RESUMO
The tRNA ligase complex (tRNA-LC) splices precursor tRNAs (pre-tRNA), and Xbp1-mRNA during the unfolded protein response (UPR). In aerobic conditions, a cysteine residue bound to two metal ions in its ancient, catalytic subunit RTCB could make the tRNA-LC susceptible to oxidative inactivation. Here, we confirm this hypothesis and reveal a co-evolutionary association between the tRNA-LC and PYROXD1, a conserved and essential oxidoreductase. We reveal that PYROXD1 preserves the activity of the mammalian tRNA-LC in pre-tRNA splicing and UPR. PYROXD1 binds the tRNA-LC in the presence of NAD(P)H and converts RTCB-bound NAD(P)H into NAD(P)+, a typical oxidative co-enzyme. However, NAD(P)+ here acts as an antioxidant and protects the tRNA-LC from oxidative inactivation, which is dependent on copper ions. Genetic variants of PYROXD1 that cause human myopathies only partially support tRNA-LC activity. Thus, we establish the tRNA-LC as an oxidation-sensitive metalloenzyme, safeguarded by the flavoprotein PYROXD1 through an unexpected redox mechanism.
Assuntos
Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , RNA Ligase (ATP)/metabolismo , RNA de Transferência/metabolismo , Animais , Antioxidantes/fisiologia , Domínio Catalítico , Feminino , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NAD/metabolismo , NADP/metabolismo , Oxirredução , Oxirredutases/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/fisiologia , RNA Ligase (ATP)/química , RNA Ligase (ATP)/genética , Splicing de RNA/genética , Splicing de RNA/fisiologia , Resposta a Proteínas não Dobradas/fisiologia , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
UFMylation involves the covalent modification of substrate proteins with UFM1 (Ubiquitin-fold modifier 1) and is important for maintaining ER homeostasis. Stalled translation triggers the UFMylation of ER-bound ribosomes and activates C53-mediated autophagy to clear toxic polypeptides. C53 contains noncanonical shuffled ATG8-interacting motifs (sAIMs) that are essential for ATG8 interaction and autophagy initiation. However, the mechanistic basis of sAIM-mediated ATG8 interaction remains unknown. Here, we show that C53 and sAIMs are conserved across eukaryotes but secondarily lost in fungi and various algal lineages. Biochemical assays showed that the unicellular alga Chlamydomonas reinhardtii has a functional UFMylation pathway, refuting the assumption that UFMylation is linked to multicellularity. Comparative structural analyses revealed that both UFM1 and ATG8 bind sAIMs in C53, but in a distinct way. Conversion of sAIMs into canonical AIMs impaired binding of C53 to UFM1, while strengthening ATG8 binding. Increased ATG8 binding led to the autoactivation of the C53 pathway and sensitization of Arabidopsis thaliana to ER stress. Altogether, our findings reveal an ancestral role of sAIMs in UFMylation-dependent fine-tuning of C53-mediated autophagy activation.
Assuntos
Peptídeos , Proteínas , Proteínas/metabolismo , Ribossomos/metabolismo , Autofagia , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismoRESUMO
BACKGROUND: Acanthamoeba spp. is the causative agent of Acanthamoeba keratitis and granulomatous amoebic encephalitis. Strathclyde minor groove binders (S-MGBs) are a promising new class of anti-infective agent that have been shown to be effective against many infectious organisms. OBJECTIVES: To synthesize and evaluate the anti-Acanthamoeba activity of a panel of S-MGBs, and therefore determine the potential of this class for further development. METHODS: A panel of 12 S-MGBs was synthesized and anti-Acanthamoeba activity was determined using an alamarBlue™-based trophocidal assay against Acanthamoeba castellanii. Cross-screening against Trypanosoma brucei brucei, Staphylococcus aureus and Escherichia coli was used to investigate selective potency. Cytotoxicity against HEK293 cells allowed for selective toxicity to be measured. DNA binding studies were carried out using native mass spectrometry and DNA thermal shift assays. RESULTS AND DISCUSSION: S-MGB-241 has an IC50 of 6.6 µM against A. castellanii, comparable to the clinically used miltefosine (5.6 µM) and negligible activity against the other organisms. It was also found to have an IC50â>â100 µM against HEK293 cells, demonstrating low cytotoxicity. S-MGB-241 binds to DNA as a dimer, albeit weakly compared to other S-MGBs previously studied. This was confirmed by DNA thermal shift assay with a ΔTmâ=â1â±â0.1°C. CONCLUSIONS: Together, these data provide confidence that S-MGBs can be further optimized to generate new, potent treatments for Acanthameoba spp. infections. In particular, S-MGB-241, has been identified as a 'hit' compound that is selectively active against A. castellanii, providing a starting point from which to begin optimization of DNA binding and potency.
