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1.
Nucleic Acids Res ; 43(4): 2102-15, 2015 Feb 27.
Artículo en Inglés | MEDLINE | ID: mdl-25662217

RESUMEN

Base J (ß-D-glucosyl-hydroxymethyluracil) replaces 1% of T in the Leishmania genome and is only found in telomeric repeats (99%) and in regions where transcription starts and stops. This highly restricted distribution must be co-determined by the thymidine hydroxylases (JBP1 and JBP2) that catalyze the initial step in J synthesis. To determine the DNA sequences recognized by JBP1/2, we used SMRT sequencing of DNA segments inserted into plasmids grown in Leishmania tarentolae. We show that SMRT sequencing recognizes base J in DNA. Leishmania DNA segments that normally contain J also picked up J when present in the plasmid, whereas control sequences did not. Even a segment of only 10 telomeric (GGGTTA) repeats was modified in the plasmid. We show that J modification usually occurs at pairs of Ts on opposite DNA strands, separated by 12 nucleotides. Modifications occur near G-rich sequences capable of forming G-quadruplexes and JBP2 is needed, as it does not occur in JBP2-null cells. We propose a model whereby de novo J insertion is mediated by JBP2. JBP1 then binds to J and hydroxylates another T 13 bp downstream (but not upstream) on the complementary strand, allowing JBP1 to maintain existing J following DNA replication.


Asunto(s)
Glucósidos/análisis , Uracilo/análogos & derivados , Proteínas de Unión al ADN/metabolismo , Glucósidos/metabolismo , Leishmania/genética , Plásmidos/genética , Proteínas Protozoarias/metabolismo , Análisis de Secuencia de ADN , Uracilo/análisis , Uracilo/metabolismo
2.
Biochemistry ; 55(37): 5201-3, 2016 09 20.
Artículo en Inglés | MEDLINE | ID: mdl-27603565

RESUMEN

We report a detailed study of a point mutation of the crucial binding site residue, D128, in the biotin-streptavidin complex. The conservative substitution, D128N, preserves the detailed structure observed for the wild-type complex but has an only minimal impact on biotin binding, even though previous experimental and computational studies suggested that a charged D128 residue was crucial for high-affinity binding. These results show clearly that the fundamental basis for streptavidin's extremely strong biotin binding affinity is more complex than assumed and illustrate some of the challenges that may arise when analyzing extremely strong ligand-protein binding interactions.


Asunto(s)
Ácido Aspártico/metabolismo , Biotina/metabolismo , Mutación , Estreptavidina/metabolismo , Sitios de Unión , Cristalografía , Estreptavidina/química
3.
J Struct Funct Genomics ; 16(2): 91-9, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25832174

RESUMEN

The methylmalonyl Co-A mutase-associated GTPase MeaB from Methylobacterium extorquens is involved in glyoxylate regulation and required for growth. In humans, mutations in the homolog methylmalonic aciduria associated protein (MMAA) cause methylmalonic aciduria, which is often fatal. The central role of MeaB from bacteria to humans suggests that MeaB is also important in other, pathogenic bacteria such as Mycobacterium tuberculosis. However, the identity of the mycobacterial MeaB homolog is presently unclear. Here, we identify the M. tuberculosis protein Rv1496 and its homologs in M. smegmatis and M. thermoresistibile as MeaB. The crystal structures of all three homologs are highly similar to MeaB and MMAA structures and reveal a characteristic three-domain homodimer with GDP bound in the G domain active site. A structure of Rv1496 obtained from a crystal grown in the presence of GTP exhibited electron density for GDP, suggesting GTPase activity. These structures identify the mycobacterial MeaB and provide a structural framework for therapeutic targeting of M. tuberculosis MeaB.


