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1.
J Biol Chem ; 294(13): 5105-5120, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30700553

RESUMO

Serine protease inhibitors of the Kunitz-bovine pancreatic trypsin inhibitor (BPTI) family are ubiquitous biological regulators of proteolysis. These small proteins are resistant to proteolysis, but can be slowly cleaved within the protease-binding loop by target proteases, thereby compromising their activity. For the human protease mesotrypsin, this cleavage is especially rapid. Here, we aimed to stabilize the Kunitz domain structure against proteolysis through disulfide engineering. Substitution within the Kunitz inhibitor domain of the amyloid precursor protein (APPI) that incorporated a new disulfide bond between residues 17 and 34 reduced proteolysis by mesotrypsin 74-fold. Similar disulfide engineering of tissue factor pathway inhibitor-1 Kunitz domain 1 (KD1TFPI1) and bikunin Kunitz domain 2 (KD2bikunin) likewise stabilized these inhibitors against mesotrypsin proteolysis 17- and 6.6-fold, respectively. Crystal structures of disulfide-engineered APPI and KD1TFPI1 variants in a complex with mesotrypsin at 1.5 and 2.0 Å resolution, respectively, confirmed the formation of well-ordered disulfide bonds positioned to stabilize the binding loop. Long all-atom molecular dynamics simulations of disulfide-engineered Kunitz domains and their complexes with mesotrypsin revealed conformational stabilization of the primed side of the inhibitor-binding loop by the engineered disulfide, along with global suppression of conformational dynamics in the Kunitz domain. Our findings suggest that the Cys-17-Cys-34 disulfide slows proteolysis by dampening conformational fluctuations in the binding loop and minimizing motion at the enzyme-inhibitor interface. The generalizable approach developed here for the stabilization against proteolysis of Kunitz domains, which can serve as important scaffolds for therapeutics, may thus find applications in drug development.


Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Aprotinina/metabolismo , Tripsina/metabolismo , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/genética , Animais , Aprotinina/química , Aprotinina/genética , Cristalografia por Raios X , Dissulfetos/química , Dissulfetos/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Engenharia de Proteínas , Proteólise , Tripsina/química
2.
Bioconjug Chem ; 29(12): 4140-4148, 2018 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-30453738

RESUMO

Fluorescence imaging has currently emerged as one of the most frequently used noninvasive imaging technologies to selectively monitor biological processes in living systems. In past decades, gold nanoclusters (Au NCs) has received increasing attraction because of their intrinsic fluorescence and their inherent biocompatibility. As a stabilizing and reducing agent, an abundant, sustainable, and widely used polypeptide derived drug molecule, aprotinin (Ap), is selected for the synthesis of Au nanoclusters (Ap-Au NCs) due to characteristic bioactivity, excellent biocompatibility, biodegradability, and non-allergenic character. Herein, Ap encapsulated Au NCs with desirable red fluorescence was facilely produced for the first time, which were subsequently used for cell imaging and detection of various analytes. Much interestingly, dynamically subcellular targeting  from the cytoplasm to the nucleus in HeLa cells was observed. Besides, it has shown that, the selective and quantitative detection of trypsin has been established by using Ap-Au NCs. Finally, Ap-Au NCs were readily used for quantitative detection of mercury and copper. The photoluminescence of the Ap-Au NCs was quenched with the addition of the aforementioned analytes. This study not only  discusses a multifunctional nanomaterial  for cell imaging, dynamically nuclear targeting and biosensing, but also opens crucial insights on the integration of funtional biomolecule with metal nanoclusters intended for extensively biomedical applications.


Assuntos
Aprotinina/química , Núcleo Celular/química , Corantes Fluorescentes/química , Ouro/química , Metais Pesados/análise , Nanoestruturas/química , Tripsina/análise , Células HeLa , Humanos
3.
Biophys J ; 114(9): 2040-2043, 2018 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-29742397

RESUMO

Anharmonicity in time-dependent conformational fluctuations is noted to be a key feature of functional dynamics of biomolecules. Although anharmonic events are rare, long-timescale (µs-ms and beyond) simulations facilitate probing of such events. We have previously developed quasi-anharmonic analysis to resolve higher-order spatial correlations and characterize anharmonicity in biomolecular simulations. In this article, we have extended this toolbox to resolve higher-order temporal correlations and built a scalable Python package called anharmonic conformational analysis (ANCA). ANCA has modules to: 1) measure anharmonicity in the form of higher-order statistics and its variation as a function of time, 2) output a storyboard representation of the simulations to identify key anharmonic conformational events, and 3) identify putative anharmonic conformational substates and visualization of transitions between these substates.