RESUMO
In this application of native mass spectrometry (nMS) to investigate complexes formed by molecular glues (MGs), we have demonstrated its efficiency in delineating stoichiometric rearrangements of E3 ligases that occur during targeted protein degradation (TPD). MGs stabilise interactions between an E3 ligase and a protein of interest (POI) targeted for degradation, and these ternary interactions are challenging to characterise. We have shown that nMS can unambiguously identify complexes formed between the CRBN : DDB1 E3 ligase and the POI GSPT1 upon the addition of lenalidomide, pomalidomide or thalidomide. Ternary complex formation was also identified involving the DCAF15 : DDA1 : DDB1 E3 ligase in the presence of MG (E7820 or indisulam) and POI RBM39. Moreover, we uncovered that the DCAF15 : DDA1 : DDB1 E3 ligase self-associates into dimers and trimers when analysed alone at low salt concentrations (100 mM ammonium acetate) which dissociate into single copies of the complex at higher salt concentrations (500 mM ammonium acetate), or upon the addition of MG and POI, forming a 1 : 1 : 1 ternary complex. This work demonstrates the strength of nMS in TPD research, reveals novel binding mechanisms of the DCAF15 E3 ligase, and its self-association into dimers and trimers at reduced salt concentration during structural analysis.
Assuntos
Espectrometria de Massas , Ubiquitina-Proteína Ligases , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/química , Espectrometria de Massas/métodos , Talidomida/química , Talidomida/análogos & derivados , Humanos , Lenalidomida/química , Multimerização Proteica , Ligação ProteicaRESUMO
Liquid-liquid phase separation (LLPS) is a process by which biomacromolecules, particularly proteins, condense into a dense phase that resembles liquid droplets. Dysregulation of LLPS is implicated in disease, yet the relationship between protein conformational changes and LLPS remains difficult to discern. This is due to the high flexibility and disordered nature of many proteins that phase separate under physiological conditions and their tendency to oligomerize. Here, we demonstrate that ion mobility mass spectrometry (IM-MS) overcomes these limitations. We used IM-MS to investigate the conformational states of full-length ubiquilin-2 (UBQLN2) protein, LLPS of which is driven by high-salt concentration and reversed by noncovalent interactions with ubiquitin (Ub). IM-MS revealed that UBQLN2 exists as a mixture of monomers and dimers and that increasing salt concentration causes the UBQLN2 dimers to undergo a subtle shift toward extended conformations. UBQLN2 binds to Ub in 2:1 and 2:2 UBQLN2/Ub complexes, which have compact geometries compared to free UBQLN2 dimers. Together, these results suggest that extended conformations of UBQLN2 are correlated with UBQLN2's ability to phase separate. Overall, delineating protein conformations that are implicit in LLPS will greatly increase understanding of the phase separation process, both in normal cell physiology and disease states.
