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1.
Structure ; 28(3): 324-335.e4, 2020 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-31864811

RESUMO

The family of cystathionine-ß-synthase (CBS)-pair domain divalent metal cation transport mediators (CNNMs) is composed of four integral membrane proteins associated with Mg2+ transport. Structurally, CNNMs contain large cytosolic regions composed of a CBS-pair and a cyclic nucleotide-binding homology (CNBH) domain. How these regulate Mg2+ transport activity is unknown. Here, we determined the crystal structures of cytosolic fragments in two conformations: Mg2+-ATP-analog bound and ligand free. The structures reveal open and closed conformations with functionally important contacts not observed in structures of the individual domains. We also identified a second Mg2+-binding region in the CBS-pair domain and a different dimerization interface for the CNBH domain. Analytical ultracentrifugation and isothermal titration calorimetry experiments revealed a tight correlation between Mg2+-ATP binding and protein dimerization. Mutations that blocked either function prevented cellular Mg2+ efflux activity. The results suggest Mg2+ efflux is regulated by conformational changes associated with Mg2+-ATP binding to CNNM CBS-pair domains.

2.
Sci Rep ; 9(1): 16023, 2019 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-31690758

RESUMO

The ability of high-resolution NMR spectroscopy to readout the response of molecular interactions at multiple atomic sites presents a unique capability to define thermodynamic equilibrium constants and kinetic rate constants for complex, multiple-step biological interactions. Nonetheless, the extraction of the relevant equilibrium binding and rate constants requires the appropriate analysis of not only a readout that follows the equilibrium concentrations of typical binding titration curves, but also the lineshapes of NMR spectra. To best take advantage of NMR data for characterizing molecular interactions, we developed NmrLineGuru, a software tool with a user-friendly graphical user interface (GUI) to model two-state, three-state, and four-state binding processes. Application of NmrLineGuru is through stand-alone GUIs, with no dependency on other software and no scripted input. NMR spectra can be fitted or simulated starting with user-specified input parameters and a chosen kinetic model. The ability to both simulate and fit NMR spectra provides the user the opportunity to not only determine the binding parameters that best reproduce the measured NMR spectra for the selected kinetic model, but to also query the possibility that alternative models agree with the data. NmrLineGuru is shown to provide an accurate, quantitative analysis of complex molecular interactions.

3.
Nat Chem Biol ; 14(8): 811-820, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29988068

RESUMO

Chaperones TAPBPR and tapasin associate with class I major histocompatibility complexes (MHC-I) to promote optimization (editing) of peptide cargo. Here, we use solution NMR to investigate the mechanism of peptide exchange. We identify TAPBPR-induced conformational changes on conserved MHC-I molecular surfaces, consistent with our independently determined X-ray structure of the complex. Dynamics present in the empty MHC-I are stabilized by TAPBPR and become progressively dampened with increasing peptide occupancy. Incoming peptides are recognized according to the global stability of the final pMHC-I product and anneal in a native-like conformation to be edited by TAPBPR. Our results demonstrate an inverse relationship between MHC-I peptide occupancy and TAPBPR binding affinity, wherein the lifetime and structural features of transiently bound peptides control the regulation of a conformational switch located near the TAPBPR binding site, which triggers TAPBPR release. These results suggest a similar mechanism for the function of tapasin in the peptide-loading complex.