Assuntos
Simulação de Dinâmica Molecular , Animais , Aprotinina/química , Aprotinina/metabolismo , Bovinos , Movimento , Conformação Proteica
4.
J Chem Phys ; 148(5): 055101, 2018 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-29421894

RESUMO

We use Markov state models (MSMs) to analyze the dynamics of a ß-hairpin-forming peptide in Monte Carlo (MC) simulations with interacting protein crowders, for two different types of crowder proteins [bovine pancreatic trypsin inhibitor (BPTI) and GB1]. In these systems, at the temperature used, the peptide can be folded or unfolded and bound or unbound to crowder molecules. Four or five major free-energy minima can be identified. To estimate the dominant MC relaxation times of the peptide, we build MSMs using a range of different time resolutions or lag times. We show that stable relaxation-time estimates can be obtained from the MSM eigenfunctions through fits to autocorrelation data. The eigenfunctions remain sufficiently accurate to permit stable relaxation-time estimation down to small lag times, at which point simple estimates based on the corresponding eigenvalues have large systematic uncertainties. The presence of the crowders has a stabilizing effect on the peptide, especially with BPTI crowders, which can be attributed to a reduced unfolding rate ku, while the folding rate kf is left largely unchanged.


Assuntos
Aprotinina/química , Simulação de Dinâmica Molecular , Peptídeos/química , Receptores de GABA-B/química , Animais , Bovinos , Cadeias de Markov , Método de Monte Carlo , Dobramento de Proteína , Temperatura Ambiente
5.
J Mol Biol ; 430(15): 2196-2202, 2018 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-29258819

RESUMO

A fully quantitative theory connecting protein conformation and optical spectroscopy would facilitate deeper insights into biophysical and simulation studies of protein dynamics and folding. The web server DichroCalc (http://comp.chem.nottingham.ac.uk/dichrocalc) allows one to compute from first principles the electronic circular dichroism spectrum of a (modeled or experimental) protein structure or ensemble of structures. The regular, repeating, chiral nature of secondary structure elements leads to intense bands in the far-ultraviolet (UV). The near-UV bands are much weaker and have been challenging to compute theoretically. We report some advances in the accuracy of calculations in the near-UV, realized through the consideration of the vibrational structure of the electronic transitions of aromatic side chains. The improvements have been assessed over a set of diverse proteins. We illustrate them using bovine pancreatic trypsin inhibitor and present a new, detailed analysis of the interactions which are most important in determining the near-UV circular dichroism spectrum.


Assuntos
Dicroísmo Circular , Biologia Computacional/métodos , Conformação Proteica , Espectrofotometria Ultravioleta , Animais , Aprotinina/química , Bovinos , Internet , Modelos Moleculares
6.
Bull Exp Biol Med ; 163(6): 742-744, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29063327

RESUMO

Liposomes containing aprotinin were produced by the phase inversion technique and purified by gel filtration. Aprotinin inclusion was assessed fluorescence labeling of the protein. The size of obtained liposomes was 212±35 nm and aprotinin concentration in the liposomal suspension was 3000 KIU/ml. The efficiency of aprotinin inclusion into liposomes was 31.9%.