Asunto(s)
Proteínas Bacterianas/química , GTP Fosfohidrolasas/química , Mycobacterium tuberculosis/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/aislamiento & purificación , Mycobacterium tuberculosis/genética
4.
Biochemistry ; 51(2): 597-607, 2012 Jan 17.
Artículo en Inglés | MEDLINE | ID: mdl-22145986

RESUMEN

We report a point mutation in the second contact shell of the high-affinity streptavidin-biotin complex that appears to reduce binding affinity through transmitted effects on equilibrium dynamics. The Y54F streptavidin mutation causes a 75-fold loss of binding affinity with 73-fold faster dissociation, a large loss of binding enthalpy (ΔΔH = 3.4 kcal/mol at 37 °C), and a small gain in binding entropy (TΔΔS = 0.7 kcal/mol). The removed Y54 hydroxyl is replaced by a water molecule in the bound structure, but there are no observable changes in structure in the first contact shell and no additional changes surrounding the mutation. Molecular dynamics simulations reveal a large increase in the atomic fluctuation amplitudes for W79, a key biotin contact residue, compared to the fluctuation amplitudes in the wild-type. The increased W79 atomic fluctuation amplitudes are caused by loss of water-mediated hydrogen bonds between the Y54 hydroxyl group and peptide backbone atoms in and near W79. We propose that the increased atomic fluctuation amplitudes diminish the integrity of the W79-biotin interaction and represents a loosening of the "tryptophan collar" that is critical to the slow dissociation and high affinity of streptavidin-biotin binding. These results illustrate how changes in protein dynamics distal to the ligand binding pocket can have a profound impact on ligand binding, even when equilibrium structure is unperturbed.


Asunto(s)
Biotina/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación Puntual , Estreptavidina/química , Estreptavidina/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Cinética , Simulación de Dinámica Molecular , Proteínas Mutantes/genética , Unión Proteica , Conformación Proteica , Estreptavidina/genética , Termodinámica
5.
Langmuir ; 28(4): 2107-12, 2012 Jan 31.
Artículo en Inglés | MEDLINE | ID: mdl-22148958

RESUMEN

To fully develop techniques that provide an accurate description of protein structure at a surface, we must start with a relatively simple model system before moving to increasingly complex systems. In this study, X-ray photoelectron spectroscopy (XPS), sum frequency generation spectroscopy (SFG), near-edge X-ray adsorption fine structure (NEXAFS) spectroscopy, and time-of-flight secondary ion mass spectrometry (ToF-SIMS) were used to probe the orientation of Protein G B1 (6 kDa) immobilized onto both amine (NH(3)(+)) and carboxyl (COO(-)) functionalized gold. Previously, we have shown that we could successfully control orientation of a similar Protein G fragment via a cysteine-maleimide bond. In this investigation, to induce opposite end-on orientations, a charge distribution was created within the Protein G B1 fragment by first substituting specific negatively charged amino acids with neutral amino acids and then immobilizing the protein onto two oppositely charged self-assembled monolayer (SAM) surfaces (NH(3)(+) and COO(-)). Protein coverage, on both surfaces, was monitored by the change in the atomic % N, as determined by XPS. Spectral features within the SFG spectra, acquired for the protein adsorbed onto a NH(3)(+)-SAM surface, indicates that this electrostatic interaction does induce the protein to form an oriented monolayer on the SAM substrate. This corresponded to the polarization dependence of the spectral feature related to the NEXAFS N(1s)-to-π* transition of the ß-sheet peptide bonds within the protein layer. ToF-SIMS data demonstrated a clear separation between the two samples based on the intensity differences of secondary ions stemming from amino acids located asymmetrically within Protein G B1 (methionine: 62 and 105 m/z; tyrosine: 107 and 137 m/z; leucine: 86 m/z). For a more quantitative examination of orientation, we developed a ratio comparing the sum of the intensities of secondary-ions stemming from the amino acid residues at either end of the protein. The 2-fold increase in this ratio, observed between the protein covered NH(3)(+) and COO(-) SAMs, indicates opposite orientations of the Protein G B1 fragment on the two different surfaces.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Inmovilizadas/química , Fenómenos Ópticos , Espectrometría de Masa de Ion Secundario , Electricidad Estática , Espectroscopía de Absorción de Rayos X , Modelos Moleculares , Estructura Terciaria de Proteína , Factores de Tiempo
6.
Biochemistry ; 49(22): 4568-70, 2010 Jun 08.
Artículo en Inglés | MEDLINE | ID: mdl-20462252

