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1.
Anal Chem ; 93(9): 4246-4254, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33592142

RESUMO

The data analysis practices associated with hydrogen-deuterium exchange mass spectrometry (HX-MS) lag far behind that of most other MS-based protein analysis tools. A reliance on external tools from other fields and a persistent need for manual data validation restrict this powerful technology to the expert user. Here, we provide an extensive upgrade to the HX data analysis suite available in the Mass Spec Studio in the form of two new apps (HX-PIPE and HX-DEAL), completing a workflow that provides an HX-tailored peptide identification capability, accelerated validation routines, automated spectral deconvolution strategies, and a rich set of exportable graphics and statistical reports. With these new tools, we demonstrate that the peptide identifications obtained from undeuterated samples generated at the start of a project contain information that helps predict and control the extent of manual validation required. We also uncover a large fraction of HX-usable peptides that remains unidentified in most experiments. We show that automated spectral deconvolution routines can identify exchange regimes in a project-wide manner, although they remain difficult to accurately assign in all scenarios. Taken together, these new tools provide a robust and complete solution suitable for the analysis of high-complexity HX-MS data.

2.
Nat Commun ; 11(1): 6233, 2020 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-33277478

RESUMO

The KEOPS complex, which is conserved across archaea and eukaryotes, is composed of four core subunits; Pcc1, Kae1, Bud32 and Cgi121. KEOPS is crucial for the fitness of all organisms examined. In humans, pathogenic mutations in KEOPS genes lead to Galloway-Mowat syndrome, an autosomal-recessive disease causing childhood lethality. Kae1 catalyzes the universal and essential tRNA modification N6-threonylcarbamoyl adenosine, but the precise roles of all other KEOPS subunits remain an enigma. Here we show using structure-guided studies that Cgi121 recruits tRNA to KEOPS by binding to its 3' CCA tail. A composite model of KEOPS bound to tRNA reveals that all KEOPS subunits form an extended tRNA-binding surface that we have validated in vitro and in vivo to mediate the interaction with the tRNA substrate and its modification. These findings provide a framework for understanding the inner workings of KEOPS and delineate why all KEOPS subunits are essential.


Assuntos
Proteínas Arqueais/química , Methanocaldococcus/metabolismo , Complexos Multiproteicos/química , RNA de Transferência/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Cristalografia por Raios X , Methanocaldococcus/genética , Modelos Moleculares , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA de Transferência de Lisina/química , RNA de Transferência de Lisina/genética , RNA de Transferência de Lisina/metabolismo
3.
J Proteomics ; 225: 103844, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32480078

RESUMO

Structural Mass Spectrometry (SMS) provides a comprehensive toolbox for the analysis of protein structure and function. It offers multiple sources of structural information that are increasingly useful for integrative structural modeling of complex protein systems. As MS-based structural workflows scale to larger systems, consistent and coherent data interpretation resources are needed to better support modeling. Unlike the proteomics community, practitioners of SMS lack adequate computational tools. Here, we review new developments in the Mass Spec Studio: an expandable ecosystem of workflows for the analysis of complementary SMS techniques with linkages to modeling. Current functionality in the Studio (version 2) supports three major SMS workflows (crosslinking, hydrogen/deuterium exchange and covalent labelling) and two pipelines for structural modeling, with a special focus on data integration. The Mass Spec Studio is an architecture focused on rapid and robust extension of functionality by a community of developers. SIGNIFICANCE: This review surveys the new data analysis capabilities within the Mass Spec Studio, a rich framework for rapid software development specifically targeting the community of structural proteomics and structural mass spectrometry. Updates to crosslinking, hydrogen/deuterium-exchange and covalent labeling apps are provided as well as a utility for translating such analyses into restraints that support integrative structural modeling. These new capabilities, together with the underlying design tools and content, provide the community with a wealth of resources to tackle complex structural problem and design new approaches to data analysis.


Assuntos
Ecossistema , Proteínas , Espectrometria de Massas , Proteômica , Software
4.
PLoS Pathog ; 13(3): e1006244, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28257520

RESUMO

Lactoferrin binding protein B (LbpB) is a bi-lobed outer membrane-bound lipoprotein that comprises part of the lactoferrin (Lf) receptor complex in Neisseria meningitidis and other Gram-negative pathogens. Recent studies have demonstrated that LbpB plays a role in protecting the bacteria from cationic antimicrobial peptides due to large regions rich in anionic residues in the C-terminal lobe. Relative to its homolog, transferrin-binding protein B (TbpB), there currently is little evidence for its role in iron acquisition and relatively little structural and biophysical information on its interaction with Lf. In this study, a combination of crosslinking and deuterium exchange coupled to mass spectrometry, information-driven computational docking, bio-layer interferometry, and site-directed mutagenesis was used to probe LbpB:hLf complexes. The formation of a 1:1 complex of iron-loaded Lf and LbpB involves an interaction between the Lf C-lobe and LbpB N-lobe, comparable to TbpB, consistent with a potential role in iron acquisition. The Lf N-lobe is also capable of binding to negatively charged regions of the LbpB C-lobe and possibly other sites such that a variety of higher order complexes are formed. Our results are consistent with LbpB serving dual roles focused primarily on iron acquisition when exposed to limited levels of iron-loaded Lf on the mucosal surface and effectively binding apo Lf when exposed to high levels at sites of inflammation.


Assuntos
Proteína B de Ligação a Transferrina/química , Proteína B de Ligação a Transferrina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Interferometria , Ferro/metabolismo , Espectrometria de Massas , Modelos Moleculares , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Neisseria meningitidis/química , Neisseria meningitidis/metabolismo , Ligação Proteica
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