RÉSUMÉ
The final stages of the charged residue mechanism/model (CRM) for ion generation via electrospray ionization (ESI) involves the binding of excess charge onto analyte species. Ions of both polarities can bind to the analyte with an excess of ions of the same polarity as the droplet. For large biomolecule/biocomplex ions, which are commonly the species of interest in native mass spectrometry (MS), the binding of acids and salts onto the analyte can lead to extensive broadening of ion signals due to adduction. Therefore, heating step(s) to facilitate desolvation and salt adduct removal are commonplace. In this work, we describe an approach to study the final stages of CRM using gas-phase ion/ion reactions to generate analyte ion/salt clusters of well-defined composition, followed by gas-phase collision-induced dissociation (CID). While there are many variables that can be studied systematically via this approach, the work described herein is focused on salt clusters of the form [Na10X11]-, where X = acetate (Ac-), chloride (Cl-), or nitrate (NO3-), in reaction with a common charge state of ubiquitin as well as several model peptides. Experiments in which equimolar quantities of each salt (i.e., NaAc, NaCl, and NaNO3) are subjected to ESI with ubiquitin (Ubi) and gas-phase ion/ion reaction studies involving [Na10X11]- and [Ubi + 6H]6+ show similar trends, in terms of the extent of sodium ion incorporation into the protein ions. Ion/ion reaction studies using model peptides show that the acetate-containing salt transfers significantly more Na+ ions into the peptide ions. Exchange of Na+ for H+ is shown to occur at the C-terminus and at up to all of the amide linkages using [Na10X11]-, whereas only the C-terminus engages in Na+/H+ exchange with [Na10Cl11]- and [Na10(NO3)11]-. In the latter cases, an additional Na+ is taken up as the excess positive charge, presumably due to solvation of the charge by multiple sites (e.g., carbonyl oxygens and basic sites).
RÉSUMÉ
Luciferases are widely used as reporter proteins in diverse fields from basic biology to medical and environmental researches. Development of luciferase applications for reporter proteins requires small size without target inhibition, appropriate genomic insertion for high expression level, and bright emission for detection sensitivity. We previously developed the minimal luciferase picALuc, but its luminescence was still dim compared to other bright luciferases in terms of expression in Escherichia coli. In this study, diverse additions of oligopeptides with charged residues (eight amino acids in total) to the C-terminus of picALuc enhanced luminescence by up to approximately 50-fold, that is, enhanced enzymatic activity. Moreover, these high luminescence activities were achieved in bacterial and mammalian expression, suggesting their further applicability in many expression systems. The finding in this study that the simple addition of oligopeptides with charged residues (or charge engineering of this kind) enhances enzymatic activity may be applied to a wide variety of enzymatic reactions and protein functions.
Sujet(s)
Escherichia coli , Luminescence , Animaux , Luciferases/génétique , Luciferases/métabolisme , Escherichia coli/génétique , Escherichia coli/métabolisme , Acides aminés , Mesures de luminescence , MammifèresRÉSUMÉ
Intrinsically disordered proteins (IDPs) are ensembles of interconverting conformers whose conformational properties are governed by several physico-chemical factors, including their amino acid composition and the arrangement of oppositely charged residues within the primary structure. In this work, we investigate the effects of charge patterning on the average compactness and shape of three model IDPs with different proline content. We model IDP ensemble conformations as ellipsoids, whose size and shape are calculated by combining data from size-exclusion chromatography and native mass spectrometry. For each model IDP, we analyzed the wild-type protein and two synthetic variants with permuted positions of charged residues, where positive and negative amino acids are either evenly distributed or segregated. We found that charge clustering induces remodeling of the conformational ensemble, promoting compaction and/or increasing spherical shape. Our data illustrate that the average shape and volume of the ensembles depend on the charge distribution. The potential effect of other factors, such as chain length, number of proline residues, and secondary structure content, is also discussed. This methodological approach is a straightforward way to model IDP average conformation and decipher the salient sequence attributes influencing IDP structural properties.