RESUMEN

We have identified a distal point mutation in streptavidin that causes a 1000-fold reduction in biotin binding affinity without disrupting the equilibrium complex structure. The F130L mutation creates a small cavity occupied by a water molecule; however, all neighboring side chain positions are preserved, and protein-biotin hydrogen bonds are unperturbed. Molecular dynamics simulations reveal a reduced mobility of biotin binding residues but no observable destabilization of protein-ligand interactions. Our combined structural and computational studies suggest that the additional water molecule may affect binding affinity through an electronic polarization effect that impacts the highly cooperative hydrogen bonding network in the biotin binding pocket.


Asunto(s)
Biotina/química , Electrones , Simulación de Dinámica Molecular , Mutación Puntual , Estreptavidina/química , Estreptavidina/metabolismo , Sitios de Unión/genética , Biotina/antagonistas & inhibidores , Biotina/metabolismo , Enlace de Hidrógeno , Leucina/química , Leucina/genética , Leucina/metabolismo , Ligandos , Fenilalanina/química , Fenilalanina/genética , Fenilalanina/metabolismo , Unión Proteica/genética , Estabilidad Proteica , Estreptavidina/antagonistas & inhibidores , Estreptavidina/genética , Termodinámica
7.
Langmuir ; 26(21): 16434-41, 2010 Nov 02.
Artículo en Inglés | MEDLINE | ID: mdl-20384305

RESUMEN

The ability to orient active proteins on surfaces is a critical aspect of many medical technologies. An important related challenge is characterizing protein orientation in these surface films. This study uses a combination of time-of-flight secondary ion mass spectrometry (ToF-SIMS), sum frequency generation (SFG) vibrational spectroscopy, and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy to characterize the orientation of surface-immobilized Protein G B1, a rigid 6 kDa domain that binds the Fc fragment of IgG. Two Protein G B1 variants with a single cysteine introduced at either end were immobilized via the cysteine thiol onto maleimide-oligo(ethylene glycol)-functionalized gold and bare gold substrates. X-ray photoelectron spectroscopy was used to measure the amount of immobilized protein, and ToF-SIMS was used to measure the amino acid composition of the exposed surface of the protein films and to confirm covalent attachment of protein thiol to the substrate maleimide groups. SFG and NEXAFS were used to characterize the ordering and orientation of peptide or side chain bonds. On both substrates and for both cysteine positions, ToF-SIMS data showed enrichment of mass peaks from amino acids located at the end of the protein opposite to the cysteine surface position as compared with nonspecifically immobilized protein, indicating end-on protein orientations. Orientation on the maleimide substrate was enhanced by increasing pH (7.0-9.5) and salt concentration (0-1.5 M NaCl). SFG spectral peaks characteristic of ordered α-helix and ß-sheet elements were observed for both variants but not for cysteine-free wild type protein on the maleimide surface. The phase of the α-helix and ß-sheet peaks indicated a predominantly upright orientation for both variants, consistent with an end-on protein binding configuration. Polarization dependence of the NEXAFS signal from the N 1s to π* transition of ß-sheet peptide bonds also indicated protein ordering, with an estimated tilt angle of inner ß-strands of 40-50° for both variants (one variant more tilted than the other), consistent with SFG results. The combined results demonstrate the power of using complementary techniques to probe protein orientation on surfaces.


