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l-DOPA plays a critical role as a precursor to dopamine and is a standard treatment for Parkinson's disease. Recent research has highlighted the potential therapeutic advantages of deuterated l-DOPA analogs having a longer biological half-life. For their spectroscopic characterization, the in-detail characterization of l-DOPA itself is necessary. This article presents a thorough examination of the vibrational spectra of l-DOPA, with a particular emphasis on chirally sensitive VOA techniques. We successfully obtained high-quality Raman and ROA spectra of l-DOPA in its cationic form, under low pH conditions, and at a high concentration of 100 mg/ml. These spectra cover a broad spectral range, allowing for precise comparisons with theoretical simulations. We also obtained IR and VCD spectra, but they faced limitations due to the narrow accessible spectral region. Exploration of l-DOPA's conformational landscape revealed its intrinsic flexibility, with multiple coexisting conformations. To characterize these conformations, we employed two methods: one involved potential energy surface scans with implicit solvation, and the other utilized molecular dynamics simulations with explicit solvation. Comparing ROA spectra from different conformer groups and applying spectral decomposition proved crucial in determining the correct conformer ratios. The use of explicit solvation significantly improved the quality of the final simulated spectral profiles. The accurate determination of conformer ratios, rather than solely relying on the number of averaged spectra, played a crucial role in simulation accuracy. In conclusion, our study offers valuable insights into the structure and conformational behavior of l-DOPA and represents a valuable resource for subsequent spectroscopic studies of its deuterated analogs.
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Levodopa , Espectrometría Raman , Rotación Óptica , Espectrometría Raman/métodos , Conformación Molecular , Simulación de Dinámica MolecularRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Structural studies of sugars in solution are challenging for most of the traditional analytical techniques. Raman and Raman optical activity (ROA) spectroscopies were found to be extremely convenient for this purpose. However, Raman and ROA spectra of saccharides are challenging to interpret and model due to saccharides' flexibility and polarity. In this study, we present an optimized computational protocol that enables the simulation of the spectra efficiently. Our protocol, which results in good agreement with experiments, combines molecular dynamics and density functional theory calculations. It further uses a smart optimization procedure and a novel adaptable scaling function. The numerical stability and accuracy of individual computational steps are evaluated by comparing simulated and experimental spectra of d-glucose, d-glucuronic acid, N-acetyl-d-glucosamine, methyl ß-d-glucopyranoside, methyl ß-d-glucuronide, and methyl ß-N-acetyl-d-glucosaminide. Overall, our Raman and ROA simulation protocol allows one to routinely and reliably calculate the spectra of small saccharides and opens the door to advanced applications, such as complete 3-dimensional structural determination by direct interpretation of the experimental spectra.
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Working at the border between innate and adaptive immunity, natural killer (NK) cells play a key role in the immune system by protecting healthy cells and by eliminating malignantly transformed, stressed or virally infected cells. NK cell recognition of a target cell is mediated by a receptor "zipper" consisting of various activating and inhibitory receptors, including C-type lectin-like receptors. Among this major group of receptors, two of the largest rodent receptor families are the NKR-P1 and the Clr receptor families. Although these families have been shown to encode receptor-ligand pairs involved in MHC-independent self-nonself discrimination and are a target for immune evasion by tumour cells and viruses, structural mechanisms of their mutual recognition remain less well characterized. Therefore, we developed a non-viral eukaryotic expression system based on transient transfection of suspension-adapted human embryonic kidney 293 cells to produce soluble native disulphide dimers of NK cell C-type lectin-like receptor ectodomains. The expression system was optimized using green fluorescent protein and secreted alkaline phosphatase, easily quantifiable markers of recombinant protein production. We describe an application of this approach to the recombinant protein production and characterization of native rat NKR-P1B and Clr-11 proteins suitable for further structural and functional studies.