Assuntos
Regulação Alostérica , Antígenos de Histocompatibilidade Classe I/metabolismo , Imunoglobulinas/metabolismo , Proteínas de Membrana/metabolismo , Peptídeos/metabolismo , Antígenos de Histocompatibilidade Classe I/química , Humanos , Imunoglobulinas/química , Proteínas de Membrana/química , Peptídeos/química , Conformação Proteica
4.
Biochemistry ; 57(5): 872-881, 2018 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-29280621

RESUMO

Cellular membranes are heterogeneous planar lipid bilayers displaying lateral phase separation with the nanometer-scale liquid-ordered phase (also known as "lipid rafts") surrounded by the liquid-disordered phase. Many membrane-associated proteins were found to permanently integrate into the lipid rafts, which is critical for their biological function. Isoforms H and N of Ras GTPase possess a unique ability to switch their lipid domain preference depending on the type of bound guanine nucleotide (GDP or GTP). This behavior, however, has never been demonstrated in vitro in model bilayers with recombinant proteins and therefore has been attributed to the action of binding of Ras to other proteins at the membrane surface. In this paper, we report the observation of the nucleotide-dependent switch of lipid domain preferences of the semisynthetic lipidated N-Ras in lipid raft vesicles in the absence of additional proteins. To detect segregation of Ras molecules in raft and disordered lipid domains, we measured Förster resonance energy transfer between the donor fluorophore, mant, attached to the protein-bound guanine nucleotides, and the acceptor, rhodamine-conjugated lipid, localized into the liquid-disordered domains. Herein, we established that N-Ras preferentially populated raft domains when bound to mant-GDP, while losing its preference for rafts when it was associated with a GTP mimic, mant-GppNHp. At the same time, the isolated lipidated C-terminal peptide of N-Ras was found to be localized outside of the liquid-ordered rafts, most likely in the bulk-disordered lipid. Substitution of the N-terminal G domain of N-Ras with a homologous G domain of H-Ras disrupted the nucleotide-dependent lipid domain switch.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Lipopeptídeos/análise , Microdomínios da Membrana/química , Proteínas Proto-Oncogênicas p21(ras)/análise , Corantes Fluorescentes/análise , Guanosina Difosfato/metabolismo , Humanos , Nanoestruturas , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Lipossomas Unilamelares/química , ortoaminobenzoatos/metabolismo
5.
J Biomol NMR ; 70(1): 21-31, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29168021

RESUMO

NMR spectroscopy of membrane proteins involved in electron transport is difficult due to the presence of both the lipids and paramagnetic centers. Here we report the solution NMR study of the NADPH-cytochrome P450 oxidoreductase (POR) in its reduced and oxidized states. We interrogate POR, first, in its truncated soluble form (70 kDa), which is followed by experiments with the full-length protein incorporated in a lipid nanodisc (240 kDa). To overcome paramagnetic relaxation in the reduced state of POR as well as the signal broadening due to its high molecular weight, we utilized the methyl-TROSY approach. Extrinsic 13C-methyl groups were introduced by modifying the engineered surface-exposed cysteines with methyl-methanethiosulfonate. Chemical shift dispersion of the resonances from different sites in POR was sufficient to monitor differential effects of the reduction-oxidation process and conformation changes in the POR structure related to its function. Despite the high molecular weight of the POR-nanodisc complex, the surface-localized 13C-methyl probes were sufficiently mobile to allow for signal detection at 600 MHz without perdeuteration. This work demonstrates a potential of the solution methyl-TROSY in analysis of structure, dynamics, and function of POR, which may also be applicable to similar paramagnetic and flexible membrane proteins.


Assuntos
Proteínas de Membrana/química , NADPH-Ferri-Hemoproteína Redutase/química , Isótopos de Carbono , Lipídeos , Proteínas de Membrana/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Oxirredução , Ligação Proteica , Conformação Proteica , Solubilidade , Relação Estrutura-Atividade
6.
J Biomol NMR ; 67(4): 309-319, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28393280

RESUMO

Chitosan interaction with chitosanase was examined through analysis of spectral line shapes in the NMR HSQC titration experiments. We established that the substrate, chitosan hexamer, binds to the enzyme through the three-state induced-fit mechanism with fast formation of the encounter complex followed by slow isomerization of the bound-state into the final conformation. Mapping of the chemical shift perturbations in two sequential steps of the mechanism highlighted involvement of the substrate-binding subsites and the hinge region in the binding reaction. Equilibrium parameters of the three-state model agreed with the overall thermodynamic dissociation constant determined by ITC. This study presented the first kinetic evidence of the induced-fit mechanism in the glycoside hydrolases.