Asunto(s)
Proteínas del Tejido Nervioso/química , Adsorción , Cisteína/química , Glicol de Etileno/química , Oro/química , Concentración de Iones de Hidrógeno , Maleimidas/química , Estructura Molecular , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/aislamiento & purificación , Tamaño de la Partícula , Espectrometría de Masa de Ion Secundario , Análisis Espectral , Compuestos de Sulfhidrilo/química , Propiedades de Superficie
8.
Parasitol Int ; 67(4): 476-480, 2018 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-29609036

RESUMEN

Leishmania infantum is responsible for human and canine leishmaniasis in the Mediterranean basin, where the major vector is Phlebotomus perniciosus. Because isolation of sufficient parasites from the sand fly gut is technically challenging, axenic cultivation of promastigotes is routinely used to obtain material for biochemical and genetic analyses. Here, we report the use of Spliced Leader RNA-seq (SL-seq) to compare transcript abundance in cultured promastigotes and those obtained from the whole midgut of the sand fly 5 days after infection. SL-seq allows for amplification of RNA from the parasite avoiding contamination with RNA from the gut of the insect. The study has been performed by means of a single technical replicate comparing pools of samples obtained from sand fly-derived (sfPro) and axenic culture promastigotes (acPro). Although there was a moderate correlation (R2 = 0.83) in gene expression, 793 genes showed significantly different (≥2-fold, p <0.05) mRNA levels in sand fly-derived promastigotes and in culture, of which 31 were up-regulated ≥8-fold (p < 10-8 in most cases). These included several genes that are typically up-regulated during metacyclogenesis, suggesting that sand fly-derived promastigotes contain a substantial number of metacyclics, and/or that their differentiation status as metacyclics is more advanced in these populations. Infection experiments and studies evaluating the proportion of metacyclic promastigotes in culture and within the sand fly gut, previously reported by us, support the last hypothesis.


Asunto(s)
Leishmania infantum/genética , Leishmania infantum/aislamiento & purificación , Estadios del Ciclo de Vida/genética , Phlebotomus/parasitología , Animales , Cultivo Axénico/métodos , Vectores de Enfermedades , Perfilación de la Expresión Génica/métodos , Intestinos/parasitología , Leishmania infantum/fisiología , Leishmaniasis/etnología , Leishmaniasis/parasitología , Phlebotomus/anatomía & histología , Análisis de Secuencia de ARN/métodos
9.
Methods Mol Biol ; 1201: 207-19, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25388116

RESUMEN

High-throughput sequencing of cDNA copies of mRNA (RNA-seq) provides a digital read-out of mRNA levels over several orders of magnitude, as well as mapping the transcripts to the nucleotide level. Here we describe an RNA-seq approach that exploits the 39-nucleotide mini-exon or spliced leader (SL) sequence found at the 5' end of all Leishmania (and other trypanosomatid) mRNAs.


Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Leishmania/genética , ARN Mensajero , Análisis de Secuencia de ARN/métodos , Regulación de la Expresión Génica , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/genética , ARN Protozoario , ARN Lider Empalmado
10.
Tuberculosis (Edinb) ; 95(2): 142-8, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25613812

RESUMEN

High-resolution three-dimensional structures of essential Mycobacterium tuberculosis (Mtb) proteins provide templates for TB drug design, but are available for only a small fraction of the Mtb proteome. Here we evaluate an intra-genus "homolog-rescue" strategy to increase the structural information available for TB drug discovery by using mycobacterial homologs with conserved active sites. Of 179 potential TB drug targets selected for x-ray structure determination, only 16 yielded a crystal structure. By adding 1675 homologs from nine other mycobacterial species to the pipeline, structures representing an additional 52 otherwise intractable targets were solved. To determine whether these homolog structures would be useful surrogates in TB drug design, we compared the active sites of 106 pairs of Mtb and non-TB mycobacterial (NTM) enzyme homologs with experimentally determined structures, using three metrics of active site similarity, including superposition of continuous pharmacophoric property distributions. Pair-wise structural comparisons revealed that 19/22 pairs with >55% overall sequence identity had active site Cα RMSD <1 Å, >85% side chain identity, and ≥80% PSAPF (similarity based on pharmacophoric properties) indicating highly conserved active site shape and chemistry. Applying these results to the 52 NTM structures described above, 41 shared >55% sequence identity with the Mtb target, thus increasing the effective structural coverage of the 179 Mtb targets over three-fold (from 9% to 32%). The utility of these structures in TB drug design can be tested by designing inhibitors using the homolog structure and assaying the cognate Mtb enzyme; a promising test case, Mtb cytidylate kinase, is described. The homolog-rescue strategy evaluated here for TB is also generalizable to drug targets for other diseases.