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Proteína Similar al Receptor de Calcitonina/genética , Subfamilia B de Receptores Similares a Lectina de Células NK/genética , Ingeniería de Proteínas/métodos , Animales , Proteína Similar al Receptor de Calcitonina/química , Proteína Similar al Receptor de Calcitonina/metabolismo , Células HEK293 , Humanos , Subfamilia B de Receptores Similares a Lectina de Células NK/química , Subfamilia B de Receptores Similares a Lectina de Células NK/metabolismo , Dominios Proteicos , Multimerización de Proteína , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
We have investigated structural changes of peptides related to antimicrobial peptide Halictine-1 (HAL-1) induced by interaction with various membrane-mimicking models with the aim to identify a mechanism of the peptide mode of action and to find a correlation between changes of primary/secondary structure and biological activity. Modifications in the HAL-1 amino acid sequence at particular positions, causing an increase of amphipathicity (Arg/Lys exchange), restricted mobility (insertion of Pro) and consequent changes in antimicrobial and hemolytic activity, led to different behavior towards model membranes. Secondary structure changes induced by peptide-membrane interaction were studied by circular dichroism, infrared spectroscopy, and fluorescence spectroscopy. The experimental results were complemented by molecular dynamics calculations. An α-helical structure has been found to be necessary but not completely sufficient for the HAL-1 peptides antimicrobial action. The role of alternative conformations (such as ß-sheet, PPII or 310-helix) also seems to be important. A mechanism of the peptide mode of action probably involves formation of peptide assemblies (possibly membrane pores), which disrupt bacterial membrane and, consequently, allow membrane penetration.
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Antibacterianos/química , Péptidos Catiónicos Antimicrobianos/química , Membrana Dobles de Lípidos/química , Fosfatidilcolinas/química , Fosfatidilgliceroles/química , Secuencia de Aminoácidos , Antibacterianos/metabolismo , Péptidos Catiónicos Antimicrobianos/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Simulación de Dinámica Molecular , Permeabilidad , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina betaRESUMEN
The protein sequences found in nature represent a tiny fraction of the potential sequences that could be constructed from the 20-amino-acid alphabet. To help define the properties that shaped proteins to stand out from the space of possible alternatives, we conducted a systematic computational and experimental exploration of random (unevolved) sequences in comparison with biological proteins. In our study, combinations of secondary structure, disorder, and aggregation predictions are accompanied by experimental characterization of selected proteins. We found that the overall secondary structure and physicochemical properties of random and biological sequences are very similar. Moreover, random sequences can be well-tolerated by living cells. Contrary to early hypotheses about the toxicity of random and disordered proteins, we found that random sequences with high disorder have low aggregation propensity (unlike random sequences with high structural content) and were particularly well-tolerated. This direct structure content/aggregation propensity dependence differentiates random and biological proteins. Our study indicates that while random sequences can be both structured and disordered, the properties of the latter make them better suited as progenitors (in both in vivo and in vitro settings) for further evolution of complex, soluble, three-dimensional scaffolds that can perform specific biochemical tasks.
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Modelos Moleculares , Biblioteca de Péptidos , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Secuencia de Aminoácidos , Dicroismo Circular , Biología Computacional , Bases de Datos de Proteínas , Conjuntos de Datos como Asunto , Resonancia Magnética Nuclear Biomolecular , Agregado de Proteínas , Pliegue de Proteína , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/toxicidad , SolubilidadRESUMEN
The Raman spectra of 20 proteinogenic amino acids were recorded in the solution, glass phase (as drop coating deposition Raman (DCDR) samples) and crystalline forms in the wide spectral range of 200-3200cm-1. The most apparent spectral differences between the Raman spectra of the crystalline forms, glass phases and aqueous solutions of amino acids were briefly discussed and described in the frame of published works. The possible density dependencies of spectral bands were noted. In some cases, a strong influence of the sample density, as well as of the organization of the water envelope, was observed. The most apparent changes were observed for Ser and Thr. Nevertheless, for the majority of amino acids, the DCDR sample form is an intermediate between the solution and crystalline forms. In contrast, aromatic amino acids have only a small sensitivity to the form of the sample. Our reference set of Raman spectra is useful for revealing discrepancies between the SERS and solid/solution spectra of amino acids. We also found that some previously published Raman spectra of polycrystalline samples resemble glassy state rather than crystalline spectra. Therefore, this reference set of spectra will find application in every branch of Raman spectroscopy where the spectra of biomolecules are collected from coatings.