Assuntos
Aprotinina/química , Lipossomos/química , Nanosferas/química , Carbocianinas/química , Colesterol/química , Composição de Medicamentos , Corantes Fluorescentes/química , Tamanho da Partícula , Fosfatidilcolinas/química , Espectrometria de Fluorescência , Coloração e Rotulagem/métodos
7.
J Phys Chem B ; 121(40): 9358-9365, 2017 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-28911225

RESUMO

The study of the function of proteins on a quantitative level requires consideration of the water molecules in and around the protein. This requirement presents a major computational challenge due to the fact that the insertion of water molecules can have a very high activation barrier and would require a long simulation time. Recently, we developed a water flooding (WF) approach which is based on a postprocessing Monte Carlo ranking of possible water configurations. This approach appears to provide a very effective way for assessing the insertion free energies and determining the most likely configurations of the internal water molecules. Although the WF approach was used effectively in modeling challenging systems that have not been addressed reliably by other microscopic approaches, it was not validated by a comparison to the more rigorous grand canonical Monte Carlo (GCMC) method. Here we validate the WF approach by comparing its performance to that of the GCMC method. It is found that the WF approach reproduces the GCMC results in well-defined test cases but does so much faster. This established the WF approach as a useful strategy for finding correct water configurations in proteins and thus to provide a powerful way for studies of the functions of proteins.


Assuntos
Aprotinina/química , Método de Monte Carlo , Água/química , Animais , Bovinos , Nuclease do Micrococo/química , Modelos Químicos , Staphylococcus
8.
J Am Soc Mass Spectrom ; 28(7): 1450-1461, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28585116

RESUMO

Charge reduction in the gas phase provides a direct means of manipulating protein charge state, and when coupled to ion mobility mass spectrometry (IM-MS), it is possible to monitor the effect of charge on protein conformation in the absence of solution. Use of the electron transfer reagent 1,3-dicyanobenzene, coupled with IM-MS, allows us to monitor the effect of charge reduction on the conformation of two proteins deliberately chosen from opposite sides of the order to disorder continuum: bovine pancreatic trypsin inhibitor (BPTI) and beta casein. The ordered BPTI presents compact conformers for each of three charge states accompanied by narrow collision cross-section distributions (TWCCSDN2→He). Upon reduction of BPTI, irrespective of precursor charge state, the TWCCSN2→He decreases to a similar distribution as found for the nESI generated ion of identical charge. The behavior of beta casein upon charge reduction is more complex. It presents over a wide charge state range (9-28), and intermediate charge states (13-18) have broad TWCCSDN2→He with multiple conformations, where both compaction and rearrangement are seen. Further, we see that the TWCCSDN2→He of the latter charge states are even affected by the presence of radical anions. Overall, we conclude that the flexible nature of some proteins result in broad conformational distributions comprised of many families, even for single charge states, and the barrier between different states can be easily overcome by an alteration of the net charge. Graphical Abstract ᅟ.


Assuntos
Espectrometria de Massas/métodos , Modelos Químicos , Proteínas/química , Proteínas/metabolismo , Aprotinina/química , Aprotinina/metabolismo , Caseínas/química , Caseínas/metabolismo , Conformação Proteica
9.
Artigo em Inglês | MEDLINE | ID: mdl-28472911

RESUMO

OBJECTIVES: The objectives of the present investigation were to prepare recombinant human insulin entrapped Eudragit-S100 microspheres containing protease inhibitors and to precisely analyze the outcome of different formulation variables on the microspheres properties using a response surface methodology to develop an optimized formulation with desirable features. METHODS: A central composite design was employed to produce microspheres of therapeutic protein by w/o/w multiple emulsion solvent evaporation technique using Eudragit S-100 as coating material and polyvinyl alcohol as a stabilizer. The effect of formulation variables (independent variables) that is levels of Eudragit S-100 (X1), therapeutic protein (X2), volumes of inner aqueous phase (X3) and external aqueous phase (X4) on dependant variables, that are encapsulation efficiency (Y1), drug release at pH 1.2 after 2 h (Y2) and drug release at pH 7.4 after of 2 h (Y3) were evaluated. RESULTS: The significant terms in the mathematical models were generated for each response parameter using multiple linear regression analysis and analysis of variance. All the formulation variables except the volume of external aqueous phase (X4) exerted a significant effect (P <0.05) on drug encapsulation efficiency (Y1) whereas first two variables, namely the levels of Eudragit S-100 (X1) and therapeutic protein (X2) materialized as the determining factors which significantly influenced drug release at pH 1.2 after 2 h (Y2) and drug release at pH 7.4 after of 2 h (Y3). The formulation was numerically optimized by framing the constraints on the dependent and independent variables using the desirability approach. The experimental values for Y1 and Y2 of optimized formulation were found to be 77.65% and 3.64%, respectively which were quite closer to results suggested by software. CONCLUSION: The results recorded indicate that the recombinant human insulin loaded Eudragit S-100 microspheres containing aprotinin have the benefits of higher loading efficiency, pH responsive and prolonged release characteristics, which may help to carry insulin to the optimum site of absorption.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Insulina/administração & dosagem , Insulina/farmacocinética , Microesferas , Ácidos Polimetacrílicos/administração & dosagem , Ácidos Polimetacrílicos/farmacocinética , Administração Oral , Animais , Aprotinina/administração & dosagem , Aprotinina/química , Aprotinina/farmacocinética , Bovinos , Composição de Medicamentos , Liberação Controlada de Fármacos/fisiologia , Humanos , Insulina/química , Ácidos Polimetacrílicos/química , Propriedades de Superfície
10.
Biomaterials ; 135: 1-9, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28477492