Assuntos
Glicosídeo Hidrolases/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Quitosana/química , Quitosana/metabolismo , Glicosídeo Hidrolases/química , Cinética , Ligantes , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína
7.
Biochemistry ; 55(43): 5973-5976, 2016 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-27741572

RESUMO

NADPH-cytochrome P450 oxidoreductase (CYPOR) was shown to undergo large conformational rearrangements in its functional cycle. Using a new Förster resonance energy transfer (FRET) approach based on femtosecond transient absorption spectroscopy (TA), we determined the donor-acceptor distance distribution in the reduced and oxidized states of CYPOR. The unmatched time resolution of TA allowed the quantitative assessment of the donor-acceptor FRET, indicating that CYPOR assumes a closed conformation in both reduced and oxidized states in the absence of the redox partner. The described ultrafast TA measurements of FRET with readily available red-infrared fluorescent labels open new opportunities for structural studies in chromophore-rich proteins and their complexes.


Assuntos
NADPH-Ferri-Hemoproteína Redutase/química , Cristalografia por Raios X , Transferência Ressonante de Energia de Fluorescência , Conformação Proteica
8.
J Fluoresc ; 26(2): 379-83, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26662810

RESUMO

Supported phospholipid bilayers are a convenient model of cellular membranes in studies of membrane biophysics and protein-lipid interactions. Traditionally, supported lipid bilayers are formed on a flat surface of a glass slide to be observed through fluorescence microscopes. This paper describes a method to enable fluorescence detection from the supported lipid bilayers using standard horizontal-beam spectrofluorometers instead of the microscopes. In the proposed approach, the supported lipid bilayers are formed on the inner optical surfaces of the standard fluorescence microcell. To enable observation of the bilayer absorbed on the cell wall, the microcell is placed in a standard fluorometer cell holder and specifically oriented to expose the inner cell walls to both excitation and emission channels with a help of the custom cell adaptor. The signal intensity from supported bilayers doped with 1 % (mol) of rhodamine-labeled lipid in the standard 3-mm optical microcell was equivalent to fluorescence of the 70-80 nM reference solution of rhodamine recorded in a commercial microcell adaptor. Because no modifications to the instruments are required in this method, a variety of steady-state and time-domain fluorescence measurements of the supported phospholipid bilayers may be performed with the spectral resolution using standard horizontal-beam spectrofluorometers.


Assuntos
Bicamadas Lipídicas/química , Fosfolipídeos/análise , Rodaminas/química , Espectrometria de Fluorescência/métodos , Fluorescência
9.
Biophys J ; 109(5): 1000-8, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26331257

RESUMO

Ras GTPase is a molecular switch controlling a number of cellular pathways including growth, proliferation, differentiation, and apoptosis. Recent reports indicated that Ras undergoes dimerization at the membrane surface through protein-protein interactions. If firmly established this property of Ras would require profound reassessment of a large amount of published data and modification of the Ras signaling paradigm. One proposed mechanism of dimerization involves formation of salt bridges between the two GTPase domains (G domains) leading to formation of a compact dimer as observed in Ras crystal structures. In this work, we interrogated the intrinsic ability of Ras to self-associate in solution by creating conditions of high local concentration through irreversibly tethering the two G domains together at their unstructured C-terminal tails. We evaluated possible self-association in this inverted tandem conjugate via analysis of the time-domain fluorescence anisotropy and NMR chemical shift perturbations. We did not observe the increased rotational correlation time expected for the G domain dimer. Variation of the ionic strength (to modulate stability of the salt bridges) did not affect the rotational correlation time in the tandem further supporting independent rotational diffusion of two G domains. In a parallel line of experiments to detect and map weak self-association of the G domains, we analyzed NMR chemical shifts perturbations at a number of sites near the crystallographic dimer interface. The nearly complete lack of chemical shift perturbations in the tandem construct supported a simple model with the independent G domains repelled from each other by their overall negative charge. These results lead us to the conclusion that self-association of the G domains cannot be responsible for homodimerization of Ras reported in the literature.