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Aminoácidos/análisis , Aminoácidos/química , Espectrometría Raman/métodos , CristalizaciónRESUMEN
Mycobacterium tuberculosis (MTb), the causative agent of tuberculosis, can persist in macrophages for decades, maintaining its basic metabolic activities. Phosphoenolpyruvate carboxykinase (Pck; EC 4.1.1.32) is a key player in central carbon metabolism regulation. In replicating MTb, Pck is associated with gluconeogenesis, but in non-replicating MTb, it also catalyzes the reverse anaplerotic reaction. Here, we explored the role of selected cysteine residues in function of MTb Pck under different redox conditions. Using mass spectrometry analysis we confirmed formation of S-S bridge between cysteines C391 and C397 localized in the C-terminal subdomain. Molecular dynamics simulations of C391-C397 bridged model indicated local conformation changes needed for formation of the disulfide. Further, we used circular dichroism and Raman spectroscopy to analyze the influence of C391 and C397 mutations on Pck secondary and tertiary structures, and on enzyme activity and specificity. We demonstrate the regulatory role of C391 and C397 that form the S-S bridge and in the reduced form stabilize Pck tertiary structure and conformation for gluconeogenic and anaplerotic reactions.
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Biocatálisis , Cisteína/metabolismo , Mycobacterium tuberculosis/enzimología , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Disulfuros/metabolismo , Estabilidad de Enzimas , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Mutación/genética , Fosfoenolpiruvato Carboxiquinasa (ATP)/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Especificidad por Sustrato , Espectrometría de Masas en TándemRESUMEN
Ligand binding of neutral progesterone, basic propranolol, and acidic warfarin to human α1-acid glycoprotein (AGP) was investigated by Raman spectroscopy. The binding itself is characterized by a uniform conformational shift in which a tryptophan residue is involved. Slight differences corresponding to different contacts of the individual ligands inside the ß-barrel are described. Results are compared with in silico ligand docking into the available crystal structure of deglycosylated AGP using quantum/molecular mechanics. Calculated binding energies are -18.2, -14.5, and -11.5 kcal/mol for warfarin, propranolol, and progesterone, respectively. These calculations are consistent with Raman difference spectroscopy; nevertheless, minor discrepancies in the precise positions of the ligands point to structural differences between deglycosylated and native AGP. Thermal dynamics of AGP with/without bounded warfarin was followed by Raman spectroscopy in a temperature range of 10-95 °C and analyzed by principal component analysis. With increasing temperature, a slight decrease of α-helical content is observed that coincides with an increase in ß-sheet content. Above 45 °C, also ß-strands tend to unfold, and the observed decrease in ß-sheet coincides with an increase of ß-turns accompanied by a conformational shift of the nearby disulfide bridge from high-energy trans-gauche-trans to more relaxed gauche-gauche-trans. This major rearrangement in the vicinity of the bridge is not only characterized by unfolding of the ß-sheet but also by subsequent ligand release. Hereby, ligand binding alters the protein dynamics, and the more rigid protein-ligand complex shows an improved thermal stability, a finding that contributes to the reported chaperone-like function of AGP.