RESUMO

Aprotinin is a broad-spectrum serine protease inhibitor used in the clinic as an anti-fibrinolytic agent in fibrin-based tissue sealants. However, upon re-exposure, some patients suffer from hypersensitivity immune reactions likely related to the bovine origin of aprotinin. Here, we aimed to develop a human-derived substitute to aprotinin. Based on sequence homology analyses, we identified the Kunitz-type protease inhibitor (KPI) domain of human amyloid-ß A4 precursor protein as being a potential candidate. While KPI has a lower intrinsic anti-fibrinolytic activity than aprotinin, we reasoned that its efficacy is additionally limited by its fast release from fibrin material, just as aprotinin's is. Thus, we engineered KPI variants for controlled retention in fibrin biomaterials, using either covalent binding through incorporation of a substrate for the coagulation transglutaminase Factor XIIIa or through engineering of extracellular matrix protein super-affinity domains for sequestration into fibrin. We showed that both engineered KPI variants significantly slowed plasmin-mediated fibrinolysis in vitro, outperforming aprotinin. In vivo, our best engineered KPI variant (incorporating the transglutaminase substrate) extended fibrin matrix longevity by 50%, at a dose at which aprotinin did not show efficacy, thus qualifying it as a competitive substitute of aprotinin in fibrin sealants.


Assuntos
Materiais Biocompatíveis/química , Fibrina/química , Inibidores de Serino Proteinase/química , Aprotinina/química , Fibrinolisina/química , Humanos , Engenharia de Proteínas/métodos
11.
Sci Rep ; 7: 41205, 2017 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-28266637

RESUMO

We report a thermodynamic and structural analysis of six extensively simplified bovine pancreatic trypsin inhibitor (BPTI) variants containing 19-24 alanines out of 58 residues. Differential scanning calorimetry indicated a two-state thermal unfolding, typical of a native protein with densely packed interior. Surprisingly, increasing the number of alanines induced enthalpy stabilization, which was however over-compensated by entropy destabilization. X-ray crystallography indicated that the alanine substitutions caused the recruitment of novel water molecules facilitating the formation of protein-water hydrogen bonds and improving the hydration shells around the alanine's methyl groups, both of which presumably contributed to enthalpy stabilization. There was a strong correlation between the number of water molecules and the thermodynamic parameters. Overall, our results demonstrate that, in contrast to our initial expectation, a protein sequence in which over 40% of the residues are alanines can retain a densely packed structure and undergo thermal denaturation with a large enthalpy change, mainly contributed by hydration.


Assuntos
Alanina/química , Substituição de Aminoácidos , Aprotinina/química , Aprotinina/genética , Animais , Varredura Diferencial de Calorimetria , Bovinos , Cristalografia por Raios X , Ligações de Hidrogênio , Modelos Moleculares , Desnaturação Proteica , Estabilidade Proteica , Termodinâmica , Água/química
12.
J Virol ; 91(10)2017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28298600

