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1.
Biomol NMR Assign ; 16(2): 391-398, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36083574

RESUMO

Monoclonal antibodies (mAbs) therapeutics are the largest and fastest growing class of biologic drugs, amongst which, the vast majority are immunoglobulin G1 (IgG1). Their antigen binding abilities are used for the treatment of immunologic diseases, cancer therapy, reversal of drug effects, and targeting viruses and bacteria. The high importance of therapeutic mAbs and their derivatives has called for the generation of well-characterized standards for method development and calibration. One such standard, the NISTmAb RM 8621 based on the antibody motavizumab, has been developed by the National Institute of Standards and Technologies (NIST) in the US. Here, we present the resonance assignment of the single chain variable fragment, NISTmAb-scFv, that was engineered by linking the variable domains of the heavy and light chains of the NISTmAb. Also, addition of a peptide, corresponding to the target antigen of motavizumab, to samples of NISTmAb-scFv has induced chemical shift perturbations on residues lining the antigen binding interface thereby indicating proper folding of the NISTmAb-scFv.


Assuntos
Produtos Biológicos , Anticorpos de Cadeia Única , Anticorpos Monoclonais/química , Imunoglobulina G/química , Ressonância Magnética Nuclear Biomolecular
2.
ACS Omega ; 5(49): 31845-31857, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-33344838

RESUMO

Product excipients are used to confer a number of desirable properties on the drug substance to maintain or improve stability and facilitate drug delivery. This is especially important for products where the active pharmaceutical ingredient (API) is a recombinant protein. In this study, we aimed to determine if excipients and formulation conditions affect the structure and/or modulate the dynamics of the protein API of filgrastim products. Samples of uniformly labeled 15N-Met-granulocyte-colony stimulating factor (GCSF) were prepared at 100 µM (near formulation concentration) with various concentrations of individual components (polysorbate-20 and -80, sorbitol) and three pH values. Nuclear magnetic resonance (NMR) spectroscopy techniques were applied to measure chemical shift perturbation (CSP) to detect structural changes, and relaxation parameters (T 1, T 2, and heteronuclear Overhauser effect) were measured to probe the effects on protein backbone motions. In parallel, the same solution conditions were subjected to protein thermal unfolding studies monitored by circular dichroism spectropolarimetry (CD). Detergents (polysorbate-20 and 80) do not induce any observable changes on the protein structure and do not modify its dynamics at formulation concentration. Lowering pH to 4.0, a condition known to stabilize the conformation of filgrastim, as well as the addition of sorbitol produced changes of the fast motion dynamics in the nanosecond and picosecond timescale. NMR-derived order parameters, which measure the local conformational entropy of the protein backbone, show that lowering pH leads to a compaction of the four-helix bundle while the addition of sorbitol relaxes helices B and C, thereby reducing the mobility of loop CD. CSPs and measurements of protein dynamics via NMR-derived order parameters provide a description in structural and motional terms at an atomic resolution on how formulation components contribute to the stabilization of filgrastim products.

3.
Anal Chem ; 92(9): 6366-6373, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32267681

RESUMO

The use of NMR spectroscopy has emerged as a premier tool to characterize the higher order structure of protein therapeutics and in particular IgG1 monoclonal antibodies (mAbs). Due to their large size, traditional 1H-15N correlation experiments have proven exceedingly difficult to implement on mAbs, and a number of alternative techniques have been proposed, including the one-dimensional (1D) 1H protein fingerprint by line shape enhancement (PROFILE) method and the two-dimensional (2D) 1H-13C methyl correlation-based approach. Both 1D and 2D approaches have relative strengths and weaknesses, related to the inherent sensitivity and resolution of the respective methods. To further increase the utility of NMR to the biopharmaceutical community, harmonized criteria for decision making in employing 1D and 2D approaches for mAb characterization are warranted. To this end, we have conducted an interlaboratory comparative study of the 1D PROFILE and 2D methyl methods on several mAbs samples to determine the degree to which each method is suited to detect spectral difference between the samples and the degree to which results from each correlate with one another. Results from the study demonstrate both methods provide statistical data highly comparable to one another and that each method is capable of complementing the limitations commonly associated with the other, thus providing a better overall picture of higher order structure.


