ABSTRACT
Knotted proteins are fascinating natural biomolecules whose backbones entangle themselves in a knot. Their particular knotted configurations provide them with a wide range of topological features. However, their folding/unfolding mechanisms, stability, and function are poorly understood. In the present work, native trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) was used for characterizing structural features of two model knotted proteins: a Gordian 52 knot ubiquitin C-terminal hydrolase (UCH) and a Stevedore 61 knot (α-haloacid dehalogenase, DehI). Experimental results showed structural transitions of UCH and DehI as a function of solution composition (0-50% MeOH) and temperature (T â¼20-95 °C). An increase in the protein charge states and collision cross sections (â¼2750-8750 Å2 and â¼3250-15,385 Å2 for UCH and DehI, respectively) with the solution organic content (OC) and temperature suggested a three-step unfolding pathway with at least four structural transitions. Results also showed that the integrity of the UCH knot core was more resistant to thermal unfolding when compared to DehI; however, both knot cores can be disrupted with the increase in the solution OC. Additional enzymatic digestion experiments using carboxypeptidase Y combined with molecular dynamics simulations showed that the knot core was preserved between Glu20 and Glu188 and Arg89 and His304 residues for UCH and DehI, respectively, where disruption of the knot core led to structural collapse followed by unfolding events. This work highlights the potential of solution OC and temperature studies combined with native TIMS-MS for the comprehensive characterization of knotted proteins to gain a better understanding of their structural transitions.
ABSTRACT
α-Synuclein is an intrinsically disordered protein that plays a critical role in the pathogenesis of neurodegenerative disorders, such as Parkinson's disease. Proteomics studies of human brain samples have associated the modification of the O-linked N-acetyl-glucosamine (O-GlcNAc) to several synucleinopathies; in particular, the position of the O-GlcNAc can regulate protein aggregation and subsequent cell toxicity. There is a need for site specific O-GlcNAc α-synuclein screening tools to direct better therapeutic strategies. In the present work, for the first time, the potential of fast, high-resolution trapped ion mobility spectrometry (TIMS) preseparation in tandem with mass spectrometry assisted by an electromagnetostatic (EMS) cell, capable of electron capture dissociation (ECD), and ultraviolet photodissociation (213 nm UVPD) is illustrated for the characterization of α-synuclein positional glycoforms: T72, T75, T81, and S87 modified with a single O-GlcNAc. Top-down 213 nm UVPD and ECD MS/MS experiments of the intact proteoforms showed specific product ions for each α-synuclein glycoforms associated with the O-GlcNAc position with a sequence coverage of â¼68 and â¼82%, respectively. TIMS-MS profiles of α-synuclein and the four glycoforms exhibited large structural heterogeneity and signature patterns across the 8+-15+ charge state distribution; however, while the α-synuclein positional glycoforms showed signature mobility profiles, they were only partially separated in the mobility domain. Moreover, a middle-down approach based on the Val40-Phe94 (55 residues) chymotrypsin proteolytic product using tandem TIMS-q-ECD-TOF MS/MS permitted the separation of the parent positional isomeric glycoforms. The ECD fragmentation of the ion mobility and m/z separated isomeric Val40-Phe94 proteolytic peptides with single O-GlcNAc in the T72, T75, T81, and S87 positions provided the O-GlcNAc confirmation and positional assignment with a sequence coverage of â¼80%. This method enables the high-throughput screening of positional glycoforms and further enhances the structural mass spectrometry toolbox with fast, high-resolution mobility separations and 213 nm UVPD and ECD fragmentation capabilities.