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Orosomucoide/química , Orosomucoide/metabolismo , Progesterona/metabolismo , Propranolol/metabolismo , Warfarina/metabolismo , Sitios de Unión , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Progesterona/química , Propranolol/química , Unión Proteica , Estabilidad Proteica , Estructura Secundaria de Proteína , Espectrometría Raman , Termodinámica , Triptófano/metabolismo , Warfarina/químicaRESUMEN
The transcriptional activity of the serum response factor (SRF) protein is triggered by its binding to a 10-base-pair DNA consensus sequence designated the CArG box, which is the core sequence of the serum response element (SRE). Sequence-specific recognition of the CArG box by a core domain of 100 amino acid residues of SRF (core-SRF) was asserted to depend almost exclusively on the intrinsic SRE conformation and on the degree of protein-induced SRE bending. Nevertheless, this paradigm was invalidated by a temperature-dependent Raman spectroscopy study of 20-mer oligonucleotides involved in bonding interactions with core-SRF that reproduced both wild type and mutated c-fos SREs. Indeed, the SRE moieties that are complexed with core-SRF exhibit permanent interconversion dynamics between bent and linear conformers. Thus, sequence-specific recognition of the CArG box by core-SRF cannot be explained only in terms of the three-dimensional structure of the SRE. A particular dynamic pairing process discriminates between the wild type and mutated complexes. Specific oscillations of the phosphate charge network of the SRE govern the recognition between both partners rather than an intrinsic set of conformations of the SRE.
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ADN/química , Oligonucleótidos/química , Fosfatos/química , Elemento de Respuesta al Suero/genética , Factor de Respuesta Sérica/química , Sitios de Unión , Humanos , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Motivos de Nucleótidos , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Factor de Respuesta Sérica/genética , Espectrometría Raman , Electricidad Estática , Termodinámica , Transcripción GenéticaRESUMEN
MADS box family transcription factors are involved in signal transduction and development control through DNA specific sequence recognition. The DNA binding domain of these proteins contains a conservative 55-60 amino acid sequence which defines the membership of this large family. Here we present a thorough study of the MADS segment of serum response factor (MADS(SRF)). Fluorescence, UV-absorption, and Raman spectroscopy studies were performed in order to disclose its behavior and basic functional properties in an aqueous environment. The secondary structure of MADS(SRF) estimated by analysis of Raman spectra and supported by CD has revealed only the C-terminal part as homologous with those of free core-SRF, while the N-terminal part has lost the stable α-helical structure found in both the free core-SRF and its specific complex with DNA. The three tyrosine residues of the MADS(SRF) were used as spectroscopic inner probes. The effect of environmental conditions, especially pH variations and addition of variously charged quenchers, on their spectra was examined. Two-component fluorescence quenching was revealed using factor analysis and corresponding Stern-Volmer constants determined. Factor analysis of absorbance and fluorescence pH titration led to determination of three dissociation constants pKa1 = 6.4 ± 0.2, pKa2 = 7.3 ± 0.2, and pKa3 = 9.6 ± 0.6. Critical comparison of all experiments identified the deprotonation of His193 hydrogen bonded to Tyr195 as a candidate for pKa1 (and that of Tyr158 as a candidate for pKa2). Within MADS(SRF), His193 is a key intermediary between the N-terminal primary DNA binding element and the hydrophobic C-terminal protein dimerization element.
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Factor de Respuesta Sérica/química , Tirosina/química , Secuencia de Aminoácidos , Humanos , Concentración de Iones de Hidrógeno , Modelos Moleculares , Péptidos/síntesis química , Péptidos/química , Estructura Secundaria de Proteína , Factor de Respuesta Sérica/metabolismo , Espectrofotometría Ultravioleta , Espectrometría Raman , Tirosina/metabolismoRESUMEN
Didehydroroflamycoin (DDHR), a recently isolated member of the polyene macrolide family, was shown to have antibacterial and antifungal activity. However, its mechanism of action has not been investigated. Antibiotics from this family are amphiphilic; thus, they have membrane activity, their biological action is localized in the membrane, and the membrane composition and physical properties facilitate the recognition of a particular compound by the target organism. In this work, we use model lipid membranes comprised of giant unilamellar vesicles (GUVs) for a systematic study of the action of DDHR. In parallel, experiments are conducted using filipin III and amphotericin B, other members of the family, and the behavior observed for DDHR is described in the context of that of these two heavily studied compounds. The study shows that DDHR disrupts membranes via two different mechanisms and that the involvement of these mechanisms depends on the presence of cholesterol. The leakage assays performed in GUVs and the conductance measurements using black lipid membranes (BLM) reveal that the pores that develop in the absence of cholesterol are transient and their size is dependent on the DDHR concentration. In contrast, cholesterol promotes the formation of more defined structures that are temporally stable.