Asunto(s)
Antituberculosos/farmacología , Diseño de Fármacos , Terapia Molecular Dirigida/métodos , Mycobacterium tuberculosis/efectos de los fármacos , Antituberculosos/química , Proteínas Bacterianas/química , Biología Computacional/métodos , Cristalografía por Rayos X/métodos , Bases de Datos de Proteínas , Activación Enzimática , Genómica/métodos , Humanos , Modelos Moleculares , Mycobacterium/clasificación , Mycobacterium/enzimología , Mycobacterium/genética , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Relación Estructura-Actividad Cuantitativa , Especificidad de la Especie
11.
J Biomed Mater Res A ; 69(3): 525-34, 2004 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-15127399

RESUMEN

Structural changes of proteins during adsorption to biomaterials affect the presentation of molecular binding sites and, ultimately, biomaterial performance. We have applied fluorescence resonance energy transfer (FRET) spectroscopy to study structural changes of the cell adhesion protein, fibronectin (Fn), following adsorption to model hydrophilic and hydrophobic surfaces. Fn was labeled with donor and acceptor fluorophores using two labeling schemes and intramolecular energy transfer was calibrated against measured structural changes of Fn in denaturing solutions. FRET was then applied to measure Fn's structure on surfaces. Based on FRET, Fn underwent greater extension of its dimer arms on hydrophilic glass than on hydrophobic fluoroalklysilane-derivatized glass (fluorosilane), and this extension was insensitive to molecular packing over a range of adsorption concentrations. Fn's conformation on glass better promoted cell attachment than on fluorosilane; the roles of both global structural changes (movements of modules) and local structural changes (disruption of secondary structure) on Fn's cell integrin binding activity are discussed. Based on previous FRET work, we compare Fn's conformations on these surfaces with its conformations in fibroblast culture. FRET is unique in allowing direct comparison of protein structure between biomaterial surfaces and cell culture.


Asunto(s)
Materiales Biocompatibles/química , Fibronectinas/química , Transferencia Resonante de Energía de Fluorescencia/métodos , Conformación Proteica , Adsorción , Animales , Materiales Biocompatibles/metabolismo , Adhesión Celular , Fibronectinas/metabolismo , Colorantes Fluorescentes/metabolismo , Fluoruros/química , Vidrio/química , Guanidina/metabolismo , Humanos , Ratones , Células 3T3 NIH/citología , Células 3T3 NIH/metabolismo , Desnaturalización Proteica , Silanos/química , Propiedades de Superficie
12.
Mol Biochem Parasitol ; 196(1): 9-11, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-25064607

RESUMEN

Base J is a DNA modification found in the genome of Trypanosoma brucei and all other kinetoplastids analyzed, where it replaces a small fraction of Ts, mainly in telomeric and chromosome-internal transcription initiation and termination regions. The synthesis of base J is a two-step process whereby a specific T is converted to HOMedU (hydroxymethyldeoxyuridine) and subsequently glucosylated to generate J. The thymidine hydroxylases (JPB1 and JBP2) that catalyze the first step have been characterized, but the identity of the glucosyltransferase catalyzing the second step has proven elusive. Recent bioinformatic analysis by Iyer et al. (Nucleic Acids Res 2013;41:7635) suggested that Tb927.10.6900 encodes the glucosyltransferase (HmdUGT) responsible for converting HOMedU to J in T. brucei. We now present experimental evidence to validate this hypothesis; null mutants of Tb927.10.6900 are unable to synthesize base J. Orthologues from related kinetoplastids show only modest conservation, with several insertion sequences found in those from Leishmania and related genera.