RESUMO

The mosquito-transmitted dengue virus (DENV) infects millions of people in tropical and subtropical regions. Maturation of DENV particles requires proper cleavage of the viral polyprotein, including processing of 8 of the 13 substrate cleavage sites by dengue virus NS2B/NS3 protease. With no available direct-acting antiviral targeting DENV, NS2/NS3 protease is a promising target for inhibitor design. Current design efforts focus on the nonprime side of the DENV protease active site, resulting in highly hydrophilic and nonspecific scaffolds. However, the prime side also significantly modulates DENV protease binding affinity, as revealed by engineering the binding loop of aprotinin, a small protein with high affinity for DENV protease. In this study, we designed a series of cyclic peptides interacting with both sides of the active site as inhibitors of dengue virus protease. The design was based on two aprotinin loops and aimed to leverage both key specific interactions of substrate sequences and the entropic advantage driving aprotinin's high affinity. By optimizing the cyclization linker, length, and amino acid sequence, the tightest cyclic peptide achieved a Ki value of 2.9 µM against DENV3 wild-type (WT) protease. These inhibitors provide proof of concept that both sides of DENV protease active site can be exploited to potentially achieve specificity and lower hydrophilicity in the design of inhibitors targeting DENV.IMPORTANCE Viruses of the flaviviral family, including DENV and Zika virus transmitted by Aedes aegypti, continue to be a threat to global health by causing major outbreaks in tropical and subtropical regions, with no available direct-acting antivirals for treatment. A better understanding of the molecular requirements for the design of potent and specific inhibitors against flaviviral proteins will contribute to the development of targeted therapies for infections by these viruses. The cyclic peptides reported here as DENV protease inhibitors provide novel scaffolds that enable exploiting the prime side of the protease active site, with the aim of achieving better specificity and lower hydrophilicity than those of current scaffolds in the design of antiflaviviral inhibitors.


Assuntos
Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Inibidores de Proteases/farmacologia , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Proteínas não Estruturais Virais/antagonistas & inibidores , Sequência de Aminoácidos , Antivirais/síntese química , Antivirais/metabolismo , Aprotinina/química , Aprotinina/metabolismo , Aprotinina/farmacologia , Domínio Catalítico , Simulação por Computador , Vírus da Dengue/química , Vírus da Dengue/enzimologia , Descoberta de Drogas/métodos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Peptídeos Cíclicos/síntese química , Inibidores de Proteases/síntese química , Inibidores de Proteases/metabolismo , Ligação Proteica , Proteínas não Estruturais Virais/química
13.
Ann Clin Biochem ; 54(2): 293-296, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27705885

RESUMO

Background One of the main challenges in the measurement of glucagon is the premise that it is unstable in human plasma. Traditionally, protease inhibitors have been used to prevent its degradation; however, their use is controversial. Here, we investigated the optimal method of sample collection for glucagon, with measurement by liquid chromatography tandem mass spectrometry (LC-MS/MS) and two commercially available immunoassays. Methods Blood from healthy fasting volunteers (n = 10) was processed under a variety of preanalytical conditions including collection in EDTA vs. lithium heparin tubes and the addition of aprotinin and/or a dipeptidyl-peptidase IV (DPPIV) inhibitor. Additionally, the effect of freeze thaw was assessed. Plasma glucagon concentrations were measured by LC-MS/MS and two commercially available immunoassays (HTRF® sandwich immunoassay, Cisbio and Milliplex MAP Human Metabolic Hormone Panel, Merck Millipore). Results A systematic bias of Milliplex > LC-MS/MS > HTRF was noted and plasma glucagon concentrations were significantly different between methods (Milliplex vs. LC-MS/MS P < 0.01; Milliplex vs. HTRF P < 0.0001; LC-MS/MS vs. HTRF P < 0.001). The addition of aprotinin, DPPIV inhibitor or a combination of aprotinin and DPPIV inhibitor had no effect on plasma glucagon concentrations when compared to 'non-stabilized' samples or each other. Whether samples were taken in EDTA tubes or lithium heparin tubes made no difference to plasma glucagon concentrations. These findings were consistent for all three methods. Plasma glucagon concentrations were not significantly different after two freeze-thaw cycles (performed on samples in EDTA tubes containing aprotinin and DPPIV inhibitor). Conclusions This study demonstrates that glucagon is stable in both EDTA and lithium heparin tubes when stored at -80℃. Furthermore, the addition of aprotinin and DPPIV inhibitors is unnecessary.