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Antibacterianos/química , Antifúngicos/química , Filipina/análogos & derivados , Membrana Dobles de Lípidos/química , Macrólidos/química , Liposomas Unilamelares/química , Anfotericina B/química , Colesterol/química , Filipina/química , CinéticaRESUMEN
BACKGROUND: The handling of cerebrospinal fluid (CSF) affects the biomarker quantification used to diagnose Alzheimer's disease (AD). Only specialized centers can test for AD markers. The precise timing and freezing is required to correctly measure these biomarkers. Therefore, the effects of CSF storage temperature and repeated freeze/thaw cycles on CSF stability were investigated. METHODS: Drop coating deposition Raman spectroscopy in combination with principal component analysis was used to analyze CSF and its dialyzed form (ELISA confirmed the removal of up to 80% of the AD markers). The advantage of this approach is that no prior knowledge of the biomarkers is necessary and that both the concentration and the protein structure of intact CSF are analyzed. RESULTS: Dialyzed CSF was stable for up to 5 h after its collection, while native CSF started to denature nearly immediately. Most of the unstable proteins were denatured within 24 h. The dialyzed CSF was not affected by freeze/thaw cycles, but the native CSF exhibited significant progressive changes, even after the first freezing. The mechanism as well as the resulting structures of the freeze-denatured proteins differed from those of the temporally denatured proteins, although both protein sets began with the same initial proteins. CONCLUSIONS: CSF must be processed immediately, within 5 h of collection. Flash cooling is recommended for freezing CSF, but any freeze/thaw cycle will affect the protein component of CSF.
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Biomarcadores/líquido cefalorraquídeo , Congelación , Espectrometría Raman , Enfermedad de Alzheimer/diagnóstico , Péptidos beta-Amiloides/líquido cefalorraquídeo , Humanos , Análisis de Componente Principal , Proteínas tau/líquido cefalorraquídeoRESUMEN
The high sensitivity to pH of a short segment (an octamer) of serum response factor (SRF), an important member of the MADS box family of transcription factors, was investigated by Raman scattering, infrared and circular dichroism spectroscopies. Molecular dynamics (MD) and density functional theory (DFT) calculations enabled interpretation of spectral changes in close detail. Although there was a negligible difference between spectra in acidic and neutral environments, the spectrum in basic pH was substantially different. The major changes were attributed to the deprotonation of tyrosine. The secondary structure of the SRF octamer fragment was estimated experimentally as well as predicted theoretically by MD. All techniques proved that it exists in a dynamical equilibrium among several conformations mostly close to ß turn, unordered conformations, and extended structure, in contrast to the stable secondary structure it possesses as a part of SRF. Generally, this approach represents a useful tool for the study of various short oligopeptides.