Asunto(s)
Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Timina/análogos & derivados , Timina/metabolismo , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/metabolismo , Eliminación de Gen , Redes y Vías Metabólicas/genética , Trypanosoma brucei brucei/genética
13.
Mol Biochem Parasitol ; 191(2): 53-7, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-24080031

RESUMEN

Regions of transcription initiation and termination in kinetoplastid protists lack known eukaryotic promoter and terminator elements, although epigenetic marks such as histone variants and the modified DNA base J have been localized to these regions in Trypanosoma brucei, Trypanosoma cruzi, and/or Leishmania major. Phenotypes of base J mutants vary significantly across trypanosomatids, implying divergence in the epigenetic networks governing transcription during evolution. Here, we demonstrate that the histone variants H2A.Z and H2B.V are essential in L. major using a powerful quantitative plasmid segregation-based test. In contrast, H3.V is not essential for viability or normal growth in Leishmania. Steady-state transcript levels and the efficiency of transcription termination at convergent strand switch regions (SSRs) in H3V-null parasites were comparable to WT parasites. Our genetic tests show a conservation of histone variant phenotypes between L. major and T. brucei, unlike the diversity of phenotypes associated with genetic manipulation of the DNA base J modification.


Asunto(s)
Evolución Molecular , Histonas/metabolismo , Kinetoplastida/metabolismo , Leishmania major/metabolismo , Proteínas Protozoarias/metabolismo , Histonas/química , Histonas/genética , Kinetoplastida/química , Kinetoplastida/clasificación , Kinetoplastida/genética , Leishmania major/química , Leishmania major/clasificación , Leishmania major/genética , Estructura Terciaria de Proteína , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Especificidad de la Especie
14.
PLoS One ; 8(1): e53851, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23382856

RESUMEN

BACKGROUND: The genus Burkholderia includes pathogenic gram-negative bacteria that cause melioidosis, glanders, and pulmonary infections of patients with cancer and cystic fibrosis. Drug resistance has made development of new antimicrobials critical. Many approaches to discovering new antimicrobials, such as structure-based drug design and whole cell phenotypic screens followed by lead refinement, require high-resolution structures of proteins essential to the parasite. METHODOLOGY/PRINCIPAL FINDINGS: We experimentally identified 406 putative essential genes in B. thailandensis, a low-virulence species phylogenetically similar to B. pseudomallei, the causative agent of melioidosis, using saturation-level transposon mutagenesis and next-generation sequencing (Tn-seq). We selected 315 protein products of these genes based on structure-determination criteria, such as excluding very large and/or integral membrane proteins, and entered them into the Seattle Structural Genomics Center for Infection Disease (SSGCID) structure determination pipeline. To maximize structural coverage of these targets, we applied an "ortholog rescue" strategy for those producing insoluble or difficult to crystallize proteins, resulting in the addition of 387 orthologs (or paralogs) from seven other Burkholderia species into the SSGCID pipeline. This structural genomics approach yielded structures from 31 putative essential targets from B. thailandensis, and 25 orthologs from other Burkholderia species, yielding an overall structural coverage for 49 of the 406 essential gene families, with a total of 88 depositions into the Protein Data Bank. Of these, 25 proteins have properties of a potential antimicrobial drug target i.e., no close human homolog, part of an essential metabolic pathway, and a deep binding pocket. We describe the structures of several potential drug targets in detail. CONCLUSIONS/SIGNIFICANCE: This collection of structures, solubility and experimental essentiality data provides a resource for development of drugs against infections and diseases caused by Burkholderia. All expression clones and proteins created in this study are freely available by request.