Assuntos
Imunoglobulina G/análise , Ressonância Magnética Nuclear Biomolecular , Isótopos de Carbono , Prótons
4.
J Pharm Biomed Anal ; 163: 144-152, 2019 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-30296716

RESUMO

The advent of monoclonal antibody biosimilar products has stimulated the development of analytical methods that can better characterize an important quality attribute, namely the higher order structure (HOS). Here, we propose a simple approach based on heteronuclear 2D NMR techniques at natural abundance for generating spectral fingerprints of the HOS at high resolution. We show that the proposed method can assess the HOS of six therapeutic products, adalimumab (Humira®), bevacizumab (Avastin®), infliximab (Remicade®), rituximab (Rituxan®), trastuzumab (Herceptin®), and Etanercept (Enbrel®). After treatment with immobilized papain, the purified fragments (Fab and Fc) were analyzed by 2D proton-nitrogen and proton-carbon NMR correlations. All Fab and Fc fragments produced high-resolution 2D-NMR spectra from which assessment of their higher order structure can be performed in the context of comparability studies. In particular, the two different sequences of Fc fragments could be unambiguously distinguished. The results show that it is possible to obtain structurally dependent information at amino acid resolution of these important therapeutic agents.


Assuntos
Medicamentos Biossimilares/química , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/química , Ressonância Magnética Nuclear Biomolecular/métodos , Adalimumab/química , Bevacizumab/química , Etanercepte/química , Estudos de Viabilidade , Infliximab/química , Ressonância Magnética Nuclear Biomolecular/instrumentação , Papaína/química , Estrutura Secundária de Proteína , Rituximab/química , Trastuzumab/química
5.
MAbs ; 11(1): 94-105, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30570405

RESUMO

The increased interest in using monoclonal antibodies (mAbs) as a platform for biopharmaceuticals has led to the need for new analytical techniques that can precisely assess physicochemical properties of these large and very complex drugs for the purpose of correctly identifying quality attributes (QA). One QA, higher order structure (HOS), is unique to biopharmaceuticals and essential for establishing consistency in biopharmaceutical manufacturing, detecting process-related variations from manufacturing changes and establishing comparability between biologic products. To address this measurement challenge, two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) methods were introduced that allow for the precise atomic-level comparison of the HOS between two proteins, including mAbs. Here, an inter-laboratory comparison involving 26 industrial, government and academic laboratories worldwide was performed as a benchmark using the NISTmAb, from the National Institute of Standards and Technology (NIST), to facilitate the translation of the 2D-NMR method into routine use for biopharmaceutical product development. Two-dimensional 1H,15N and 1H,13C NMR spectra were acquired with harmonized experimental protocols on the unlabeled Fab domain and a uniformly enriched-15N, 20%-13C-enriched system suitability sample derived from the NISTmAb. Chemometric analyses from over 400 spectral maps acquired on 39 different NMR spectrometers ranging from 500 MHz to 900 MHz demonstrate spectral fingerprints that are fit-for-purpose for the assessment of HOS. The 2D-NMR method is shown to provide the measurement reliability needed to move the technique from an emerging technology to a harmonized, routine measurement that can be generally applied with great confidence to high precision assessments of the HOS of mAb-based biotherapeutics.