Subject(s)
Parkinson Disease , alpha-Synuclein , Humans , alpha-Synuclein/chemistry , Tandem Mass Spectrometry/methods , Parkinson Disease/metabolism , Peptides/metabolism , Proteolysis , Peptide Hydrolases/metabolismABSTRACT
There is a growing demand for lower-cost, benchtop analytical instruments with complementary separation capabilities for the screening and characterization of biological samples. In this study, we report on the custom integration of trapped ion mobility spectrometry and ultraviolet photodissociation capabilities in a commercial Paul quadrupolar ion trap multistage mass spectrometer (TIMS-QIT-MSn UVPD platform). A gated TIMS operation allowed for the accumulation of ion mobility separated ion in the QIT, followed by a mass analysis (MS1 scan) or m/z isolation, followed by selected collision induced dissociation (CID) or ultraviolet photodissociation (UVPD) and a mass analysis (MS2 scan). The analytical potential of this platform for the analysis of complex and labile biological samples is illustrated for the case of positional isomers with varying PTM location of the histone H4 tryptic peptide 4-17 singly and doubly acetylated and the histone H3.1 tail (1-50) singly trimethylated. For all cases, a baseline ion mobility precursor molecular ion preseparation was obtained. The tandem CID and UVPD MS2 allowed for effective sequence confirmation as well as the identification of reporter fragment ions associated with the PTM location; a higher sequence coverage was obtained using UVPD when compared to CID. Different from previous IMS-MS implementation, the novel TIMS-QIT-MSn UVPD platform offers a lower-cost alternative for the structural characterization of biological molecules that can be widely disseminated in clinical laboratories.
Subject(s)
Ion Mobility Spectrometry , Ultraviolet Rays , Mass Spectrometry/methods , Peptides/chemistry , Ions , HistonesABSTRACT
Here, we reported a spontaneous reaction between anticancer drug doxorubicin and GTP or dGTP. Incubation of doxorubicin with GTP or dGTP at 37 °C or above yields a covalent product: the doxorubicin-GTP or -dGTP conjugate where a covalent bond is formed between the C14 position of doxorubicin and the 2-amino group of guanine. Density functional theory calculations show the feasibility of this spontaneous reaction. Fluorescence imaging studies demonstrate that the doxorubicin-GTP and -dGTP conjugates cannot enter nuclei although they rapidly accumulate in human SK-OV-3 and NCI/ADR-RES cells. Consequently, the doxorubicin-GTP and -dGTP conjugates are less cytotoxic than doxorubicin. We also demonstrate that doxorubicin binds to ATP, GTP, and other nucleotides with a dissociation constant (Kd) in the sub-millimolar range. Since human cells contain millimolar levels of ATP and GTP, these results suggest that doxorubicin may target ATP and GTP, energy molecules that support essential processes in living organisms.
Subject(s)
Antineoplastic Agents , Humans , Antineoplastic Agents/pharmacology , Doxorubicin/pharmacology , Deoxyguanine Nucleotides/metabolism , Guanosine Triphosphate/metabolism , Adenosine TriphosphateABSTRACT
Post-translational modifications (PTMs) on intact histones play a major role in regulating chromatin dynamics and influence biological processes such as DNA transcription, replication, and repair. The nature and position of each histone PTM is crucial to decipher how this information is translated into biological response. In the present work, the potential of a novel tandem top-"double-down" approachâultraviolet photodissociation followed by mobility and mass-selected electron capture dissociation and mass spectrometry (UVPD-TIMS-q-ECD-ToF MS/MS)âis illustrated for the characterization of HeLa derived intact histone H4 proteoforms. The comparison between q-ECD-ToF MS/MS spectra and traditional Fourier-transform-ion cyclotron resonance-ECD MS/MS spectra of a H4 standard showed a similar sequence coverage (â¼75%) with significant faster data acquisition in the ToF MS/MS platform (â¼3 vs â¼15 min). Multiple mass shifts (e.g., 14 and 42 Da) were observed for the HeLa derived H4 proteoforms for which the top-down UVPD and ECD fragmentation analysis were consistent in detecting the presence of acetylated PTMs at the N-terminus and Lys5, Lys8, Lys12, and Lys16 residues, as well as methylated, dimethylated, and trimethylated PTMs at the Lys20 residue with a high sequence coverage (â¼90%). The presented top-down results are in good agreement with bottom-up TIMS ToF MS/MS experiments and allowed for additional description of PTMs at the N-terminus. The integration of a 213 nm UV laser in the present platform allowed for UVPD events prior to the ion mobility-mass precursor separation for collision-induced dissociation (CID)/ECD-ToF MS. Selected c305+ UVPD fragments, from different H4 proteoforms (e.g., Ac + Me2, 2Ac + Me2 and 3Ac + Me2), exhibited multiple IMS bands for which similar CID/ECD fragmentation patterns per IMS band pointed toward the presence of conformers, adopting the same PTM distribution, with a clear assignment of the PTM localization for each of the c305+ UVPD fragment H4 proteoforms. These results were consistent with the biological "zip" model, where acetylation proceeds in the Lys16 to Lys5 direction. This novel platform further enhances the structural toolbox with alternative fragmentation mechanisms (UVPD, CID, and ECD) in tandem with fast, high-resolution mobility separations and shows great promise for global proteoform analysis.