Assuntos
Multimerização Proteica , Proteínas ras/química , Difusão , Modelos Moleculares , Estrutura Terciária de Proteína
10.
PLoS One ; 9(7): e101227, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24984078

RESUMO

The Fluorescence2D is free software that allows analysis of two-dimensional fluorescence spectra obtained using the accelerated "triangular" acquisition schemes. The software is a combination of Python and MATLAB-based programs that perform conversion of the triangular data, display of the two-dimensional spectra, extraction of 1D slices at different wavelengths, and output in various graphic formats.


Assuntos
Software , Espectrometria de Fluorescência/métodos , Humanos , Proteínas Proto-Oncogênicas p21(ras)/química
11.
J Biol Chem ; 289(26): 18110-20, 2014 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-24817117

RESUMO

N-acetyl-d-quinovosamine (2-acetamido-2,6-dideoxy-d-glucose, QuiNAc) occurs in the polysaccharide structures of many Gram-negative bacteria. In the biosynthesis of QuiNAc-containing polysaccharides, UDP-QuiNAc is the hypothetical donor of the QuiNAc residue. Biosynthesis of UDP-QuiNAc has been proposed to occur by 4,6-dehydration of UDP-N-acetyl-d-glucosamine (UDP-GlcNAc) to UDP-2-acetamido-2,6-dideoxy-d-xylo-4-hexulose followed by reduction of this 4-keto intermediate to UDP-QuiNAc. Several specific dehydratases are known to catalyze the first proposed step. A specific reductase for the last step has not been demonstrated in vitro, but previous mutant analysis suggested that Rhizobium etli gene wreQ might encode this reductase. Therefore, this gene was cloned and expressed in Escherichia coli, and the resulting His6-tagged WreQ protein was purified. It was tested for 4-reductase activity by adding it and NAD(P)H to reaction mixtures in which 4,6-dehydratase WbpM had acted on the precursor substrate UDP-GlcNAc. Thin layer chromatography of the nucleotide sugars in the mixture at various stages of the reaction showed that WbpM converted UDP-GlcNAc completely to what was shown to be its 4-keto-6-deoxy derivative by NMR and that addition of WreQ and NADH led to formation of a third compound. Combined gas chromatography-mass spectrometry analysis of acid hydrolysates of the final reaction mixture showed that a quinovosamine moiety had been synthesized after WreQ addition. The two-step reaction progress also was monitored in real time by NMR. The final UDP-sugar product after WreQ addition was purified and determined to be UDP-d-QuiNAc by one-dimensional and two-dimensional NMR experiments. These results confirmed that WreQ has UDP-2-acetamido-2,6-dideoxy-d-xylo-4-hexulose 4-reductase activity, completing a pathway for UDP-d-QuiNAc synthesis in vitro.


Assuntos
Acetilglucosamina/química , Proteínas de Bactérias/química , Oxirredutases/química , Rhizobium etli/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Biocatálise , Oxirredutases/genética , Oxirredutases/isolamento & purificação , Oxirredutases/metabolismo , Rhizobium etli/genética
12.
J Immunol ; 192(6): 2885-91, 2014 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-24523505