Assuntos
Anticorpos Monoclonais/química , Biofarmácia/normas , Laboratórios/normas , Espectroscopia de Ressonância Magnética/métodos , Humanos , Reprodutibilidade dos Testes
6.
J Pharm Biomed Anal ; 141: 229-233, 2017 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-28454057

RESUMO

The higher order structure (HOS) of biotherapeutics is a critical quality attribute that can be evaluated by nuclear magnetic resonance (NMR) spectroscopy at atomic resolution. NMR spectral mapping of HOS can be used to establish HOS consistency of a biologic across manufacturing changes or to compare a biosimilar to an innovator reference product. A previous inter-laboratory study performed using filgrastim drug products demonstrated that two-dimensional (2D)-NMR 1HN-15NH heteronuclear correlation spectroscopy is a highly robust and precise method for mapping the HOS of biologic drugs at natural abundance using high sensitivity NMR 'cold probes.' Here, the applicability of the 2D-NMR method to fingerprint the HOS of filgrastim products is demonstrated using lower sensitivity, room temperature NMR probes. Combined chemical shift deviation and principal component analysis are used to illustrate the performance and inter-laboratory precision of the 2D-NMR method when implemented on room temperature probes.


Assuntos
Espectroscopia de Ressonância Magnética , Filgrastim , Temperatura
8.
Anal Biochem ; 490: 52-4, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26278168

RESUMO

N-sulfonated oversulfated chondroitin sulfate (NS-OSCS), recently reported as a potential threat to the heparin supply, was prepared along with its intermediate derivatives. All compounds were spiked into marketplace heparin and subjected to United States Pharmacopeia (USP) identification assays for heparin (proton nuclear magnetic resonance [(1)H NMR], chromatographic identity, % galactosamine [%GalN], anti-factor IIa potency, and anti-factor Xa/IIa ratio). The U.S. Food and Drug Administration (FDA) strong-anionic exchange high-performance liquid chromatography (SAX-HPLC) method resolved NS-OSCS from heparin and OSCS and had a limit of detection of 0.26% (w/w) NS-OSCS. The %GalN test was sensitive to the presence of NS-OSCS in heparin. Therefore, current USP heparin monograph tests (i.e., SAX-HPLC and %GalN) detect the presence of NS-OSCS in heparin.


Assuntos
Anticoagulantes/química , Sulfatos de Condroitina/análise , Contaminação de Medicamentos , Heparina/química , Indicadores e Reagentes/análise , Resinas de Troca Aniônica , Anticoagulantes/farmacologia , Sulfatos de Condroitina/química , Sulfatos de Condroitina/toxicidade , Cromatografia Líquida de Alta Pressão , Dimetilformamida/química , Contaminação de Medicamentos/prevenção & controle , Galactosamina/análise , Heparina/farmacologia , Hidrazinas/química , Indicadores e Reagentes/química , Indicadores e Reagentes/toxicidade , Limite de Detecção , Espectroscopia de Prótons por Ressonância Magnética , Controle de Qualidade , Estados Unidos , United States Food and Drug Administration
9.
Biochemistry ; 53(37): 5907-15, 2014 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-25162988

RESUMO

Three-dimensional domain swapping is a mode of self-interaction that can give rise to altered functional states and has been identified as the trigger event in some protein deposition diseases, yet rates of interconversion between oligomeric states are usually slow, with the requirement for transient disruption of an extensive network of interactions giving rise to a large kinetic barrier. Here we demonstrate that the cytoplasmic domain of the Escherichia coli GlpG rhomboid protease undergoes slow dimerization via domain swapping and that micromolar concentrations of micelles can be used to enhance monomer-dimer exchange rates by more than 1000-fold. Detergents bearing a phosphocholine headgroup are shown to be true catalysts, with hexadecylphosphocholine reducing the 26 kcal/mol free energy barrier by >11 kcal/mol while preserving the 5 kcal/mol difference between monomer and dimer states. Catalysis involves the formation of a micelle-bound intermediate with a partially unfolded structure that is primed for domain swapping. Taken together, these results are the first to demonstrate true catalysis for domain swapping, by using micelles that work in a chaperonin-like fashion to unfold a kinetically trapped state and allow access to the domain-swapped form.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Endopeptidases/química , Endopeptidases/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Catálise , Dicroísmo Circular , Citoplasma/metabolismo , Detergentes/química , Cinética , Micelas , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Fosforilcolina/química , Conformação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína
10.
Anal Bioanal Chem ; 406(26): 6559-67, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24264620