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Simulación de Dinámica Molecular , Protones , Factor de Respuesta Sérica/química , Tirosina/químicaRESUMEN
Raman microscopy permits structural analysis of protein crystals in situ in hanging drops, allowing for comparison with Raman measurements in solution. Nevertheless, the two methods sometimes reveal subtle differences in structure that are often ascribed to the water layer surrounding the protein. The novel method of drop-coating deposition Raman spectropscopy (DCDR) exploits an intermediate phase that, although nominally "dry," has been shown to preserve protein structural features present in solution. The potential of this new approach to bridge the structural gap between proteins in solution and in crystals is explored here with extrinsic protein PsbP of photosystem II from Spinacia oleracea. In the high-resolution (1.98 Å) x-ray crystal structure of PsbP reported here, several segments of the protein chain are present but unresolved. Analysis of the three kinds of Raman spectra of PsbP suggests that most of the subtle differences can indeed be attributed to the water envelope, which is shown here to have a similar Raman intensity in glassy and crystal states. Using molecular dynamics simulations cross-validated by Raman solution data, two unresolved segments of the PsbP crystal structure were modeled as loops, and the amino terminus was inferred to contain an additional beta segment. The complete PsbP structure was compared with that of the PsbP-like protein CyanoP, which plays a more peripheral role in photosystem II function. The comparison suggests possible interaction surfaces of PsbP with higher-plant photosystem II. This work provides the first complete structural picture of this key protein, and it represents the first systematic comparison of Raman data from solution, glassy, and crystalline states of a protein.
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Complejo de Proteína del Fotosistema II/química , Proteínas de Plantas/química , Spinacia oleracea/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Espectrometría RamanRESUMEN
RNase L, a key enzyme in the host defense system, is activated by the binding of 2'-5'-linked oligoadenylates (2-5A) to the N-terminal ankyrin repeat domain, which causes the inactive monomer to form a catalytically active homodimer. We focused on the structural changes of human RNase L as a result of interactions with four different activators: natural 2-5 pA(4) and three tetramers with 3'-end AMP units replaced with ribo-, arabino- and xylo-configured phosphonate analogs of AMP (pA(3)X). The extent of the RNase L dimerization and its cleavage activity upon binding of all these activators were similar. A drop-coating deposition Raman (DCDR) spectroscopy possessed uniform spectral changes upon binding of all of the tetramers, which verified the same binding mechanism. The estimated secondary structural composition of monomeric RNase L is 44% α-helix, 28% ß-sheet, 17% ß-turns and 11% of unordered structures, whereas dimerization causes a slight decrease in α-helix and increase in ß-sheet (ca. 2%) content. The dimerization affects at least three Tyr, five Phe and two Trp residues. The α-ß structural switch may fix domain positions in the hinge region (residues ca. 336-363) during homodimer formation.
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Endorribonucleasas/química , Nucleótidos de Adenina/química , Nucleótidos de Adenina/metabolismo , Repetición de Anquirina , Endorribonucleasas/metabolismo , Humanos , Conformación Proteica , Multimerización de Proteína , Espectrometría RamanRESUMEN
Receptors belonging to NKR-P1 family and their specific Clr ligands form an alternative missing self recognition system critical in immunity against tumors and viruses, elimination of tumor cells subjected to genotoxic stress, activation of T cell dependent immune response, and hypertension. The three-dimensional structure of the extracellular domain of the mouse natural killer (NK) cell receptor mNKR-P1Aex has been determined by X-ray diffraction. The core of the C-type lectin domain (CTLD) is homologous to the other CTLD receptors whereas one quarter of the domain forms an extended loop interacting tightly with a neighboring loop in the crystal. This domain swapping mechanism results in a compact interaction interface. A second dimerization interface resembles the known arrangement of other CTLD NK receptors. A functional dimeric form of the receptor is suggested, with the loop, evolutionarily conserved within this family, proposed to participate in interactions with ligands.