Subject(s)
Histones , Tandem Mass Spectrometry , Humans , Histones/chemistry , Tandem Mass Spectrometry/methods , Electrons , Protein Processing, Post-Translational , Fourier AnalysisABSTRACT
The mammalian high mobility group protein AT-hook 2 (HMGA2) is an intrinsically disordered DNA-binding protein expressed during embryogenesis. In the present work, the conformational and binding dynamics of HMGA2 and HMGA2 in complex with a 22-nt (DNA22) and a 50-nt (DNA50) AT-rich DNA hairpin were investigated using trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) under native starting solvent conditions (e.g., 100 mM aqueous NH4Ac) and collision-induced unfolding/dissociation (CIU/CID) as well as solution fluorescence anisotropy to assess the role of the DNA ligand when binding to the HMGA2 protein. CIU-TIMS-CID-MS/MS experiments showed a significant reduction of the conformational space and charge-state distribution accompanied by an energy stability increase of the native HMGA2 upon DNA binding. Fluorescence anisotropy experiments and CIU-TIMS-CID-MS/MS demonstrated for the first time that HMGA2 binds with high affinity to the minor groove of AT-rich DNA oligomers and with lower affinity to the major groove of AT-rich DNA oligomers (minor groove occupied by a minor groove binder Hoechst 33258). The HMGA2·DNA22 complex (18.2 kDa) 1:1 and 1:2 stoichiometry suggests that two of the AT-hook sites are accessible for DNA binding, while the other AT-hook site is probably coordinated by the C-terminal motif peptide (CTMP). The HMGA2 transition from disordered to ordered upon DNA binding is driven by the interaction of the three basic AT-hook residues with the minor and/or major grooves of AT-rich DNA oligomers.
Subject(s)
HMGA2 Protein , Ion Mobility Spectrometry , Animals , DNA/chemistry , HMGA2 Protein/chemistry , HMGA2 Protein/metabolism , Mammals/genetics , Mammals/metabolism , Tandem Mass SpectrometryABSTRACT
Trapped ion mobility spectrometry (TIMS) when coupled with mass spectrometry (MS) offers great advantages for the separation of isobaric, isomeric, and/or conformeric species. In the present work, we report the advantages of coupling TIMS with a low-cost, ultraviolet photodissociation (UVPD) linear ion trap operated at few mbars prior to time-of-flight (ToF) MS analysis for the effective characterization of isobaric, isomeric, and/or conformeric species based on mobility-selected fragmentation patterns. These three traditional challenges to MS-based separations are illustrated for the case of biologically relevant model systems: H3.1 histone tail PTM isobars (K4Me3/K18Ac), lanthipeptide regioisomers (overlapping/nonoverlapping ring patterns), and a model peptide conformer (angiotensin I). The sequential nature of the TIMS operation allows for effective synchronization with the ToF MS scans, in addition to parallel operation between the TIMS and the UVPD trap. Inspection of the mobility-selected UVPD MS spectra showed that for all three cases considered, unique fragmentation patterns (fingerprints) were observed per mobility band. Different from other IMS-UVPD implementations, the higher resolution of the TIMS device allowed for high mobility resolving power (R > 100) and effective mobility separation. The mobility selected UVPD MS provided high sequence coverage (>85%) with a fragmentation efficiency up to â¼40%.