RESUMO

The FDA has approved more than 100 protein and peptide drugs with hundreds more in the pipeline (Lanthier et al. in Nat Rev Drug Discov 7(9):733-737, 2008). Many of these originator biologic products are now coming off patent and are being manufactured by alternate methods than the innovator as follow-on drugs. Because changes to the production method often lead to subtle differences (e.g., degradation products, different posttranslational modifications or impurities) in the therapeutic (Schiestl et al. in Nat Biotechnol 29(4):310-312, 2011), there is a critical need to define techniques to test and insure the quality of these drugs. In addition, the emergence of protein therapeutics manufactured by unapproved methodologies presents an ongoing and growing regulatory challenge. In this work, high-resolution mass spectrometry was used to determine the presence or absence of posttranslational modifications for one FDA-approved and three foreign-sourced, unapproved filgrastim products. Circular dichroism (CD) was used to compare the secondary structure and probe the temperature stability of these products. Native 2D (1)H,(15)N-heteronuclear singular quantum coherence (HSQC) NMR test was applied to these samples to compare the higher-order structure of the four products. Finally, a cell proliferation assay was performed on the filgrastims to compare their bioactivity, and stressed filgrastim was tested in the bioassay to better understand the effects of changes in protein structure on activity. The results showed that orthogonal approaches are capable of characterizing the physiochemical properties of this protein drug and assessing the impact of structural changes on filgrastim purity and potency.


Assuntos
Fator Estimulador de Colônias de Granulócitos/química , Fator Estimulador de Colônias de Granulócitos/farmacologia , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dicroísmo Circular , Filgrastim , Espectrometria de Massas/métodos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia
11.
J Pharm Biomed Anal ; 85: 99-107, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23917037

RESUMO

During the 2007-2008 heparin crisis it was found that the United States Pharmacopeia (USP) testing monograph for heparin sodium or low molecular weight heparins did not detect the presence of the contaminant, oversulfated chondroitin sulfate (OSCS). In response to this concern, new tests and specifications were developed by the Food and Drug Administration (FDA) and USP and put in place to detect not only the contaminant OSCS, but also to improve assurance of quality and purity of these drug products. The USP monographs for the low molecular weight heparins (LMWHs) approved for use in the United States (dalteparin, tinzaparin and enoxaparin) are also undergoing revision to include many of the same tests used for heparin sodium, including; one-dimensional (1D) 500 MHz (1)H NMR, SAX-HPLC, percent galactosamine in total hexosamine and anticoagulation time assays with purified Factor IIa or Factor Xa. These tests represent orthogonal approaches for heparin identification, measurement of bioactivity and for detection of process impurities or contaminants in these drug products. Here we describe results from a survey of multiple lots from three types of LMWHs in the US market which were collected after the 2009 heparin sodium monograph revision. In addition, innovator and generic versions of formulated enoxaparin products purchased in 2011 are compared using these tests and found to be highly similar within the discriminating power of the assays applied.


Assuntos
Contaminação de Medicamentos , Heparina de Baixo Peso Molecular/análise , Cromatografia Líquida de Alta Pressão , Eletroforese Capilar , Enoxaparina/farmacologia , Heparina de Baixo Peso Molecular/farmacologia , Heparina de Baixo Peso Molecular/normas , Espectroscopia de Ressonância Magnética
12.
J Pharm Biomed Anal ; 85: 108-17, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23917038

