RESUMEN
BACKGROUND: A mutually beneficial need exists between postdoctoral scholars (postdocs) who want to grow their science communication, networking, and teaching skills and those in the general health sciences research community who want to learn more about specialized topics. Recognizing this need, interdepartmental teams at two public universities began offering postdocs a teaching opportunity at their health sciences libraries, which serve as discipline-neutral learning spaces for researchers. CASE PRESENTATION: At the University of Pittsburgh (Pitt) and Virginia Commonwealth University (VCU), postdocs are invited to submit talk proposals on "how to do something" related to the health sciences. Selected postdoc speakers conduct one-hour talks, get science communication and teaching support, have their talks uploaded to YouTube, and receive feedback from attendees. CONCLUSIONS: Postdoc participants appreciated being able to participate in this program, and attendees strongly indicated that the talks are of value. At VCU, surveys of the 25 talks from 2015-2018 showed that 91% of attendees believed they had a better understanding of the topic because of their attendance, and 85% planned to use the knowledge they gained. More than a year after their talks, several postdocs across both institutions informed the coordinators that they were subsequently contacted for advice or further discussion, with 2 postdocs stating that it helped them with job opportunities. This model can be easily adapted at other health sciences libraries to benefit their academic communities.
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Investigación Biomédica , Servicios de Biblioteca , Investigadores/educación , HumanosRESUMEN
The Thermotoga maritima arginine-binding protein (TmArgBP) has been modified to create a reagentless fluorescent protein biosensor. Two design methods for biosensor construction are compared: 1) solvent accessibility of environmentally-sensitive probes and 2) fluorescence deactivation due to photo-induced electron transfer (PET). Nine single cysteine TmArgBP mutants were created and labeled with three different environmentally sensitive fluorescent probes. These mutants demonstrated limited changes in fluorescence emission upon the addition of arginine. In contrast, the PET-based biosensor provides significant enhancements over the traditional approach and provides a fluorescence quenching mechanism that was capable of providing quantitative detection of arginine. Site-directed mutagenesis of TmArgBP was used to create attachment points for the fluorescent probe (K145C) and for an internal aromatic residue (D18X) to serve as the PET quencher. Both tyrosine and tryptophan, but not phenylalanine, were able to quench the emission of the fluorescent probe by more than 80% upon the addition of arginine. The dissociation constant for arginine ranged from 0.87 to 1.5 µM across the different sensors. This PET-based strategy provides a simple and broadly applicable approach for the analytical detection of small molecules that may be applied to any protein that exhibits conformational switching in a ligand dependent manner.
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Arginina/análisis , Técnicas Biosensibles/métodos , Colorantes Fluorescentes/química , Proteínas de Unión Periplasmáticas/metabolismo , Thermotoga maritima/metabolismo , Arginina/genética , Arginina/metabolismo , Proteínas Bacterianas , Sitios de Unión , Fluorescencia , Conformación Molecular , Mutagénesis Sitio-Dirigida , Mutación/genética , Proteínas de Unión Periplasmáticas/química , Proteínas de Unión Periplasmáticas/genética , Unión Proteica , Espectrometría de Fluorescencia , Thermotoga maritima/genética , Thermotoga maritima/crecimiento & desarrollo , Triptófano/química , Triptófano/genética , Triptófano/metabolismoRESUMEN
The intracellular pathogen Toxoplasma gondii is a purine auxotroph that relies on purine salvage for proliferation. We have optimized T. gondii purine nucleoside phosphorylase (TgPNP) stability and crystallized TgPNP with phosphate and immucillin-H, a transition-state analogue that has high affinity for the enzyme. Immucillin-H bound to TgPNP with a dissociation constant of 370 pM, the highest affinity of 11 immucillins selected to probe the catalytic site. The specificity for transition-state analogues indicated an early dissociative transition state for TgPNP. Compared to Plasmodium falciparum PNP, large substituents surrounding the 5'-hydroxyl group of inhibitors demonstrate reduced capacity for TgPNP inhibition. Catalytic discrimination against large 5' groups is consistent with the inability of TgPNP to catalyze the phosphorolysis of 5'-methylthioinosine to hypoxanthine. In contrast to mammalian PNP, the 2'-hydroxyl group is crucial for inhibitor binding in the catalytic site of TgPNP. This first crystal structure of TgPNP describes the basis for discrimination against 5'-methylthioinosine and similarly 5'-hydroxy-substituted immucillins; structural differences reflect the unique adaptations of purine salvage pathways of Apicomplexa.