Subject(s)
Ion Mobility Spectrometry , Peptides , Ion Mobility Spectrometry/methods , Isomerism , Mass Spectrometry/methodsABSTRACT
Although it is widely accepted that protein function is largely dependent on its structure, intrinsically disordered proteins (IDPs) lack defined structure but are essential in proper cellular processes. Mammalian high mobility group proteins (HMGA) are one such example of IDPs that perform a number of crucial nuclear activities and have been highly studied due to their involvement in the proliferation of a variety of disease and cancers. Traditional structural characterization methods have had limited success in understanding HMGA proteins and their ability to coordinate to DNA. Ion mobility spectrometry and mass spectrometry provide insights into the diversity and heterogeneity of structures adopted by IDPs and are employed here to interrogate HMGA2 in its unbound states and bound to two DNA hairpins. The broad distribution of collision cross sections observed for the apo-protein are restricted when HMGA2 is bound to DNA, suggesting that increased protein organization is promoted in the holo-form. Ultraviolet photodissociation was utilized to probe the changes in structures for the compact and elongated structures of HMGA2 by analyzing backbone cleavage propensities and solvent accessibility based on charge-site analysis, which revealed a spectrum of conformational possibilities. Namely, preferential binding of the DNA hairpins with the second of three AT-hooks of HMGA2 is suggested based on the suppression of backbone fragmentation and distribution of DNA-containing protein fragments.
Subject(s)
Intrinsically Disordered Proteins , Ion Mobility Spectrometry , Animals , DNA/chemistry , Intrinsically Disordered Proteins/chemistry , Mammals , Mass Spectrometry , Molecular ConformationABSTRACT
The mammalian high mobility group protein AT-hook 2 (HMGA2) houses three motifs that preferentially bind short stretches of AT-rich DNA regions. These DNA binding motifs, known as 'AT-hooks', are traditionally characterized as being unstructured. Upon binding to AT-rich DNA, they form ordered assemblies. It is this disordered-to-ordered transition that has implicated HMGA2 as a protein actively involved in many biological processes, with abnormal HMGA expression linked to a variety of health problems including diabetes, obesity, and oncogenesis. In the current work, the solution binding dynamics of the three 'AT-hook' peptides (ATHPs) with AT-rich DNA hairpin substrates were studied using DNA UV melting studies, fluorescence spectroscopy, native ion mobility spectrometry-mass spectrometry (IMS-MS), solution isothermal titration calorimetry (ITC) and molecular modeling. Results showed that the ATHPs bind to the DNA to form a single, 1:1 and 2:1, 'key-locked' conformational ensemble. The molecular models showed that 1:1 and 2:1 complex formation is driven by the capacity of the ATHPs to bind to the minor and major grooves of the AT-rich DNA oligomers. Complementary solution ITC results confirmed that the 2:1 stoichiometry of ATHP: DNA is originated under native conditions in solution.
Subject(s)
AT-Hook Motifs , DNA , Animals , DNA/chemistry , High Mobility Group Proteins/metabolism , Mammals/genetics , Nucleic Acid Denaturation , Peptides/geneticsABSTRACT
Lanthipeptides belong to the family of ribosomally synthesized and post-translationally modified peptides (RiPPs). The (methyl)lanthionine cross-links characteristic to lanthipeptides are essential for their stability and bioactivities. In most bacteria, lanthipeptides are maturated from single precursor peptides encoded in the corresponding biosynthetic gene clusters. However, cyanobacteria engage in combinatorial biosynthesis and encode as many as 80 substrate peptides with highly diverse sequences that are modified by a single lanthionine synthetase into lanthipeptides of different lengths and ring patterns. It is puzzling how a single enzyme could exert control over the cyclization processes of such a wide range of substrates. Here, we used a library of ProcA3.3 precursor peptide variants and show that it is not the enzyme ProcM but rather its substrate sequences that determine the regioselectivity of lanthionine formation. We also demonstrate the utility of trapped ion mobility spectrometry-tandem mass spectrometry (TIMS-MS/MS) as a fast and convenient method to efficiently separate lanthipeptide constitutional isomers, particularly in cases where the isomers cannot be resolved by conventional liquid chromatography. Our data allowed identification of factors that are important for the cyclization outcome, but also showed that there are no easily identifiable predictive rules for all sequences. Our findings provide a platform for future deep learning approaches to allow such prediction of ring patterns of products of combinatorial biosynthesis.