Sujet(s)
Protéines intrinsèquement désordonnées , Acides aminés/composition chimique , Protéines intrinsèquement désordonnées/composition chimique , Proline , Conformation des protéines , Structure secondaire des protéinesRÉSUMÉ
Protein phase separation is a major governing factor in multiple cellular processes, such as RNA metabolism and those involving RNA-binding proteins. Despite many key observations, the exact structural characteristics of proteins involved in the phase separation process are still not fully deciphered. In this work, we show that proteins harbouring sequence regions with specific charged residue patterns are significantly associated with liquid-liquid phase separation. In particular, regions with repetitive arrays of alternating charges show the strongest association, whereas segments with generally high charge density and single α-helices also show detectable but weaker connections.
Sujet(s)
Protéines , Protéines/composition chimiqueRÉSUMÉ
Human organic anion transporting polypeptide 1B3 (OATP1B3, gene symbol SLCO1B3) is a liver-specific uptake transporter. Its function was reported to be largely affected by some positively charged amino acid residues. However, so far the effect of naturally occurring genetic variants of charged residues on OATP1B3's function has not been explored yet. Therefore, in the present study nonsynonymous single nucleotide variants that led to the replacement of charged residues of OATP1B3 were investigated. Our results demonstrated that rare coding variants c.542G > A (p.R181H) and c.592G > A (p.D198N) had a great effect on the function of OATP1B3 mainly due to their influence on protein's surface expression. Further mutation studies showed that a negatively charged residue at position 198 was indispensable to the proper expression of OATP1B3 on the plasma membrane, while a positively charged reside at position 181 was not a must. Structural modeling indicated that R181 is located at the center of putative transmembrane domain 4 (TM4) and its side chain faces towards TM2 instead of towards the substrate translocation pathway, whereas D198 is located at the border of TM4 and intracellular loop 2 and may electrostatically repulse negatively charged phospholipid head groups. In conclusion, our results indicated that rare coding variants that cause changes of charged amino acid residues might have large influence on the function and expression of OATP1B3.
Sujet(s)
Foie/métabolisme , Mutation , Membre 1B3 de la famille des transporteurs d'anions organiques appartenant aux transporteurs de solutés/génétique , Membre 1B3 de la famille des transporteurs d'anions organiques appartenant aux transporteurs de solutés/métabolisme , Substitution d'acide aminé , Transport biologique , Cellules cultivées , Humains , Foie/cytologie , Mutagenèse dirigée/méthodes , Domaines protéiques , Relation structure-activitéRÉSUMÉ
Surface charge residues have been recognized as one of the stability determinants in protein. In this study, we sought to compare and analyse the stability and conformational dynamics of staphylococcal lipase mutants with surface lysine mutation using computational and experimental methods. Three highly mutable and exposed lysine residues (Lys91, Lys177, Lys325) were targeted to generate six mutant lipases in silico. The model structures were simulated in water environment at 25 °C. Our simulations showed that the stability was compromised when Lys177 was substituted while mutation at position 91 and 325 improved the stability. To illustrate the putative alterations of enzyme stability in the stabilising mutants, we characterized single mutant K325G and double mutant K91A/K325G. Both mutants showed a 5 °C change in optimal temperature compared to their wild type. Single mutant K325G rendered a longer half-life at 25 °C (T1/2 = 21 h) while double mutant K91A/K325G retained only 40% of relative activity after 12 h incubation. The optimal pH for mutant K325G was shifted from 8 to 9 and similar substrate preference was observed for the wild type and two mutants. Our findings indicate that surface lysine mutation alters the enzymatic behaviour and, thus, rationalizes the functional effects of surface exposed lysine in conformational stability and activity of this lipase.