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Inhibidores Enzimáticos/química , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Purina-Nucleósido Fosforilasa/química , Purina-Nucleósido Fosforilasa/metabolismo , Toxoplasma/enzimología , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Cinética , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/genética , Nucleósidos de Purina/química , Nucleósidos de Purina/metabolismo , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Purina-Nucleósido Fosforilasa/genética , Pirimidinonas/química , Especificidad por Sustrato , Toxoplasma/química , Toxoplasma/genéticaRESUMEN
Plasmodium parasites rely upon purine salvage for survival. Plasmodium purine nucleoside phosphorylase is part of the streamlined Plasmodium purine salvage pathway that leads to the phosphorylysis of both purines and 5'-methylthiopurines, byproducts of polyamine synthesis. We have explored structural features in Plasmodium falciparum purine nucleoside phosphorylase (PfPNP) that affect efficiency of catalysis as well as those that make it suitable for dual specificity. We used site directed mutagenesis to identify residues critical for PfPNP catalytic activity as well as critical residues within a hydrophobic pocket required for accommodation of the 5'-methylthio group. Kinetic analysis data shows that several mutants had disrupted binding of the 5'-methylthio group while retaining activity for inosine. A triple PfPNP mutant that mimics Toxoplasma gondii PNP had significant loss of 5'-methylthio activity with retention of inosine activity. Crystallographic investigation of the triple mutant PfPNP with Tyr160Phe, Val66Ile, andVal73Ile in complex with the transition state inhibitor immucillin H reveals fewer hydrogen bond interactions for the inhibitor in the hydrophobic pocket.
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Metiltioinosina/análogos & derivados , Plasmodium falciparum/enzimología , Purina-Nucleósido Fosforilasa/química , Purina-Nucleósido Fosforilasa/metabolismo , Secuencia de Aminoácidos , Dominio Catalítico , Cinética , Metiltioinosina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Purina-Nucleósido Fosforilasa/genética , Pirimidinonas/metabolismo , Pirimidinonas/farmacología , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The Thermotoga maritima arginine binding protein (TmArgBP) is a member of the periplasmic binding protein superfamily. As a highly thermostable protein, TmArgBP has been investigated for the potential to serve as a protein scaffold for the development of fluorescent protein biosensors. To establish a relationship between structural dynamics and ligand binding capabilities, we constructed single tryptophan mutants to probe the arginine binding pocket. Trp residues placed around the binding pocket reveal a strong dependence on fluorescence emission of the protein with arginine for all but one of the mutants. Using these data, we calculated dissociation constants of 1.9-3.3 µM for arginine. Stern-Volmer quenching analysis demonstrated that the protein undergoes a large conformational change upon ligand binding, which is a common feature of this protein superfamily. While still active at room temperature, time-resolved intensity and anisotropy decay data suggest that the protein exists as a highly rigid structure under these conditions. Interestingly, TmArgBP exists as a dimer at room temperature in both the presence and absence of arginine, as determined by asymmetric flow field flow fractionation (AF4) and supported by native gel-electrophoresis and time-resolved anisotropy. Our data on dynamics and stability will contribute to our understanding of hyperthermophilic proteins and their potential biotechnological applications.
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Proteínas de Unión Periplasmáticas/genética , Thermotoga maritima , Triptófano/genética , Arginina/química , Sitios de Unión/genética , Fluorescencia , Polarización de Fluorescencia , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteínas de Unión Periplasmáticas/química , Unión Proteica , Multimerización de Proteína , Triptófano/químicaRESUMEN
Infrared spectroscopy (IR) is commonly used to study secondary structure of both peptides and proteins. The amide I band is very sensitive to peptide secondary structure, and the conformation of a peptide can be probed at the residue level by introducing site-specific isotope-labels into the peptide backbone. The replacement of a carbonyl (12)C with a (13)C results in a approximately 40 cm(-1) shift in the amide I' band. The amide I bands of specifically labeled helices should vary systematically as a function of the number and relative spacing of the labeled residues; thus one should be able to describe the conformation of a polypeptide in substantial detail by probing the changes in IR spectra as a function of the number and positioning of isotope labels. In this study, we report IR spectra of a series of differently labeled helical peptides. A series of 25mer peptides were synthesized based on the repeat sequence (AAAAK)(n). We have varied the number and spacing of the labels on each peptide and studied the changes in the (12)C and (13)C amide I' band due to label position. Our results indicate that changing the number of labels changes the frequency and intensity of both the (12)C and the (13)C amide mode. We also found that varying the spacing between labels causes these amide peaks to shift. Isotope labeling, combined with IR spectroscopy and theoretical predictions, may generate a description of peptide backbone conformations at the residue level.