Subject(s)
Alanine/analogs & derivatives , Peptides/chemistry , Peptides/metabolism , Sulfides/chemistry , Alanine/chemistry , Amino Acid Sequence , Models, Molecular , Protein ConformationABSTRACT
Lasso peptides are members of the natural product superfamily of ribosomally synthesized and post-translationally modified peptides (RiPPs). Here, we describe the first lasso peptide originating from a biosynthetic gene cluster belonging to a unique lasso peptide subclade defined by the presence of a bifunctional protein harboring both a leader peptidase (B2) and an ABC transporter (D) domain. Bioinformatic analysis revealed that these clusters also encode homologues of the NisR/NisK regulatory system and the NisF/NisE/NisG immunity factors, which are usually associated with the clusters of antimicrobial class I lanthipeptides, such as nisin, another distinct RiPP subfamily. The cluster enabling the heterologous production of the lasso peptide cochonodin I in E. coli originated from Streptococcus suis LSS65, and the threaded structure of cochonodin I was evidenced through extensive MS/MS analysis and stability assays. It was shown that the ABC transporter domain from SsuB2/D is not essential for lasso peptide maturation. By extensive genome mining dedicated exclusively to other lasso peptide biosynthetic gene clusters featuring bifunctional B2/D proteins, it was furthermore revealed that many bacteria associated with human or animal microbiota hold the biosynthetic potential to produce cochonodin-like lasso peptides, implying that these natural products might play roles in human and animal health.
Subject(s)
ATP-Binding Cassette Transporters/genetics , Bacterial Proteins/genetics , Membrane Proteins/genetics , Serine Endopeptidases/genetics , Streptococcus suis/genetics , Computational Biology , Multigene Family , Protein Processing, Post-TranslationalABSTRACT
Comprehensive characterization of post-translationally modified histone proteoforms is challenging due to their high isobaric and isomeric content. Trapped ion mobility spectrometry (TIMS), implemented on a quadrupole/time-of-flight (Q-ToF) mass spectrometer, has shown great promise in discriminating isomeric complete histone tails. The absence of electron activated dissociation (ExD) in the current platform prevents the comprehensive characterization of unknown histone proteoforms. In the present work, we report for the first time the use of an electromagnetostatic (EMS) cell devised for nonergodic dissociation based on electron capture dissociation (ECD), implemented within a nESI-TIMS-Q-ToF mass spectrometer for the characterization of acetylated (AcK18 and AcK27) and trimethylated (TriMetK4, TriMetK9 and TriMetK27) complete histone tails. The integration of the EMS cell in a TIMS-q-TOF MS permitted fast mobility-selected top-down ECD fragmentation with near 10% efficiency overall. The potential of this coupling was illustrated using isobaric (AcK18/TriMetK4) and isomeric (AcK18/AcK27 and TriMetK4/TriMetK9) binary H3 histone tail mixtures, and the H3.1 TriMetK27 histone tail structural diversity (e.g., three IMS bands at z = 7+). The binary isobaric and isomeric mixtures can be separated in the mobility domain with RIMS > 100 and the nonergodic ECD fragmentation permitted the PTM localization (sequence coverage of â¼86%). Differences in the ECD patterns per mobility band of the z = 7+ H3 TriMetK27 molecular ions suggested that the charge location is responsible for the structural differences observed in the mobility domain. This coupling further enhances the structural toolbox with fast, high resolution mobility separations in tandem with nonergodic fragmentation for effective proteoform differentiation.