Sujet(s)
Protéines bactériennes/composition chimique , Triacylglycerol lipase/composition chimique , Staphylococcus/enzymologie , Substitution d'acide aminé , Protéines bactériennes/génétique , Stabilité enzymatique , Température élevée , Triacylglycerol lipase/génétique , Lysine/composition chimique , Lysine/génétique , Mutation faux-sens , Domaines protéiques , Staphylococcus/génétiqueRÉSUMÉ
KEY POINTS: The Arg271Gln mutation of the glycine receptor (GlyR) causes hereditary hyperekplexia. This mutation dramatically compromises GlyR function; however, the underlying mechanism is not yet known. This study, by employing function and computation methods, proposes that charged residues (including the Arg residue) at the pore extracellular half from each of the five subunits of the homomeric α1 GlyR, create an electrostatic repulsive potential to widen the pore, thereby facilitating channel opening. This mechanism explains how the Arg271Gln mutation, in which the positively charged Arg residue is substituted by the neutral Gln residue, compromises GlyR function. This study furthers our understanding of the biophysical mechanism underlying the Arg271Gln mutation compromising GlyR function. ABSTRACT: The R271(19')Q mutation in the α1 subunit of the glycine receptor (GlyR) chloride channel causes hereditary hyperekplexia. This mutation dramatically compromises channel function; however, the underlying mechanism is not yet known. The R271 residue is located at the extracellular half of the channel pore. In this study, an Arg-scanning mutagenesis was performed at the pore extracellular half from the 262(10') to the 272(20') position on the background of the α1 GlyR carrying the hyperekplexia-causing mutation R271(19')Q. It was found that the placement of the Arg residue rescued channel function to an extent inversely correlated with the distance between the residue and the pore central axis (perpendicular to the plane of the lipid bilayer). Accordingly, it was hypothesized that the placed Arg residues from each of the five subunits of the homomeric α1 GlyR create an electrostatic repulsive potential to widen the pore, thereby facilitating channel opening. This hypothesis was quantitatively verified by theoretical computation via exploiting basic laws of electrostatics and thermodynamics, and further supported by more experimental findings that the placement of another positively charged Lys residue or even a negatively charged Asp residue also rescued channel function in the same manner. This study provides a novel mechanism via which charged residues in the pore region facilitate channel gating, not only for the disease-causing 19'R residue in the GlyR, but also potentially for charged residues in the same region of other ion channels.
Sujet(s)
Hyperekplexie , Récepteur de la glycine , Humains , Raideur musculaire , Récepteur de la glycine/génétique , Récepteur de la glycine/métabolisme , Électricité statique , Transmission synaptiqueRÉSUMÉ
Ion-ion interactions (salt bridges) between favorable pairs of charged residues are important for the conformational stability of proteins. Molecular dynamic (MD) simulations are useful for elucidating the interactions among charged residues fluctuating in solution. However, the quality of MD results depends strongly on the force fields used. In this study, we compared the strengths of salt bridges among force fields by performing MD simulations using the CutA1 protein (trimer) from the hyperthermophile Pyrococcus horikoshii (PhCutA1), which has an unusually large proportion of charged residues. The force fields Chemistry at HARvard Macromolecular Mechanics (Charmm)27, Assisted Model Building and Energy Refinement (Amber)99sb, Amber14sb, GROningen Molecular Simulation (Gromos)43a1, and Gromos53a6 were used in combination with two different water models, tip3p (for Charmm27, Amber99sb, and Amber14sb) and simple point charge/extended (for Amber99sb, Gromos43a1, and Gromos53a6), yielding a total of six combinations. The RMSDs of all Cα atoms of PhCutA1 were similar among force fields, except for Charmm27, during 400-ns MD simulations at 300 K; however, the radius of gyration (Rg ) was greater for Amber99sb and shorter for Gromos43a1. The average strengths of salt bridges for each positively charged residue did not differ greatly among force fields, but the strengths at specific sites within the structure depended sensitively on the force field used. In the case of the Gromos group, positively charged residues could engage in favorable interactions with many more charged residues than in the other force fields, especially in loop regions; consequently, the apparent strength at each site was lower.