RESUMO

Although conformational changes in TCRs and peptide Ags presented by MHC protein (pMHC) molecules often occur upon binding, their relationship to intrinsic flexibility and role in ligand selectivity are poorly understood. In this study, we used nuclear magnetic resonance to study TCR-pMHC binding, examining recognition of the QL9/H-2L(d) complex by the 2C TCR. Although the majority of the CDR loops of the 2C TCR rigidify upon binding, the CDR3ß loop remains mobile within the TCR-pMHC interface. Remarkably, the region of the QL9 peptide that interfaces with CDR3ß is also mobile in the free pMHC and in the TCR-pMHC complex. Determination of conformational exchange kinetics revealed that the motions of CDR3ß and QL9 are closely matched. The matching of conformational exchange in the free proteins and its persistence in the complex enhances the thermodynamic and kinetic stability of the TCR-pMHC complex and provides a mechanism for facile binding. We thus propose that matching of structural fluctuations is a component of how TCRs scan among potential ligands for those that can bind with sufficient stability to enable T cell signaling.


Assuntos
Regiões Determinantes de Complementaridade/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Oligopeptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/metabolismo , Cinética , Ligantes , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Conformação Molecular , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Ligação Proteica/imunologia , Conformação Proteica , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/metabolismo
13.
J Am Chem Soc ; 135(25): 9377-83, 2013 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-23705660

RESUMO

Proteins exist in a delicate balance between the native and unfolded states, where thermodynamic stability may be sacrificed to attain the flexibility required for efficient catalysis, binding, or allosteric control. Partition-defective 6 (Par-6) regulates the Par polarity complex by transmitting a GTPase signal through the Cdc42/Rac interaction binding PSD-95/Dlg/ZO-1 (CRIB-PDZ) module that alters PDZ ligand binding. Allosteric activation of the PDZ is achieved by local rearrangement of the L164 and K165 side chains to stabilize the interdomain CRIB:PDZ interface and reposition a conserved element of the ligand binding pocket. However, microsecond to millisecond dynamics measurements revealed that L164/K165 exchange requires a larger rearrangement than expected. The margin of thermodynamic stability for the PDZ domain is modest (∼3 kcal/mol) and further reduced by transient interactions with the disordered CRIB domain. Measurements of local structural stability revealed that tertiary contacts within the PDZ are disrupted by a partial unfolding transition that enables interconversion of the L/K switch. The unexpected participation of partial PDZ unfolding in the allosteric mechanism of Par-6 suggests that native-state unfolding may be essential for the function of other marginally stable proteins.


Assuntos
Proteínas de Drosophila/química , Domínios PDZ , Proteína Quinase C/química , Regulação Alostérica , Desdobramento de Proteína , Termodinâmica
15.
J Biol Chem ; 287(53): 44289-300, 2012 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-23135272

RESUMO

Conformational flexibility between structural ensembles is an essential component of enzyme function. Although the broad dynamical landscape of proteins is known to promote a number of functional events on multiple time scales, it is yet unknown whether structural and functional enzyme homologues rely on the same concerted residue motions to perform their catalytic function. It is hypothesized that networks of contiguous and flexible residue motions occurring on the biologically relevant millisecond time scale evolved to promote and/or preserve optimal enzyme catalysis. In this study, we use a combination of NMR relaxation dispersion, model-free analysis, and ligand titration experiments to successfully capture and compare the role of conformational flexibility between two structural homologues of the pancreatic ribonuclease family: RNase A and eosinophil cationic protein (or RNase 3). In addition to conserving the same catalytic residues and structural fold, both homologues show similar yet functionally distinct clusters of millisecond dynamics, suggesting that conformational flexibility can be conserved among analogous protein folds displaying low sequence identity. Our work shows that the reduced conformational flexibility of eosinophil cationic protein can be dynamically and functionally reproduced in the RNase A scaffold upon creation of a chimeric hybrid between the two proteins. These results support the hypothesis that conformational flexibility is partly required for catalytic function in homologous enzyme folds, further highlighting the importance of dynamic residue sectors in the structural organization of proteins.