RESUMO

Tacrolimus (FK506) is a potent, narrow therapeutic index, immunosuppressive drug used to avoid organ rejection in patients that have undergone organ transplantation. Recent clinical reports suggested a significant reduction in the tacrolimus concentration/dose ratio in the plasma of liver and kidney recipients when the reference listed drug was substituted with a generic drug. In response to these concerns about switching between tacrolimus from different approved manufacturers during treatment, the FDA initiated purity, potency and quality studies of the innovator and generic tacrolimus products available in the US marketplace. A combination of analytical methods, including mass spectrometry (LC-MS), nuclear magnetic resonance (NMR) and bioactivity assay were developed and validated to assess the quality of tacrolimus. These tests measured the identity, impurities and activity of tacrolimus from active pharmaceutical ingredient (API) sources and with formulated drug product from five different approved manufactures. In addition, some testing was performed on tacrolimus capsules obtained from a non US approved Indian source. The data obtained showed no discernible difference in the impurity profiles and potency between the generic and innovator tacrolimus products.


Assuntos
Cromatografia Líquida/métodos , Espectroscopia de Ressonância Magnética/métodos , Espectrometria de Massas/métodos , Tacrolimo/análise , Contaminação de Medicamentos , Tacrolimo/farmacologia , Tacrolimo/normas
13.
J Biol Chem ; 288(1): 247-54, 2013 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-23184955

RESUMO

Enzymatic addition of GalNAc to isotopically labeled IFNα2a produced in Escherichia coli yielded the O-linked glycoprotein GalNAcα-[(13)C,(15)N]IFNα2a. The three-dimensional structure of GalNAcα-IFNα2a has been determined in solution by NMR spectroscopy at high resolution. Proton-nitrogen heteronuclear Overhauser enhancement measurements revealed that the addition of a single monosaccharide unit at Thr-106 significantly slowed motions of the glycosylation loop on the nanosecond time scale. Subsequent addition of a Gal unit produced Gal(ß1,3)GalNAcα-[(13)C,(15)N]IFNα2a. This extension resulted in a further decrease in the dynamics of this loop. The methodology used here allowed the first such description of the structure and dynamics of an O-glycoprotein and opens the way to the study of this class of proteins.


Assuntos
Acetilgalactosamina/química , Interferon-alfa/metabolismo , Polissacarídeos/química , Treonina/química , Acetilgalactosamina/genética , Biologia Computacional/métodos , Dissulfetos/química , Escherichia coli/metabolismo , Glicoproteínas/química , Glicosilação , Humanos , Interferon alfa-2 , Interferons/química , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Conformação Molecular , Peptídeos/química , Conformação Proteica , Proteínas Recombinantes/metabolismo
14.
Biochemistry ; 51(39): 7794-803, 2012 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-22963263

RESUMO

Rhomboid proteins comprise the largest class of intramembrane protease known, being conserved from bacteria to humans. The functional status of these proteases is typically assessed through direct or indirect detection of peptide cleavage products. Although these assays can report on the ability of a rhomboid to catalyze peptide bond cleavage, differences in measured hydrolysis rates can reflect changes in the structure and activity of catalytic residues, as well as the ability of the substrate to access the active site. Here we show that a highly reactive and sterically unencumbered fluorophosphonate activity-based protein profiling probe can be used to report on the catalytic integrity of active site residues in the Escherichia coli GlpG protein. We used results obtained with this probe on GlpG in proteomic samples, in combination with a conventional assay of proteolytic function on purified samples, to identify residues that are located on the cytoplasmic side of the lipid bilayer that are required for maximal proteolytic activity. Regions tested include the 90-residue aqueous-exposed N-terminus that encompasses a globular structure that we have determined by solution nuclear magnetic resonance, along with residues on the cytoplasmic side of the transmembrane domain core. While in most cases mutation or elimination of these residues did not significantly alter the catalytic status of the GlpG active site, the lipid-facing residue Arg227 was found to be important for maintaining a catalytically competent active site. In addition, we found a functionally critical region outside the transmembrane domain (TMD) core that is required for maximal protease activity. This region encompasses an additional 8-10 residues on the N-terminal side of the TMD core that precedes the first transmembrane segment and was not previously known to play a role in rhomboid function. These findings highlight the utility of the activity-based protein profiling approach for the characterization of rhomboid function.