Assuntos
Fatores de Transcrição , Ubiquitina , Conformação Proteica , Espectrometria de MassasRESUMO
RATIONALE: Protein degraders are small molecules that promote cellular degradation of a target protein. Degraders simultaneously bind to their target and an E3 ligase, bringing them into close spatial proximity, but the formation of this ternary complex is difficult to measure with many biophysical techniques. METHODS: Native mass spectrometry (nMS) is an effective label-free technique to identify the complexes formed by proteolysis-targeting chimeras (PROTACs). It can monitor the formation of ternary E3-PROTAC-target complexes and detect intermediate binary species. Experiments are described using a Synapt G2Si (Waters) equipped with a nano-electrospray ionisation source. RESULTS: The protocol describes nMS experiments for measuring the complexes formed by PROTAC molecules. It also describes how to investigate differences in the affinity of PROTAC complexes, whether a PROTAC shows specificity for a given target and whether a PROTAC shows cooperative behaviour. CONCLUSIONS: Here, we provide step-by-step instructions for the sample preparation of PROTAC complexes and their nMS interrogation to obtain optimal information on their binding modes.
Assuntos
Quimera de Direcionamento de Proteólise , Manejo de Espécimes , Proteólise , Espectrometria de MassasRESUMO
Chromosome segregation depends on sister chromatid cohesion which is established by cohesin during DNA replication. Cohesive cohesin complexes become acetylated to prevent their precocious release by WAPL before cells have reached mitosis. To obtain insight into how DNA replication, cohesion establishment and cohesin acetylation are coordinated, we analysed the interaction partners of 55 human proteins implicated in these processes by mass spectrometry. This proteomic screen revealed that on chromatin the cohesin acetyltransferase ESCO2 associates with the MCM2-7 subcomplex of the replicative Cdc45-MCM-GINS helicase. The analysis of ESCO2 mutants defective in MCM binding indicates that these interactions are required for proper recruitment of ESCO2 to chromatin, cohesin acetylation during DNA replication, and centromeric cohesion. We propose that MCM binding enables ESCO2 to travel with replisomes to acetylate cohesive cohesin complexes in the vicinity of replication forks so that these complexes can be protected from precocious release by WAPL Our results also indicate that ESCO1 and ESCO2 have distinct functions in maintaining cohesion between chromosome arms and centromeres, respectively.
Assuntos
Acetiltransferases/metabolismo , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , Acetilação , Proteínas de Ciclo Celular/metabolismo , Humanos , Mitose/genética , CoesinasRESUMO
Myoglobin (Mb) can react with hydrogen peroxide (H2 O2 ) to form a highly active intermediate compound and catalyse oxidation reactions. To enhance this activity, known as pseudo-peroxidase activity, previous studies have focused on the modification of key amino acid residues of Mb or the heme cofactor. In this work, the Mb scaffold (apo-Mb) was systematically reconstituted with a set of cofactors based on six metal ions and two ligands. These Mb variants were fully characterised by UV-Vis spectroscopy, circular dichroism (CD) spectroscopy, inductively coupled plasma mass spectrometry (ICP-MS) and native mass spectrometry (nMS). The steady-state kinetics of guaiacol oxidation and 2,4,6-trichlorophenol (TCP) dehalogenation catalysed by Mb variants were determined. Mb variants with iron chlorin e6 (Fe-Ce6) and manganese chlorin e6 (Mn-Ce6) cofactors were found to have improved catalytic efficiency for both guaiacol and TCP substrates in comparison with wild-type Mb, i. e. Fe-protoporphyrin IX-Mb. Furthermore, the selected cofactors were incorporated into the scaffold of a Mb mutant, swMb H64D. Enhanced peroxidase activity for both substrates were found via the reconstitution of Fe-Ce6 into the mutant scaffold.