Asunto(s)
Infecciones por Burkholderia/genética , Burkholderia pseudomallei/genética , Genómica , Redes y Vías Metabólicas/genética , Infecciones por Burkholderia/tratamiento farmacológico , Burkholderia pseudomallei/patogenicidad , Biología Computacional , Bases de Datos de Proteínas , Diseño de Fármacos , Genes Esenciales , Genoma Bacteriano , Humanos , Filogenia , Conformación Proteica
15.
Artículo en Inglés | MEDLINE | ID: mdl-23976839

RESUMEN

The ability to orient biologically active proteins on surfaces is a major challenge in the design, construction, and successful deployment of many medical technologies. As methods to orient biomolecules are developed, it is also essential to develop techniques that can an accurately determine the orientation and structure of these materials. In this study, two model protein and peptide systems are presented to highlight the strengths of three surface analysis techniques for characterizing protein films: time-of-flight secondary ion mass spectrometry (ToF-SIMS), sum-frequency generation (SFG) vibrational spectroscopy, and near-edge x-ray absorption fine structure (NEXAFS) spectroscopy. First, the orientation of Protein G B1, a rigid 6 kDa domain covalently attached to a maleimide-functionalized self-assembled monolayer, was examined using ToF-SIMS. Although the thickness of the Protein G layer was similar to the ToF-SIMS sampling depth, orientation of Protein G was successfully determined by analyzing the C2H5S+ intensity, a secondary ion derived from a methionine residue located at one end of the protein. Next, the secondary structure of a 13-mer leucine-lysine peptide (LK310) adsorbed onto hydrophilic quartz and hydrophobic fluorocarbon surfaces was examined. SFG spectra indicated that the peptide's lysine side chains were ordered on the quartz surface, while the peptide's leucine side chains were ordered on the fluorocarbon surface. NEXAFS results provided complementary information about the structure of the LK310 film and the orientations of amide bonds within the LK310 peptide.

16.
Proc Natl Acad Sci U S A ; 99(8): 5139-43, 2002 Apr 16.
Artículo en Inglés | MEDLINE | ID: mdl-11959962

RESUMEN

Evidence is emerging that mechanical stretching can alter the functional states of proteins. Fibronectin (Fn) is a large, extracellular matrix protein that is assembled by cells into elastic fibrils and subjected to contractile forces. Assembly into fibrils coincides with expression of biological recognition sites that are buried in Fn's soluble state. To investigate how supramolecular assembly of Fn into fibrillar matrix enables cells to mechanically regulate its structure, we used fluorescence resonance energy transfer (FRET) as an indicator of Fn conformation in the fibrillar matrix of NIH 3T3 fibroblasts. Fn was randomly labeled on amine residues with donor fluorophores and site-specifically labeled on cysteine residues in modules FnIII(7) and FnIII(15) with acceptor fluorophores. Intramolecular FRET was correlated with known structural changes of Fn in denaturing solution, then applied in cell culture as an indicator of Fn conformation within the matrix fibrils of NIH 3T3 fibroblasts. Based on the level of FRET, Fn in many fibrils was stretched by cells so that its dimer arms were extended and at least one FnIII module unfolded. When cytoskeletal tension was disrupted using cytochalasin D, FRET increased, indicating refolding of Fn within fibrils. These results suggest that cell-generated force is required to maintain Fn in partially unfolded conformations. The results support a model of Fn fibril elasticity based on unraveling and refolding of FnIII modules. We also observed variation of FRET between and along single fibrils, indicating variation in the degree of unfolding of Fn in fibrils. Molecular mechanisms by which mechanical force can alter the structure of Fn, converting tensile forces into biochemical cues, are discussed.


Asunto(s)
Citoesqueleto/fisiología , Fibronectinas/química , Células 3T3 , Animales , Adhesión Celular , Citocalasina D/química , Ratones , Microscopía Fluorescente , Conformación Proteica , Pliegue de Proteína , Espectrometría de Fluorescencia
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