Assuntos
Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/metabolismo , Endopeptidases/análise , Endopeptidases/metabolismo , Proteínas de Escherichia coli/análise , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana/análise , Proteínas de Membrana/metabolismo , Domínio Catalítico , Proteínas de Ligação a DNA/genética , Endopeptidases/genética , Escherichia coli/química , Escherichia coli/citologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Membrana/genética , Modelos Moleculares , Mutação , Conformação Proteica , Estrutura Terciária de Proteína , Proteólise , Proteômica
15.
Commun Integr Biol ; 4(1): 101-3, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21509194

RESUMO

Symmetric cell division in Gram-negative bacteria requires the concerted action of three Min proteins that together ensure exclusive formation of the cell division septum at the mid-point of the cell. We have recently described the structure and dynamic properties of MinE, the protein responsible for directing the cell division inhibitor complex formed by the MinC and MinD proteins away from the middle of the cell. An unexpected feature of this structure was the location of MinD-binding residues at buried, non-accessible sites in the dimeric interface. Here we elaborate on the potential role of conformational changes that might be involved to allow access to these residues, along with the interesting questions raised by these features of the MinE structure.

16.
Proc Natl Acad Sci U S A ; 107(43): 18416-21, 2010 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-20937912

RESUMO

MinE is required for the dynamic oscillation of Min proteins that restricts formation of the cytokinetic septum to the midpoint of the cell in gram negative bacteria. Critical for this oscillation is MinD-binding by MinE to stimulate MinD ATP hydrolysis, a function that had been assigned to the first ∼30 residues in MinE. Previous models based on the structure of an autonomously folded dimeric C-terminal fragment suggested that the N-terminal domain is freely accessible for interactions with MinD. We report here the solution NMR structure of the full-length MinE dimer from Neisseria gonorrhoeae, with two parts of the N-terminal domain forming an integral part of the dimerization interface. Unexpectedly, solvent accessibility is highly restricted for residues that were previously hypothesized to directly interact with MinD. To delineate the true MinD-binding region, in vitro assays for MinE-stimulated MinD activity were performed. The relative MinD-binding affinities obtained for full-length and N-terminal peptides from MinE demonstrated that residues that are buried in the dimeric interface nonetheless participate in direct interactions with MinD. According to results from NMR spin relaxation experiments, access to these buried residues may be facilitated by the presence of conformational exchange. We suggest that this concealment of MinD-binding residues by the MinE dimeric interface provides a mechanism for prevention of nonspecific interactions, particularly with the lipid membrane, to allow the free diffusion of MinE that is critical for Min protein oscillation.


Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Ciclo Celular/genética , Dimerização , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/metabolismo , Ressonância Magnética Nuclear Biomolecular , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eletricidade Estática
17.
Biochem Biophys Res Commun ; 378(2): 235-9, 2009 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-19026612

RESUMO

beta-Microseminoprotein (MSP), a 10kDa seminal plasma protein, forms a tight complex with cysteine-rich secretory protein 3 (CRISP-3) from granulocytes. The 3D structure of human MSP has been determined but there is as yet no 3D structure for CRISP-3. We have now studied the complex between human MSP and CRISP-3 with multidimensional NMR. (15)N-HSQC spectra show substantial differences between free and complexed hMSP. Using several 3D-NMR spectra of triply labeled hMSP in complex with a recombinant N-terminal domain of CRISP-3, most of the backbone of hMSP could be assigned. The data show that only one side of hMSP, comprising beta-strands 1, 4, 5, and 8 are affected by the complex formation, indicating that beta-strands 1 and 8 form the main binding surface. Based on this we present a tentative structure for the hMSP-CRISP-3 complex using the known crystal structure of triflin as a model of CRISP-3.