Assuntos
Peróxido de Hidrogênio , Mioglobina , Aminoácidos , Guaiacol , Heme/química , Peróxido de Hidrogênio/química , Manganês , Mioglobina/química , Mioglobina/genética , Mioglobina/metabolismo , Peroxidases/metabolismoRESUMO
BACKGROUND: Previously, we evaluated the intracellular mycobactericidal activity of the minor groove binder, S-MGB-364 against the clinical Mycobacterium tuberculosis (Mtb) strain HN878 in macrophages. OBJECTIVES: To assess the mycobactericidal activity of S-MGB-364 in Mtb-infected mice. Further, we investigated a plausible DNA binding mechanism of action of S-MGB-364. METHODS: The anti-TB and host immune effects of intranasal S-MGB-364 or S-MGB-364 encapsulated in non-ionic surfactant vesicles (NIV) were assessed in Mtb-infected mice by cfu enumeration, ELISA, histology, and flow cytometry. DNA binding was examined using native mass spectrometry and UV-vis thermal melt determination. S-MGB interference with DNA-centric biological events was assessed using a representative panel of Mtb and human topoisomerase I, and gyrase assays. RESULTS: S-MGB-364 bound strongly to DNA as a dimer, significantly increasing the stability of the DNA:S-MGB complex compared with DNA alone. Moreover, S-MGB-364 inhibited the relaxation of Mtb topoisomerase I but not the human form. In macrophages, S-MGB-364 or S-MGB-364-NIV did not cause DNA damage as shown by the low γ-H2AX expression. Importantly, in the lungs, the intranasal administration of S-MGB-364 or S-MGB-364-NIV formulation in Mtb-infected mice was non-toxic and resulted in a â¼1 log cfu reduction in mycobacterial burden, reduced the expression of proinflammatory cytokines/chemokines, altered immune cell recruitment, and importantly reduced recruitment of neutrophils. CONCLUSIONS: Together, these data provide proof of concept for S-MGBs as novel anti-TB therapeutics in vivo.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Animais , Antituberculosos/farmacologia , Imunidade , Macrófagos/microbiologia , Camundongos , Tuberculose/tratamento farmacológico , Tuberculose/microbiologiaRESUMO
The neglected tropical disease leishmaniasis, caused by Leishmania spp., is becoming more problematic due to the emergence of drug-resistant strains. Therefore, new drugs to treat leishmaniasis, with novel mechanisms of action, are urgently required. Strathclyde minor groove binders (S-MGBs) are an emerging class of anti-infective agent that have been shown to have potent activity against various bacteria, viruses, fungi and parasites. Herein, it is shown that S-MGBs have potent activity against L. donovani, and that an N-oxide derivation of the tertiary amine tail of typical S-MGBs leads to selective anti-leishmanial activity. Additionally, using S-MGB-219, the N-oxide derivation is shown to retain strong binding to DNA as a 2:1 dimer. These findings support the further study of anti-leishmanial S-MGBs as novel therapeutics.
Assuntos
Leishmania , Óxidos , Aminas , DNA/metabolismo , Leishmania/metabolismoRESUMO
Protein phosphorylation regulates key processes in all organisms. In Gram-positive bacteria, protein arginine phosphorylation plays a central role in protein quality control by regulating transcription factors and marking aberrant proteins for degradation. Here, we report structural, biochemical, and in vivo data of the responsible kinase, McsB, the founding member of an arginine-specific class of protein kinases. McsB differs in structure and mechanism from protein kinases that act on serine, threonine, and tyrosine residues and instead has a catalytic domain related to that of phosphagen kinases (PhKs), metabolic enzymes that phosphorylate small guanidino compounds. In McsB, the PhK-like phosphotransferase domain is structurally adapted to target protein substrates and is accompanied by a novel phosphoarginine (pArg)-binding domain that allosterically controls protein kinase activity. The identification of distinct pArg reader domains in this study points to a remarkably complex signaling system, thus challenging simplistic views of bacterial protein phosphorylation.
Assuntos
Arginina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Arginina/química , Modelos Moleculares , FosforilaçãoRESUMO
The bacterial ubiD and ubiX or the homologous fungal fdc1 and pad1 genes have been implicated in the non-oxidative reversible decarboxylation of aromatic substrates, and play a pivotal role in bacterial ubiquinone (also known as coenzyme Q) biosynthesis or microbial biodegradation of aromatic compounds, respectively. Despite biochemical studies on individual gene products, the composition and cofactor requirement of the enzyme responsible for in vivo decarboxylase activity remained unclear. Here we show that Fdc1 is solely responsible for the reversible decarboxylase activity, and that it requires a new type of cofactor: a prenylated flavin synthesized by the associated UbiX/Pad1. Atomic resolution crystal structures reveal that two distinct isomers of the oxidized cofactor can be observed, an isoalloxazine N5-iminium adduct and a N5 secondary ketimine species with markedly altered ring structure, both having azomethine ylide character. Substrate binding positions the dipolarophile enoic acid group directly above the azomethine ylide group. The structure of a covalent inhibitor-cofactor adduct suggests that 1,3-dipolar cycloaddition chemistry supports reversible decarboxylation in these enzymes. Although 1,3-dipolar cycloaddition is commonly used in organic chemistry, we propose that this presents the first example, to our knowledge, of an enzymatic 1,3-dipolar cycloaddition reaction. Our model for Fdc1/UbiD catalysis offers new routes in alkene hydrocarbon production or aryl (de)carboxylation.
