas/metabolismo , Glicerol/química , Ferro/metabolismo , Proteínas Mitocondriais , Chaperonas Moleculares/genética , Mutação , Peptídeos/química , Fenótipo , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas de Saccharomyces cerevisiae/genética , Espectrometria de Fluorescência , Ressonância de Plasmônio de Superfície , Temperatura
18.
Otolaryngol Head Neck Surg ; 132(3): 520-2, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15746877

RESUMO

Paragangliomas of the larynx are rare neuroendocrine tumors, with fewer than 70 cases reported in the literature. 1 Typically, laryngeal paragangliomas are not found in patients with multicentric or familial paragangliomas. Only 1 case of laryngeal paraganglioma has been reported in a patient with a synchronous lesion elsewhere (carotid body tumor). 2 We report an additional case of a patient with a laryngeal and multiple other paragangliomas.


Assuntos
Neoplasias de Cabeça e Pescoço/diagnóstico por imagem , Neoplasias Laríngeas/diagnóstico por imagem , Neoplasias Primárias Múltiplas/diagnóstico por imagem , Paraganglioma/diagnóstico por imagem , Humanos , Masculino , Pessoa de Meia-Idade , Tomografia Computadorizada por Raios X
19.
J Biol Chem ; 279(28): 29167-74, 2004 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-15123690

RESUMO

Isu, the scaffold for assembly of Fe-S clusters in the yeast mitochondrial matrix, is a substrate protein for the Hsp70 Ssq1 and the J-protein Jac1 in vitro. As expected for an Hsp70-substrate interaction, the formation of a stable complex between Isu and Ssq1 requires Jac1 in the presence of ATP. Here we report that a conserved tripeptide, PVK, of Isu is critical for interaction with Ssq1 because amino acid substitutions in this tripeptide inhibit both the formation of the Isu-Ssq1 complex and the ability of Isu to stimulate the ATPase activity of Ssq1. These biochemical defects correlate well with the growth defects of cells expressing mutant Isu proteins. We conclude that the Ssq1-Isu substrate interaction is critical for Fe-S cluster biogenesis in vivo. The ability of Jac1 and mutant Isu proteins to cooperatively stimulate the ATPase activity of Ssq1 was also measured. Increasing the concentration of Jac1 and mutant Isu together but not individually partially overcame the effect of the reduced affinity of the Isu mutant proteins for Ssq1. These results, along with the observation that overexpression of Jac1 was able to suppress the growth defect of an ISU mutant, support the hypothesis that Isu is "targeted" to Ssq1 by Jac1, with a preformed Jac1-Isu complex interacting with Ssq1.


Assuntos
Proteínas Ferro-Enxofre/metabolismo , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Proteínas de Choque Térmico HSP70 , Proteínas Ferro-Enxofre/genética , Substâncias Macromoleculares , Proteínas Mitocondriais/genética , Chaperonas Moleculares/genética , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
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