Assuntos
Proteína Catiônica de Eosinófilo/química , Proteína Catiônica de Eosinófilo/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Ribonuclease Pancreático/química , Ribonuclease Pancreático/metabolismo , Monofosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteína Catiônica de Eosinófilo/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Ribonuclease Pancreático/genética
16.
Biochemistry ; 51(48): 9638-46, 2012 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-23148511

RESUMO

Ras is a small monomeric GTPase acting as molecular switch in multiple cellular processes. The N-terminal G domain of Ras binds GTP or GDP accompanied by a magnesium ion, which is strictly required for GTPase activity and performs a structural role. Another ion-binding site on the opposite face of the G domain has been recently observed to specifically associate with calcium acetate in the crystal [Buhrman, G., et al. (2010) Proc. Natl. Aacd. Sci. U.S.A. 107, 4931-4936]. In this article, we report thermodynamic measurements of the affinity and specificity of the remote ion-binding site in H-Ras as observed in solution. Using (15)N-(1)H nuclear magnetic resonance spectroscopy, we determined that, in contrast to the crystalline state, the remote site in solution is specific for a divalent cation, binding both calcium and magnesium with anions playing a minimal role. The affinity of the remote site for divalent cations is in the low millimolar range and remarkably different for GDP- and GppNHp-bound forms of the G domain, indicating that the GTP-binding pocket and the remote site are allosterically coupled through the distance of more than 25 Å. Considering that the remote site is oriented toward the membrane surface in vivo, we hypothesize that its cognate biological ligand might be a positively charged group extending from a lipid or an integral membrane protein.


Assuntos
Proteína Oncogênica p21(ras)/metabolismo , Sítios de Ligação , Cátions Bivalentes , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Proteína Oncogênica p21(ras)/química , Conformação Proteica , Termodinâmica
17.
Biochemistry ; 51(43): 8583-96, 2012 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-23025283

RESUMO

Peptidyl prolyl cis-trans isomerization acts as an effective molecular timer that plays significant roles in biological and pathological processes. Enzymes such as Pin1 catalyze cis-trans isomerization, accelerating the otherwise slow isomerization rate into time scales relevant for cellular signaling. Here we have combined NMR line shape analysis, fluorescence spectroscopy, and isothermal titration calorimetry to determine the kinetic and thermodynamic parameters describing the trans-specific interaction between the binding domain of Pin1 (WW domain) and a key cis-trans molecular switch in the amyloid precursor protein cytoplasmic tail. A three-state model, in which the cis-trans isomerization equilibrium is coupled to the binding equilibrium through the trans isomer, was found to fit the data well. The trans isomer binds the WW domain with ∼22 µM affinity via very fast association (approaching the diffusion limit) and dissociation rates. The common structural and electrostatic characteristics of Pin1 substrates, which contain a phosphorylated serine/threonine-proline motif, suggest that very rapid binding kinetics are a general feature of Pin1 interactions with other substrates. The fast binding kinetics of the WW domain allows rapid response of Pin1 to the dynamic events of phosphorylation and dephosphorylation in the cell that alter the relative populations of diverse Pin1 substrates. Furthermore, our results also highlight the vastly different rates at which slow uncatalyzed cis-trans isomerization and fast isomer-specific binding events occur. These results, along with the experimental methods presented herein, should guide future experiments aimed at the thermodynamic and kinetic characterization of cis-trans molecular switches and isomer-specific interactions involved in various biological processes.


Assuntos
Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/metabolismo , Sítios de Ligação , Humanos , Isomerismo , Cinética , Modelos Moleculares , Peptidilprolil Isomerase de Interação com NIMA , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Termodinâmica , Triptofano/química , Triptofano/metabolismo
18.
J Biomol NMR ; 53(3): 257-70, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22610542