Subject(s)
Electrons , Ion Mobility Spectrometry , Cell Differentiation , Ions , Mass SpectrometryABSTRACT
Lanthipeptides are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by intramolecular thioether cross-links formed between a dehydrated serine/threonine (dSer/dThr) and a cysteine residue. Prochlorosin 2.8 (Pcn2.8) is a class II lanthipeptide that exhibits a non-overlapping thioether ring pattern, for which no biological activity has been reported yet. The variant Pcn2.8[16RGD] has been shown to bind tightly to the αvß3 integrin receptor. In the present work, tandem mass spectrometry, using collision-induced dissociation (CID) and electron capture dissociation (ECD), and trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) were used to investigate structural signatures for the non-overlapping thioether ring pattern of Pcn2.8. CID experiments on Pcn2.8 yielded bi and yj fragments between the thioether cross-links, evidencing the presence of a non-overlapping thioether ring pattern. ECD experiments of Pcn2.8 showed a significant increase of hydrogen migration events near the residues involved in the thioether rings with a more pronounced effect at the dehydrated residues as compared to the cysteine residues. The high-resolution mobility analysis, aided by site-directed mutagenesis ([P8A], [P11A], [P12A], [P8A/P11A], [P8A/P12A], [P11A/P12A], and [P8A/P11A/P12A] variants), demonstrated that Pcn2.8 adopts cis/trans-conformations at Pro8, Pro11, and Pro12 residues. These observations were complementary to recent NMR findings, for which only the Pro8 residue was evidenced to adopt cis/trans-orientations. This study highlights the analytical power of the TIMS-MS/MS workflow for the structural characterization of lanthipeptides and could be a useful tool in our understanding of the biologically important structural elements that drive the thioether cyclization process.
Subject(s)
Ion Mobility Spectrometry , Peptides/chemistry , Tandem Mass Spectrometry , Amino Acid Sequence , Protein ConformationABSTRACT
The structural elucidation of native macromolecular assemblies has been a subject of considerable interest in native mass spectrometry (MS), and more recently in tandem with ion mobility spectrometry (IMS-MS), for a better understanding of their biochemical and biophysical functions. In the present work, we describe a new generation trapped ion mobility spectrometer (TIMS), with extended mobility range (K0 = 0.185-1.84 cm2·V-1·s-1), capable of trapping high-molecular-weight (MW) macromolecular assemblies. This compact 4 cm long TIMS analyzer utilizes a convex electrode, quadrupolar geometry with increased pseudopotential penetration in the radial dimension, extending the mobility trapping to high-MW species under native state (i.e., lower charge states). The TIMS capabilities to perform variable scan rate (Sr) mobility measurements over short time (100-500 ms), high-mobility resolution, and ion-neutral collision cross-section (CCSN2) measurements are presented. The trapping capabilities of the convex electrode TIMS geometry and ease of operation over a wide gas flow, rf range, and electric field trapping range are illustrated for the first time using a comprehensive list of standards varying from CsI clusters (n = 6-73), Tuning Mix oligomers (n = 1-5), common proteins (e.g., ubiquitin, cytochrome C, lysozyme, concanavalin (n = 1-4), carbonic anhydrase, ß clamp (n = 1-4), topoisomerase IB, bovine serum albumin (n = 1-3), topoisomerase IA, alcohol dehydrogenase), IgG antibody (e.g., avastin), protein-DNA complexes, and macromolecular assemblies (e.g., GroEL and RNA polymerase (n = 1-2)) covering a wide mass (up to m/z 19â¯000) and CCS range (up to 22â¯000 Å2 with <0.6% relative standard deviation (RSD)).
Subject(s)
Ion Mobility Spectrometry , Proteins , Ions , Mass Spectrometry , UbiquitinABSTRACT
The behavior of biomolecules as a function of the solution temperature is often crucial to assessing their biological activity and function. While heat-induced changes of biomolecules are traditionally monitored using optical spectroscopy methods, their conformational changes and unfolding transitions remain challenging to interpret. In the present work, the structural transitions of bovine serum albumin (BSA) in native conditions (100 mM aqueous ammonium acetate) were investigated as a function of the starting solution temperature (T â¼ 23-70 °C) using a temperature-controlled nanoelectrospray ionization source (nESI) coupled to a trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) instrument. The charge state distribution of the monomeric BSA changed from a native-like, narrow charge state ([M + 12H]12+ to [M + 16H]16+ at â¼23 °C) and narrow mobility distribution toward an unfolded-like, broad charge state (up to [M + 46H]46+ at â¼70 °C) and broad mobility distribution. Inspection of the average charge state and collision cross section (CCS) distribution suggested a two-state unfolding transition with a melting temperature Tm â¼ 56 ± 1 °C; however, the inspection of the CCS profiles at the charge state level as a function of the solution temperature showcases at least six structural transitions (T1-T7). If the starting solution concentration is slightly increased (from 2 to 25 µM), this method can detect nonspecific BSA dimers and trimers which dissociate early (Td â¼ 34 ± 1 °C) and may disturb the melting curve of the BSA monomer. In a single experiment, this technology provides a detailed view of the solution, protein structural landscape (mobility vs solution temperature vs relative intensity for each charge state).