Assuntos
Biocatálise , Carboxiliases/metabolismo , Reação de Cicloadição , Alcenos/química , Alcenos/metabolismo , Aspergillus niger/enzimologia , Aspergillus niger/genética , Carboxiliases/química , Carboxiliases/genética , Cristalografia por Raios X , Descarboxilação , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Flavinas/biossíntese , Flavinas/química , Flavinas/metabolismo , Isomerismo , Ligantes , Modelos Moleculares , Ubiquinona/biossínteseRESUMO
The global dimensions and amplitudes of conformational fluctuations of intrinsically disordered proteins are governed, in part, by the linear segregation versus clustering of oppositely charged residues within the primary sequence. Ion mobility-mass spectrometry (IM-MS) affords unique advantages for probing the conformational consequences of the linear patterning of oppositely charged residues because it measures and separates proteins electrosprayed from solution on the basis of charge and shape. Here, we use IM-MS to measure the conformational consequences of charge patterning on the C-terminal intrinsically disordered region (p27 IDR) of the cell cycle inhibitory protein p27Kip1. We report the range of charge states and accompanying collisional cross section distributions for wild-type p27 IDR and two variants with identical amino acid compositions, κ14 and κ56, distinguished by the extent of linear mixing versus segregation of oppositely charged residues. Wild-type p27 IDR (κ31) and κ14, where the oppositely charged residues are more evenly distributed, exhibit a broad distribution of charge states. This is concordant with high degrees of conformational heterogeneity in solution. By contrast, κ56 with linear segregation of oppositely charged residues leads to limited conformational heterogeneity and a narrow distribution of charged states. Gas-phase molecular dynamics simulations demonstrate that the interplay between chain solvation and intrachain interactions (self-solvation) leads to conformational distributions that are modulated by salt concentration, with the wild-type sequence showing the most sensitivity to changes in salt concentration. These results suggest that the charge patterning within the wild-type p27 IDR may be optimized to sample both highly solvated and self-solvated conformational states.
Assuntos
Proteínas Intrinsicamente Desordenadas/química , Espectrometria de Massas , Inibidor de Quinase Dependente de Ciclina p27/química , Simulação de Dinâmica Molecular , Conformação ProteicaRESUMO
Intrinsically disordered proteins have been reported to undergo disorder-to-order transitions upon binding to their partners in the cell. The extent of the ordering upon binding and the lack of order prior to binding is difficult to visualize with classical structure determination methods. Binding of p27 to the Cdk2/cyclinâ A complex is accompanied by partial folding of p27 in the KID domain, with the retention of dynamic behavior for function, particularly in the C-terminal half of the protein. Herein, native ion mobility mass spectrometry (IM-MS) is employed to measure the intrinsic dynamic properties of p27, both in isolation and within the trimeric complex with Cdk2/cyclinâ A. The trimeric Cdk2/cyclinâ A/p27-KID complex possesses significant structural heterogeneity compared to Cdk2/cyclinâ A. These findings support the formation of a fuzzy complex in which both the N- and C-termini of p27 interact with Cdk2/cyclinâ A in multiple, closely associated states.