RESUMO

Biological function of proteins relies on conformational transitions and binding of specific ligands. Protein-ligand interactions are thermodynamically and kinetically coupled to conformational changes in protein structures as conceptualized by the models of pre-existing equilibria and induced fit. NMR spectroscopy is particularly sensitive to complex ligand-binding modes-NMR line-shape analysis can provide for thermodynamic and kinetic constants of ligand-binding equilibria with the site-specific resolution. However, broad use of line shape analysis is hampered by complexity of NMR line shapes in multi-state systems. To facilitate interpretation of such spectral patterns, I computationally explored systems where isomerization or dimerization of a protein (receptor) molecule is coupled to binding of a ligand. Through an extensive analysis of multiple exchange regimes for a family of three-state models, I identified signature features to guide an NMR experimentalist in recognizing specific interaction mechanisms. Results show that distinct multi-state models may produce very similar spectral patterns. I also discussed aggregation of a receptor as a possible source of spurious three-state line shapes and provided specific suggestions for complementary experiments that can ensure reliable mechanistic insight.


Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Proteínas/metabolismo , Software , Cinética , Ligantes , Modelos Moleculares , Ligação Proteica , Termodinâmica
19.
Biomol NMR Assign ; 6(1): 91-3, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21814767

RESUMO

The small GTPase Ras is an important signaling molecule acting as a molecular switch in eukaryotic cells. Recent findings of global conformational exchange and a putative allosteric binding site in the G domain of Ras opened an avenue to understanding novel aspects of Ras function. To facilitate detailed NMR studies of Ras in physiological solution conditions, we performed backbone resonance assignments of Ras bound to slowly hydrolysable GTP mimic, guanosine 5'-[ß, γ-imido]triphosphate at pH 7.2. Out of 163 non-proline residues of the G domain, signals from backbone amide proton, nitrogen and carbon spins of 127 residues were confidently assigned with the remaining unassigned residues mostly located at the exchange-broadened effectors interface.


Assuntos
Guanilil Imidodifosfato/metabolismo , Ressonância Magnética Nuclear Biomolecular , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Concentração de Íons de Hidrogênio , Ligação Proteica , Estrutura Terciária de Proteína
20.
J Biomol NMR ; 51(1-2): 21-34, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21947912

RESUMO

The phosphorylation-specific peptidyl-prolyl isomerase Pin1 catalyzes the isomerization of the peptide bond preceding a proline residue between cis and trans isomers. To best understand the mechanisms of Pin1 regulation, rigorous enzymatic assays of isomerization are required. However, most measures of isomerase activity require significant constraints on substrate sequence and only yield rate constants for the cis isomer, [Formula: see text] and apparent Michaelis constants, [Formula: see text]. By contrast, NMR lineshape analysis is a powerful tool for determining microscopic rates and populations of each state in a complex binding scheme. The isolated catalytic domain of Pin1 was employed as a first step towards elucidating the reaction scheme of the full-length enzyme. A 24-residue phosphopeptide derived from the amyloid precurser protein intracellular domain (AICD) phosphorylated at Thr668 served as a biologically-relevant Pin1 substrate. Specific (13)C labeling at the Pin1-targeted proline residue provided multiple reporters sensitive to individual isomer binding and on-enzyme catalysis. We have performed titration experiments and employed lineshape analysis of phosphopeptide (13)C-(1)H constant time HSQC spectra to determine [Formula: see text], [Formula: see text], [Formula: see text], and [Formula: see text] for the catalytic domain of Pin1 acting on this AICD substrate. The on-enzyme equilibrium value of [E·trans]/[E·cis] = 3.9 suggests that the catalytic domain of Pin1 is optimized to operate on this substrate near equilibrium in the cellular context. This highlights the power of lineshape analysis for determining the microscopic parameters of enzyme catalysis, and demonstrates the feasibility of future studies of Pin1-PPIase mutants to gain insights on the catalytic mechanism of this important enzyme.


Assuntos
Peptidilprolil Isomerase/química , Sítios de Ligação , Biocatálise , Domínio Catalítico , Cinética , Peptidilprolil Isomerase de Interação com NIMA , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Termodinâmica
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