Sujet(s)
Protéines d'archée/composition chimique , Simulation de dynamique moléculaire , Pyrococcus horikoshii/composition chimique , Chlorure de sodium/composition chimique , Cristallographie aux rayons X , Ions/composition chimique , Modèles moléculaires , Concentration osmolaire , Conformation des protéines , Stabilité protéiqueRÉSUMÉ
The endoplasmic reticulum (ER) membrane protein complex (EMC) is a key contributor to biogenesis and membrane integration of transmembrane proteins, but our understanding of its mechanisms and the range of EMC-dependent proteins remains incomplete. Here, we carried out an unbiased mass spectrometry (MS)-based quantitative proteomic analysis comparing membrane proteins in EMC-deficient cells to wild-type (WT) cells and identified 36 EMC-dependent membrane proteins and 171 EMC-independent membrane proteins. Of these, six EMC-dependent and six EMC-independent proteins were further independently validated. We found that a common feature among EMC-dependent proteins is that they contain transmembrane domains (TMDs) with polar and/or charged residues. Mutagenesis studies demonstrate that EMC dependency can be converted in cells by removing or introducing polar and/or charged residues within TMDs. Our studies expand the list of validated EMC-dependent and EMC-independent proteins and suggest that the EMC is involved in handling TMDs with residues challenging for membrane integration.
Sujet(s)
Réticulum endoplasmique/métabolisme , Membranes intracellulaires/métabolisme , Protéines membranaires/métabolisme , Complexes multiprotéiques/métabolisme , Protéomique , Lignée cellulaire , Cellules HEK293 , Cellules HeLa , Humains , Protéines membranaires/composition chimique , Mutagenèse/génétique , Domaines protéiques , Reproductibilité des résultats , Réponse aux protéines mal repliéesRÉSUMÉ
It remains unclear how the abundant charged residues in proteins from hyperthermophiles contribute to the stabilization of proteins. Previously, based on molecular dynamics (MD) simulations, we proposed that these charged residues decrease the entropic effect by forming salt bridges in the denatured state under physiological conditions (Yutani et al., Sci. Rep. 8, 7613 (2018)). Because the quality of MD results is strongly dependent on the force fields used, in this study we performed the MD simulations using a different force field (AMBER99SB) along with the one we used before (Gromos43a1), at the same temperatures examined previously as well as at higher temperatures. In these experiments, we used the same ionic mutant (Ec0VV6) of CutA1 from Escherichia coli as in the previous study. In MD simulations at 300 K, Lys87 and Arg88 in the loop region of Ec0VV6 formed salt bridges with different favorable pairs in different force fields. Furthermore, the helical content and radius of gyration differed slightly between two force fields. However, at a higher temperature (600 K), the average numbers of salt bridges for the six substituted residues of Ec0VV6 were 0.87 per residue for Gromos43a1 and 0.88 for AMBER99SB in 400-ns MD simulation, indicating that the values were similar despite the use of different force fields. These observations suggest that the charged residues in Ec0VV6 can form a considerable number of salt bridges, even in the denatured state with drastic fluctuation at 600 K. These results corroborate our previous proposal.
RÉSUMÉ
The ability to transfer intact proteins and protein complexes into the gas phase by electrospray ionization (ESI) has opened up numerous mass spectrometry (MS)-based avenues for exploring biomolecular structure and function. However, many details regarding the ESI process and the properties of gaseous analyte ions are difficult to decipher when relying solely on experimental data. Molecular dynamics (MD) simulations can provide additional insights into the behavior of ESI droplets and protein ions. This review is geared primarily towards experimentalists who wish to adopt MD simulations as a complementary research tool. We touch on basic points such as force fields, the choice of a proper water model, GPU-acceleration, possible artifacts, as well as shortcomings of current MD models. Following this technical overview, we highlight selected applications. Simulations on aqueous droplets confirm that "native" ESI culminates in protein ion release via the charged residue model. MD-generated charge states and collision cross sections match experimental data. Gaseous protein ions produced by native ESI retain much of their solution structure. Moving beyond classical fixed-charge algorithms, we discuss a simple strategy that captures the mobile nature of H+ within gaseous biomolecules. These mobile proton simulations confirm the high propensity of gaseous proteins to form salt bridges, as well as the occurrence of charge migration during collision-induced unfolding and dissociation. It is hoped that this review will promote the use of MD simulations in ESI-related research. We also hope to encourage the development of improved algorithms for charged droplets and gaseous biomolecular ions.