Assuntos
Modelos Moleculares , Proteínas Secretadas pela Próstata/química , Proteínas e Peptídeos Salivares/química , Proteínas de Plasma Seminal/química , Sequência de Aminoácidos , Cristalografia , Humanos , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Proteínas Secretadas pela Próstata/metabolismo , Estrutura Secundária de Proteína , Proteínas e Peptídeos Salivares/metabolismo , Proteínas de Plasma Seminal/metabolismo
18.
J Mol Biol ; 362(3): 502-15, 2006 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-16930619

RESUMO

Beta-microseminoprotein (MSP) is a small cysteine-rich protein (molecular mass about 10 kDa) first isolated from human seminal plasma and later identified in several other organisms. The function of MSP is not known, but a recent study has shown MSP to bind CRISP-3, a protein present in neutrophilic granulocytes. The amino acid sequence is highly variable between species raising the question of the evolutionary conservation of the 3D structure. Here we present NMR solution structures of both the human and the porcine MSP. The two proteins (sequence identity 51%) have a very similar 3D structure with the secondary structure elements well conserved and with most of the amino acid substitutions causing a change of charge localized to one side of the molecule. MSP is a beta-sheet-rich protein with two distinct domains. The N-terminal domain is composed of a four-stranded beta-sheet, with the strands arranged according to the Greek key-motif, and a less structured part. The C-terminal domain contains two two-stranded beta-sheets with no resemblance to known structural motifs. The two domains, connected to each other by the peptide backbone, one disulfide bond, and interactions between the N and C termini, are oriented to give the molecule a rather extended structure. This global fold differs markedly from that of a previously published structure for porcine MSP, in which the two domains have an entirely different orientation to each other. The difference probably stems from a misinterpretation of ten specific inter-domain NOEs.


Assuntos
Proteínas Secretadas pela Próstata/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Sequência Conservada , DNA Complementar/genética , Humanos , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Proteínas Secretadas pela Próstata/genética , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homologia de Sequência de Aminoácidos , Soluções , Eletricidade Estática , Suínos
19.
Biochemistry ; 44(24): 8782-9, 2005 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-15952784

RESUMO

Vitamin K-dependent protein S is a cofactor of activated protein C, a serine protease that regulates blood coagulation. Deficiency of protein S can cause venous thrombosis. Protein S has four EGF domains in tandem; domains 2-4 bind calcium with high affinity whereas domains 1-2 mediate interaction with activated protein C. We have now solved the solution structure of the EGF3-4 fragment of protein S. The linker between the two domains is similar to what has been observed in other calcium-binding EGF domains where it provides an extended conformation. Interestingly, a disagreement between NOE and RDC data revealed a conformational heterogeneity within EGF3 due to a hinge-like motion around Glu186 in the Cys-Glu-Cys sequence, the only point in the domain where flexibility is allowed. The dominant, bent conformation of EGF3 in the pair has no precedent among calcium-binding EGF domains. It is characterized by a change in the psi angle of Glu186 from 160 degrees +/- 40 degrees , as seen in ten other EGF domains, to approximately 0 degrees +/- 15 degrees . NOESY data suggest that Tyr193, a residue not conserved in other calcium-binding EGF domains (except in the homologue Gas6), induces the unique fold of EGF3. However, SAXS data, obtained on EGF1-4 and EGF2-4, showed a dominant, extended conformation in these fragments. This may be due to a counterproductive domain-domain interaction between EGF2 and EGF4 if EGF3 is in a bent conformation. We speculate that the ability of EGF3 to adopt different conformations may be of functional significance in protein-protein interactions involving protein S.


Assuntos
Cálcio/metabolismo , Osteocalcina/química , Osteocalcina/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Proteína S/química , Proteína S/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Termodinâmica
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