Assuntos
Coleta de Amostras Sanguíneas/normas , Cromatografia Líquida/normas , Glucagon/sangue , Imunoensaio/normas , Espectrometria de Massas em Tandem/normas , Anticoagulantes/química , Aprotinina/química , Inibidores da Dipeptidil Peptidase IV/química , Ácido Edético/química , Congelamento , Voluntários Saudáveis , Heparina/química , Humanos , Transição de Fase , Estabilidade Proteica , Inibidores de Serino Proteinase/química , Temperatura Ambiente
14.
J Mol Graph Model ; 71: 80-87, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27855339

RESUMO

We probe the dynamics of the Bpti and Galectin-3 proteins using molecular dynamics simulations employing three water models at different levels of resolution, viz. the atomistic TIP4P-Ewald, the coarse-grained Elba and an implicit generalised Born model. The dynamics are quantified indirectly by model-free order parameters, S2 of the backbone NH and selected side-chain bond vectors, which also have been determined experimentally through NMR relaxation measurements. For the backbone, the order parameters produced with the three solvent models agree to a large extent with experiments, giving average unsigned deviations between 0.03 and 0.06. For the side-chains, for which the experimental data is incomplete, the deviations are considerably larger with mean deviations between 0.13 and 0.17. However, for both backbone and side-chains, it is difficult to pick a winner, as all models perform equally well overall. For a more complete set of side-chain vectors, we resort to analysing the variation among the estimates from different solvent models. Unfortunately, the variations are found to be sizeable with mean deviations between 0.11 and 0.15. Implications for computational assessment of protein dynamics are discussed.


Assuntos
Galectina 3/química , Conformação Proteica , Água/química , Aprotinina/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Solventes/química
15.
J Biol Chem ; 291(51): 26304-26319, 2016 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-27810896

RESUMO

The molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. Although considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here, we examine the importance of substrate dynamics in the cleavage of Kunitz-bovine pancreatic trypsin inhibitor protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis. A 1.4-Å crystal structure of a mesotrypsin-product complex formed with a rapidly cleaved substrate reveals a dramatic conformational change in the substrate upon proteolysis. By using long all-atom molecular dynamics simulations of acyl-enzyme intermediates with proteolysis rates spanning 3 orders of magnitude, we identify global and local dynamic features of substrates on the nanosecond-microsecond time scale that correlate with enzymatic rates and explain differential susceptibility to proteolysis. By integrating multiple enhanced sampling methods for molecular dynamics, we model a viable conformational pathway between substrate-like and product-like states, linking substrate dynamics on the nanosecond-microsecond time scale with large collective substrate motions on the much slower time scale of catalysis. Our findings implicate substrate flexibility as a critical determinant of catalysis.


Assuntos
Aprotinina/química , Simulação de Dinâmica Molecular , Proteólise , Tripsina/química , Animais , Catálise , Bovinos , Cristalografia por Raios X , Domínios Proteicos
16.
J Thromb Haemost ; 14(12): 2509-2523, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27797450