Assuntos
Ciclina A/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Ciclina A/química , Quinase 2 Dependente de Ciclina/química , Inibidor de Quinase Dependente de Ciclina p27/química , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Espectrometria de Mobilidade Iônica , Espectrometria de Massas , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas , Multimerização ProteicaRESUMO
In recent years both mass spectrometry (MS) and ion mobility mass spectrometry (IM-MS) have been developed as techniques with which to study proteins that lack a fixed tertiary structure but may contain regions that form secondary structure elements transiently, namely intrinsically disordered proteins (IDPs). IM-MS is a suitable method for the study of IDPs which provides an insight to conformations that are present in solution, potentially enabling the analysis of lowly populated structural forms. Here, we describe the IM-MS data of two IDPs; α-Synuclein (α-Syn) which is implicated in Parkinson's disease, and Apolipoprotein C-II (ApoC-II) which is involved in cardiovascular diseases. We report an apparent discrepancy in the way that ApoC-II behaves in the gas phase. While most IDPs, including α-Syn, present in many charge states and a wide range of rotationally averaged collision cross sections (CCSs), ApoC-II presents in just four charge states and a very narrow range of CCSs, independent of solution conditions. Here, we compare MS and IM-MS data of both proteins, and rationalise the differences between the proteins in terms of different ionisation processes which they may adhere to.
Assuntos
Medição da Troca de Deutério/métodos , Proteínas Intrinsicamente Desordenadas/análise , Proteínas Intrinsicamente Desordenadas/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Sequência de Aminoácidos , Apolipoproteína C-II , Gases , Humanos , Dados de Sequência Molecular , Doença de Parkinson , Conformação Proteica , alfa-SinucleínaRESUMO
In the past decade, mass spectrometry (MS) coupled with electrospray ionization (ESI) has been extensively applied to the study of intact proteins and their complexes, often without the requirement of labels. Solvent conditions (for example, pH, ionic strength, and concentration) affect the observed desolvated species; the ease of altering such extrinsic factors renders ESI-MS an appropriate method by which to consider the range of conformational states that proteins may occupy, including natively folded, disordered and amyloid. Rotationally averaged collision cross sections of the ionized forms of proteins, provided by the combination of mass spectrometry and ion mobility (IM-MS), are also instructive in exploring conformational landscapes in the absence of solvent. Here, we ask the following question: "If the only technique you had was ESI-IM-MS, what information would it provide on the structural preferences of an unknown protein?" We have selected 20 different proteins, both monomeric and multimeric, ranging in mass from 2846 Da (melittin) to 150 kDa (Immunoglobulin G), and we consider how they are presented to a mass spectrometer under different solvent conditions. Mass spectrometery allows us to distinguish which of these proteins are structured (melittin, human beta defensin 1, truncated human lymphotactin, Cytochrome C, holo hemoglobin-α, ovalbumin, human transthyretin, avidin, bovine serum albumin, concanavalin, human serum amyloid protein, and Immunoglobulin G) from those that contain at least some regions of disorder (human lymphotactin, N-terminal p53, α-Synuclein, N-terminal MDM2, and p53 DNA binding domain) or denatured due to solvent conditions (ubiquitin, apo hemoglobin-α, apo hemoglobin-ß) by considering two experimental parameters: the range of charge states occupied by the protein (Δz) and the range of collision cross sections in which the protein is observed (ΔCCS). We also provide a simple model to predict the difference between the collision cross sections of the most compact and the most extended form of a given protein, based on the volume of the amino acids it contains. We compare these calculated parameters with experimental values. In addition, we consider the occupancy of conformations based on the intensities of ions in the mass spectra. This allows us to qualitatively predict the potential energy landscape of each protein. Our empirical approach to assess order or disorder is shown to be more accurate than the use of charge hydropathy plots, which are frequently used to predict disorder, and could provide an initial route to characterization. Finally, we present an ESI-IM-MS methodology to determine if a given protein is structured or disordered.