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Subfamilia B de Receptores Similares a Lectina de Células NK/química , Subfamilia B de Receptores Similares a Lectina de Células NK/metabolismo , Secuencia de Aminoácidos , Animales , Células Asesinas Naturales/metabolismo , Ratones , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Espectrometría Raman , Difracción de Rayos XRESUMEN
Receptor proteins at the cell surface regulate the ability of natural killer cells to recognize and kill a variety of aberrant target cells. The structural features determining the function of natural killer receptor proteins 1 (NKR-P1s) are largely unknown. In the present work, refined homology models are generated for the C-type lectin-like extracellular domains of rat NKR-P1A and NKR-P1B, mouse NKR-P1A, NKR-P1C, NKR-P1F, and NKR-P1G, and human NKR-P1 receptors. Experimental data on secondary structure, tertiary interactions, and thermal transitions are acquired for four of the proteins using Raman and infrared spectroscopy. The experimental and modeling results are in agreement with respect to the overall structures of the NKR-P1 receptor domains, while suggesting functionally significant local differences among species and isoforms. Two sequence regions that are conserved in all analyzed NKR-P1 receptors do not correspond to conserved structural elements as might be expected, but are represented by loop regions, one of which is arranged differently in the constructed models. This region displays high flexibility but is anchored by conserved sequences, suggesting that its position relative to the rest of the domain might be variable. This loop may contribute to ligand-binding specificity via a coupled conformational transition.
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Secuencia Conservada , Subfamilia B de Receptores Similares a Lectina de Células NK/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Humanos , Ratones , Ratones Endogámicos C57BL , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Subfamilia B de Receptores Similares a Lectina de Células NK/clasificación , Filogenia , Estructura Terciaria de Proteína , Ratas , Alineación de Secuencia , Espectroscopía Infrarroja por Transformada de Fourier , Espectrometría Raman , Homología Estructural de Proteína , TermodinámicaRESUMEN
Transcriptional activity of serum response factor (SRF) is dependent on its binding to the CC(A/T)(6)GG box (CArG box) of serum response element (SRE). By Raman spectroscopy, we carried out a comparative analysis, in solution, of the complexes obtained from the association of core-SRF with 20-mer SREs bearing wild-type and mutated c-fos CArG boxes. In case of association with the wild type c-fos CArG box, the complex does not bring out the expected Raman signature of a stable bending of the targeted SRE but keeps a bend-linear conformer oligonucleotide interconversion. The linear conformer population is larger than that of free oligonucleotide. In the core-SRF moiety of the wild-type complex a large spectral change associated with the CO-groups from Asp and/or Glu residues shows that their ionization states and the strength of their interactions decrease as compared to those of mutated non-specific complexes. Structural constraints evidenced on the free core-SRF are released in the wild-type complex and environmental heterogeneities appear in the vicinity of Tyr residues, due to higher water molecule access. The H-bonding configuration of one Tyr OH-group, in average, changes with a net transfer from H-bond acceptor character to a combined donor and acceptor character. A charge repartition distributed on both core-SRF and targeted SRE stabilizes the specific complex, allowing the two partners to experience a variety of conformations.
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Regulación de la Expresión Génica , Elemento de Respuesta al Suero , Factor de Respuesta Sérica/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Conformación Proteica , Estructura Terciaria de Proteína , Factor de Respuesta Sérica/química , Factor de Respuesta Sérica/genética , Espectrometría Raman , Tirosina/química , Tirosina/genética , Tirosina/metabolismoRESUMEN
In an attempt to prepare a library of short oligoadenylate analogues featuring both the enzyme-stable internucleotide linkage and the 5'-O-methylphosphonate moiety and thus obtain a pool of potential RNase L agonists/antagonists, we studied the spontaneous polycondensation of the adenosin-5'-O-ylmethylphosphonic acid (p(c)A), an isopolar AMP analogue, and its imidazolide derivatives employing N,N'-dicyclohexylcarbodiimide under nonaqueous conditions and uranyl ions under aqueous conditions, respectively. The RP LC-MS analyses of the reaction mixtures per se, and those obtained after the periodate treatment, along with analyses and separations by capillary zone electrophoresis, allowed us to characterize major linear and cyclic oligoadenylates obtained. The structure of selected compounds was supported, after their isolation, by NMR spectroscopy. Ab initio calculation of the model structures simulating the AMP-imidazolide and p(c)A-imidazolide offered the explanation why the latter compound exerted, in contrast to AMP-imidazolide, a very low stability in aqueous solutions.