RESUMO

Essentials Current antifibrinolytics - aminocaproic acid and tranexamic acid-can cause seizures or renal injury. KD1L17R -KT , aprotinin and tranexamic acid were tested in a modified mouse tail-amputation model. S2'-subsite variations between human and mouse factor XIa result in vastly different inhibition profiles. KD1L17R -KT reduces blood loss and D-dimer levels in mouse with unobserved seizures or renal injury. SUMMARY: Background Using tissue factor pathway inhibitor (TFPI)-2 Kunitz domain1 (KD1), we obtained a bifunctional antifibrinolytic molecule (KD1L17R -KT ) with C-terminal lysine (kringle domain binding) and P2'-residue arginine (improved specificity towards plasmin). KD1L17R -KT strongly inhibited human plasmin (hPm), with no inhibition of human kallikrein (hKLK) or factor XIa (hXIa). Furthermore, KD1L17R -KT reduced blood loss comparable to aprotinin in a mouse liver-laceration model of organ hemorrhage. However, effectiveness of these antifibrinolytic agents in a model of hemorrhage mimicking extremity trauma and their inhibition efficiencies for mouse enzymes (mPm, mKLK or mXIa) remain to be determined. Objective To determine potential differences in inhibition constants of various antifibrinolytic agents against mouse and human enzymes and test their effectiveness in a modified mouse tail-amputation hemorrhage model. Methods/Results Unexpectedly, mXIa was inhibited with ~ 17-fold increased affinity by aprotinin (Ki ~ 20 nm) and with measurable affinity for KD1L17R -KT (Ki ~ 3 µm); in contrast, KD1WT -VT inhibited hXIa or mXIa with similar affinity. Compared with hPm, mPm had ~ 3-fold reduced affinity, whereas species specificity for hKLK and mKLK was comparable for each inhibitor. S2'-subsite variations largely accounted for the observed differences. KD1L17R -KT and aprotinin were more effective than KD1WT -VT or tranexamic acid in inhibiting tPA-induced mouse plasma clot lysis. Further, KD1L17R -KT was more effective than KD1WT -VT and was comparable to aprotinin and tranexamic acid in reducing blood loss and D-dimer levels in the mouse tail-amputation model. Conclusions Inhibitor potencies differ between antifibrinolytic agents against human and mouse enzymes. KD1L17R -KT is effective in reducing blood loss in a tail-amputation model that mimics extremity injury.


Assuntos
Fator XIa/genética , Fibrinolisina/genética , Glicoproteínas/química , Calicreínas/genética , Animais , Antifibrinolíticos , Aprotinina/química , Bovinos , Produtos de Degradação da Fibrina e do Fibrinogênio/química , Fibrinólise , Glicoproteínas/genética , Hemorragia , Humanos , Leucina/química , Fígado/metabolismo , Camundongos , Modelos Moleculares , Mutação , Peptídeo Hidrolases/química , Domínios Proteicos , Convulsões , Especificidade da Espécie , Ácido Tranexâmico/química , Tripsina/química
17.
J Chem Theory Comput ; 12(12): 6118-6129, 2016 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-27792332

RESUMO

Analysis of molecular dynamics, for example using Markov models, often requires the identification of order parameters that are good indicators of the rare events, i.e. good reaction coordinates. Recently, it has been shown that the time-lagged independent component analysis (TICA) finds the linear combinations of input coordinates that optimally represent the slow kinetic modes and may serve in order to define reaction coordinates between the metastable states of the molecular system. A limitation of the method is that both computing time and memory requirements scale with the square of the number of input features. For large protein systems, this exacerbates the use of extensive feature sets such as the distances between all pairs of residues or even heavy atoms. Here we derive a hierarchical TICA (hTICA) method that approximates the full TICA solution by a hierarchical, divide-and-conquer calculation. By using hTICA on distances between heavy atoms we identify previously unknown relaxation processes in the bovine pancreatic trypsin inhibitor.


Assuntos
Aprotinina/química , Animais , Aprotinina/metabolismo , Bovinos , Cinética , Cadeias de Markov , Simulação de Dinâmica Molecular , Peptídeos/química , Peptídeos/metabolismo , Análise de Componente Principal
18.
FEBS Lett ; 590(20): 3501-3509, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27685427

RESUMO

Biophysical understanding of amorphous protein aggregation can significantly impact diverse area of biotechnology. Here, we report the time dependent salt-induced formation of amorphous aggregation as monitored by fluorescence self-quenching and compare the results with conventional methods for detecting protein aggregation [static light scattering (LS) and dynamic light scattering (DLS)]. As a model protein, we used a bovine pancreatic trypsin inhibitor (BPTI) variant extended by two glycines (C2G) at its C terminus, and three variants where three types of Solubility Controlling Peptide tags (SCP tags) made of five serines (C5S), alanines (C5A) or aspartic acids (C5D) were added to the C terminus of C2G. All variants have a native-like BPTI structure and trypsin inhibitory activity, but different solubilities controlled by the SCP tags. The BPTIs were labeled using NHS-Fluorescein (FAM) conjugated to BPTI's lysines, and we measured the changes in fluorescence intensity occurring upon the addition of NaCl. The fluorescence of all FAM-BPTIs decreased almost immediately, albeit to a different extent, upon addition of salt and became constant after 10 min for 24 h or more. On the other hand, LS and DLS signal changes were dependent on the type of tags. Namely, C2G's LS and DLS signals changed immediately, the signals of C5S and C5A tagged FAM-BPTIs increased slowly from 10 min to 24 h, and those of C5D remained constant. These observations indicated the presence of at least one intermediate step, with increased protein-protein interaction yielding a 'molecular condensation' phase. According to this model, C2G would rapidly turn from 'condensates' to aggregates, whereas C5S and C5A tagged FAM-BPTIs would do so slowly, and the soluble C5D tagged variant would remain in the molecular condensation state.