Subject(s)
Ion Mobility Spectrometry , Proteins , Mass Spectrometry , Protein Denaturation , TemperatureABSTRACT
The ring-shaped sliding clamp proteins have crucial roles in the regulation of DNA replication, recombination, and repair in all organisms. We previously showed that the Escherichia coli ß-clamp is dynamic in solution, transiently visiting conformational states in which Domain 1 at the dimer interface is more flexible and prone to unfolding. This work aims to understand how the stability of the dimer interface influences clamp-opening dynamics and clamp loading by designing and characterizing stabilizing and destabilizing mutations in the clamp. The variants with stabilizing mutations conferred similar or increased thermostability and had similar quaternary structure as compared to the wild type. These variants stimulated the ATPase function of the clamp loader, complemented cell growth of a temperature-sensitive strain, and were successfully loaded onto a DNA substrate. The L82D and L82E I272A variants with purported destabilizing mutations had decreased thermostability, did not complement the growth of a temperature-sensitive strain, and had weakened dimerization as determined by native trapped ion mobility spectrometry-mass spectrometry. The ß L82E variant had a reduced melting temperature but dimerized and complemented growth of a temperature-sensitive strain. All three clamps with destabilizing mutations had perturbed loading on DNA. Molecular dynamics simulations indicate altered hydrogen-bonding patterns at the dimer interface, and cross-correlation analysis showed the largest perturbations in the destabilized variants, consistent with the observed change in the conformations and functions of these clamps.
Subject(s)
DNA Polymerase III/metabolism , DNA, Bacterial/metabolism , Escherichia coli/metabolism , Protein Multimerization , DNA Polymerase III/genetics , Enzyme Stability , Escherichia coli/growth & development , Hydrogen Bonding , Molecular Dynamics Simulation , Mutant Proteins/chemistry , Mutation/genetics , Temperature , Templates, GeneticABSTRACT
Recently, we proposed a high-throughput screening workflow for the elucidation of agonistic or antagonistic growth hormone-releasing hormone (GHRH) potencies based on structural motif descriptors as a function of the starting solution. In the present work, we revisited the influence of solution and gas-phase GHRH molecular microenvironment using trapped ion mobility-mass spectrometry (TIMS-MS). The effect of the starting solvent composition (10 mM ammonium acetate (NH4Ac), 50% methanol (MeOH), 50% acetonitrile (MeCN), and 50% acetone (Ac)) and gas-phase modifiers (N2, N2 + MeOH, N2 + MeCN, and N2 + Ac) on the conformational states of three GHRH analogues, GHRH (1-29), MR-406, and MIA-602, is described as a function of the trapping time (100-500 ms). Changes in the mobility profiles were observed showing the dependence of the conformational states of GHRH analogues according to the molecular microenvironment in solution, suggesting the presence of solution memory effects on the gas-phase observed structures. Modifying the bath gas composition resulted in smaller mobilities that are correlated with the size and mass of the organic modifier, and more importantly led to substantial changes in relative abundances of the IMS profiles. We attributed the observed changes in the mobility profiles by a clustering/declustering mechanism between the GHRH analogue ions and the gas modifiers, redefining the free energy landscape and leading to other local minima structures. Moreover, inspection of the mobility profiles as a function of the trapping time (100-500 ms) allowed for conformational interconversions toward more stable "gas-phase" structures. These experiments enabled us to outline a more detailed description of the structures and intermediates involved in the biological activity of GHRH, MR-406, and MIA-602.