Assuntos
Espectrometria de Massas , Conformação Proteica , Proteínas/análise , Proteínas/químicaRESUMO
There is mounting evidence that crystal nucleation from supersaturated solution involves the formation and reorganization of prenucleation clusters, contradicting classical nucleation theory. One of the key unresolved issues pertains to the origin, composition, and structure of these clusters. Here, a range of amino acids and peptides is investigated using light scattering, mass spectrometry, and in situ terahertz Raman spectroscopy, showing that the presence of amorphous aggregates is a general phenomenon in supersaturated solutions. Significantly, these aggregates are found on a vast range of length scales from dimers to 30-mers to the nanometre and even micrometre scale, implying a continuous distribution throughout this range. Larger amorphous aggregates are sites of spontaneous crystal nucleation and act as intermediates for laser-induced crystal nucleation. These results are shown to be consistent with a nonclassical nucleation model in which barrierless (homogeneous) nucleation of amorphous aggregates is followed by the nucleation of crystals from solute-enriched aggregates. This provides a novel perspective on crystal nucleation and the role of nonclassical pathways.
RESUMO
Protein-protein interactions of c-Myc (MYC) are often regulated by post-translational modifications (PTMs), such as phosphorylation, and crosstalk thereof. Studying these interactions requires proteins with unique PTM patterns, which are challenging to obtain by recombinant methods. Standard peptide synthesis and native chemical ligation can produce such modified proteins, but are time-consuming and therefore typically limited to the study of individual PTMs. Herein, we report the development of flow-based methods for the rapid synthesis of phosphorylated MYC sequences (up to 84 AA), and demonstrate the versatility of this approach for the incorporation of other PTMs (N ε-methylation, sulfation, acetylation, glycosylation) and combinations thereof. Peptides containing up to seven PTMs and phosphorylation at up to five sites were successfully prepared and isolated in high yield and purity. We further produced ten PTM-decorated analogues of the MYC Transactivation Domain (TAD) to screen for binding to the tumor suppressor protein, Bin1, using heteronuclear NMR and native mass spectrometry. We determined the effects of phosphorylation and glycosylation on the strength of the MYC:Bin1 interaction, and reveal an influence of MYC sequence length on binding. Our platform for the rapid synthesis of MYC sequences up to 84 AA with distinct PTM patterns thus enables the systematic study of PTM function at a molecular level, and offers a convenient way for expedited screening of constructs.
RESUMO
In the last ten years mass spectrometry has emerged as a powerful biophysical technique capable of providing unique insights into the structure and dynamics of proteins. Part of this explosion in use involves investigations of the most recently 'discovered' subset of proteins: the so-called 'Intrinsically Disordered' or 'Natively Unstructured' proteins. A key advantage of the use of mass spectrometry to study intrinsically disordered proteins (IDPs) is its ability to test biophysical assertions made about why they differ from structured proteins. For example, from the charge state distribution presented by a protein following nano-electrospray (n-ESI) it is possible to infer the range of conformations present in solution and hence the extent of disorder; n-ESI is highly sensitive to the degree of folding at the moment of transfer from the liquid to the gas phase. The combination of mass spectrometry with ion mobility (IM-MS) provides rotationally averaged collision cross-sections of molecular ions which can be correlated with conformation; this too can be applied to IDPs. Another feature which can be monitored by IM-MS is the tendency of disordered proteins to form amyloid fibrils, the protein aggregates involved in the onset of neurodegenerative diseases such as Parkinson's and Alzheimer's. IM-MS provides a useful insight into events that occur during the early stages of aggregation including delineating the structure of the monomer, identifying oligomer distributions, and revealing mechanistic details of the aggregation process. Here we will review the use of MS and IM-MS to study IDPs using examples from our own and other laboratories.
Assuntos
Espectrometria de Massas/métodos , Proteínas/química , Animais , Medição da Troca de Deutério , Elétrons , Humanos , Proteínas/metabolismo , Proteólise , ProteômicaRESUMO
Mass spectrometry (MS) is now established as an analytical tool to interrogate the structure and dynamics of proteins and their assemblies. An array of MS-based technologies has been developed, with each providing unique information pertaining to protein structure, and forming the heart of integrative structural biology studies. This special issue includes a collection of review articles that discuss both established and emerging structural MS methodologies, along with examples of how these technologies are being deployed to interrogate protein structure and function. Combined, this collection highlights the immense potential of the structural MS toolkit in the study of molecular mechanisms underpinning cellular homeostasis and disease.