Assuntos
Aminoácidos/química , Aprotinina/química , Aprotinina/genética , Fluoresceína/química , Cloreto de Sódio/farmacologia , Animais , Bovinos , Difusão Dinâmica da Luz , Fluorescência , Mutação , Conformação Proteica/efeitos dos fármacos , Solubilidade , Coloração e Rotulagem
19.
Talanta ; 160: 761-767, 2016 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-27591673

RESUMO

Hydrophobins are one of the most active surface active proteins in nature, with an amphiphilic nature and the ability to self-assembly in elastic monolayers, the possible applications in industry are continuously increasing. However, production and purification of these proteins still remains a tedious process. We introduce here the use of polydopamine as imprinter polymer to create specific magnetic nanoparticles for the recognition of Hydrophobin HFBII from Trichoderma reesei. The protein was molecularly imprinted to magnetic nanoparticles to facilitate its specific detection and purification from liquids or carbonated beverages in the presence of other proteins. The resulting magnetic nanoparticles were successfully imprinted adsorbing till 77,4µg of HFBII hydrophobin per miligram of nanoparticles. The adsorption capacity of the imprinted nanoparticles was also tested for specificity using a mixture of five different proteins and peptides. A slight cross interaction was observed when proteins of similar molecular weight to HFBII were used. With larger proteins and peptides the interaction was very low. with other class II Hydrophobins the interaction was very similar as to HFBII.


Assuntos
Proteínas Fúngicas/química , Indóis/química , Nanopartículas de Magnetita/química , Polímeros/química , Trichoderma , Adsorção , Angiotensina II/química , Aprotinina/química , Proteínas de Transporte/química , Insulina/química , Lactoglobulinas/química , Impressão Molecular , Soroalbumina Bovina/química
20.
J Chem Theory Comput ; 12(10): 4947-4958, 2016 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-27631425

RESUMO

The graphics processing unit (GPU) has become a popular computational platform for molecular dynamics (MD) simulations of biomolecules. A significant speedup in the simulations of small- or medium-size systems using only a few computer nodes with a single or multiple GPUs has been reported. Because of GPU memory limitation and slow communication between GPUs on different computer nodes, it is not straightforward to accelerate MD simulations of large biological systems that contain a few million or more atoms on massively parallel supercomputers with GPUs. In this study, we develop a new scheme in our MD software, GENESIS, to reduce the total computational time on such computers. Computationally intensive real-space nonbonded interactions are computed mainly on GPUs in the scheme, while less intensive bonded interactions and communication-intensive reciprocal-space interactions are performed on CPUs. On the basis of the midpoint cell method as a domain decomposition scheme, we invent the single particle interaction list for reducing the GPU memory usage. Since total computational time is limited by the reciprocal-space computation, we utilize the RESPA multiple time-step integration and reduce the CPU resting time by assigning a subset of nonbonded interactions on CPUs as well as on GPUs when the reciprocal-space computation is skipped. We validated our GPU implementations in GENESIS on BPTI and a membrane protein, porin, by MD simulations and an alanine-tripeptide by REMD simulations. Benchmark calculations on TSUBAME supercomputer showed that an MD simulation of a million atoms system was scalable up to 256 computer nodes with GPUs.


Assuntos
Simulação de Dinâmica Molecular , Oligopeptídeos/química , Alanina/química , Algoritmos , Animais , Aprotinina/química , Aprotinina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bovinos , Mycobacterium smegmatis/metabolismo , Porinas/química , Porinas/metabolismo , Termodinâmica
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