Subject(s)
Growth Hormone-Releasing Hormone/chemistry , Ion Mobility Spectrometry , Models, Molecular , Protein Conformation , Solvents/chemistryABSTRACT
Lipids are a major class of molecules that play key roles in different biological processes. Understanding their biological roles and mechanisms remains analytically challenging due to their high isomeric content (e.g., varying acyl chain positions and/or double bond locations/geometries) in eukaryotic cells. In the present work, a combination of liquid chromatography (LC) followed by high resolution trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) was used to investigate common isomeric glycerophosphocholine (PC) and diacylglycerol (DG) lipid species from human plasma. The LC dimension was effective for the separation of isomeric lipid species presenting distinct double bond locations or geometries but was not able to differentiate lipid isomers with distinct acyl chain positions. High resolution TIMS-MS resulted in the identification of lipid isomers that differ in the double bond locations/geometries as well as in the position of the acyl chain with resolving power ( R) up to â¼410 ( R â¼ 320 needed on average). Extremely small structural differences exhibiting collision cross sections (CCS) of less than 1% (down to 0.2%) are sufficient for the discrimination of the isomeric lipid species using TIMS-MS. The same level of performance was maintained in the complex biological mixture for the biologically relevant PC 16:0/18:1 lipid isomers. These results suggest several advantages of using complementary LC-TIMS-MS separations for regular lipidomic analysis, with the main emphasis in the elucidation of isomer-specific lipid biological activities.
Subject(s)
Chromatography, Liquid , Diglycerides/chemistry , Diglycerides/isolation & purification , Glycerylphosphorylcholine/chemistry , Glycerylphosphorylcholine/isolation & purification , Mass Spectrometry , IsomerismABSTRACT
Microcin J25 is a ribosomal synthesized and post-translationally modified peptide (RiPP) characterized by a mechanically interlocked topology called the lasso fold. This structure provides microcin J25 a potent antimicrobial activity resulting from internalization via the siderophore receptor FhuA and further inhibition of the RNA polymerase. In the present work, nuclear magnetic resonance (NMR) and trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) were used to investigate the lasso structure of microcin J25. NMR experiments showed that the lasso peptide microcin J25 can adopt conformational states where Pro16 can be found in the cis- and trans-orientations. The high-resolution mobility analysis, aided by site-directed mutagenesis ([P7A], [P16A], and [P7A/P16A] variants), demonstrated that microcin J25 can adopt cis/cis-, cis/trans-, trans/cis-, and trans/trans-conformations at the Pro7 and Pro16 peptide bonds. It was also shown that interconversion between the conformers can occur as a function of the starting solvent conditions and ion heating (collision-induced activation, CIA) despite the lasso topology. Complementary to NMR findings, the cis-conformations at Pro7 were assigned using TIMS-MS. This study highlights the analytical power of TIMS-MS and site-directed mutagenesis for the study of biological systems with large micro-heterogeneity as a way to further increase our understanding of the receptor-binding dynamics and biological activity.
ABSTRACT
Topoisomerases are important complex enzymes that modulate DNA topology to maintain chromosome superstructure and integrity. These enzymes are involved in many cellular processes that resolve specific DNA superstructures and intermediates. The low abundance combined with the biological heterogeneity of relevant intermediates of topoisomerases makes their structural information not readily accessible using traditional structural biology tools (e.g., NMR and X-ray crystallography). In the present work, a second-generation trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) was used to study Escherichia coli topoisomerase IA (EcTopIA) and variola virus topoisomerase IB (vTopIB) as well as their complexes with a single-stranded DNA and a stem-loop DNA under native conditions. The higher trapping efficiency and extended mass range of the new, convex TIMS geometry allowed for the separation and identification of multiple conformational states for the two topoisomerases and their DNA complexes. Inspection of the conformational space of EcTopIA and vTopIB in complex with DNA showed that upon DNA binding, the number of conformational states is significantly reduced, suggesting that the DNA binding selects for a narrow range of conformers restricted by the interaction with the DNA substrate. The large microheterogeneity observed for the two DNA binding proteins suggests that they can have multiple biological functions. This work highlights the potential of TIMS-MS for the structural investigations of intrinsically disordered proteins (e.g., DNA binding proteins) as a way to gain a better understanding of the mechanisms involved in DNA substrate recognition, binding, and assembly of the catalytically active enzyme-DNA complex.