Sujet(s)
Simulation de dynamique moléculaire , Conformation des protéines , Spectrométrie de masse ESI/méthodesRÉSUMÉ
Factors that influence the charging of protein ions formed by electrospray ionization from aqueous solutions in which proteins have native structures and function were investigated. Protein ions ranging in molecular weight from 12.3 to 79.7 kDa and pI values from 5.4 to 9.6 were formed from different solutions and reacted with volatile bases of gas-phase basicities higher than that of ammonia in the cell of a Fourier-transform ion cyclotron resonance mass spectrometer. The charge-state distribution of cytochrome c ions formed from aqueous ammonium or potassium acetate is the same. Moreover, ions formed from these two solutions do not undergo proton transfer to 2-fluoropyridine, which is 8 kcal/mol more basic than ammonia. These results provide compelling evidence that proton transfer between ammonia and protein ions does not limit protein ion charge in native electrospray ionization. Both circular dichroism and ion mobility measurements indicate that there are differences in conformations of proteins in pure water and aqueous ammonium acetate, and these differences can account for the difference in the extent of charging and proton-transfer reactivities of protein ions formed from these solutions. The extent of proton transfer of the protein ions with higher gas-phase basicity bases trends with how closely the protein ions are charged to the value predicted by the Rayleigh limit for spherical water droplets approximately the same size as the proteins. These results indicate that droplet charge limits protein ion charge in native mass spectrometry and are consistent with these ions being formed by the charged residue mechanism. Graphical Abstract á .
Sujet(s)
Protéines/composition chimique , Spectrométrie de masse ESI/méthodes , Acétates/composition chimique , Ammoniac/composition chimique , Dichroïsme circulaire , Cytochromes c/composition chimique , Spectrométrie de mobilité ionique , Masse moléculaire , Protons , Pyridines/composition chimique , Solutions/composition chimique , Eau/composition chimiqueRÉSUMÉ
Infusion of NaCl solutions into an electrospray ionization (ESI) source produces [Na(n+1)Cl n ]+ and other gaseous clusters. The n = 4, 13, 22 magic number species have cuboid ground state structures and exhibit elevated abundance in ESI mass spectra. Relatively few details are known regarding the mechanisms whereby these clusters undergo collision-induced dissociation (CID). The current study examines to what extent molecular dynamics (MD) simulations can be used to garner insights into the sequence of events taking place during CID. Experiments on singly charged clusters reveal that the loss of small neutrals is the dominant fragmentation pathway. MD simulations indicate that the clusters undergo extensive structural fluctuations prior to decomposition. Consistent with the experimentally observed behavior, most of the simulated dissociation events culminate in ejection of small neutrals ([NaCl] i , with i = 1, 2, 3). The MD data reveal that the prevalence of these dissociation channels is linked to the presence of short-lived intermediates where a relatively compact core structure carries a small [NaCl] i protrusion. The latter can separate from the parent cluster via cleavage of a single Na-Cl contact. Fragmentation events of this type are kinetically favored over other dissociation channels that would require the quasi-simultaneous rupture of multiple electrostatic contacts. The CID behavior of NaCl cluster ions bears interesting analogies to that of collisionally activated protein complexes. Overall, it appears that MD simulations represent a valuable tool for deciphering the dissociation of noncovalently bound systems in the gas phase. Graphical Abstract á .
RÉSUMÉ
The bactericidal/permeability-increasing protein (BPI) with bactericidal and endotoxin-neutralizing activity is of considerable interest in clinical applications. However, the crucial residues responsible for the bactericidal activity of BPI remain elusive. In previous study, we identified the mutation of mBPI5 associated with the male infertility of mice. Here, the effects of Asp190Ala mutation on the antibacterial activity of mBPI5 have been determined. Substitution of Asp190 by alanine caused significant improvement in cytotoxic effect toward both E.coli J5 and P.aeruginosa. Liposome co-sedimentation assay showed that the ratio of Asp190Ala mutant binding to lipids increased by 8 folds. These results were well consistent with known fact that antibacterial activity of BPI is attributed to its high affinity for lipid moiety of lipopolysaccharides (LPS). The constructed structure of mBPI5 revealed that Asp190 was located close to 6 positively charged residues on the surface of N-terminal domain. When replacing Asp190 with alanine, salt linkages with Arg188 were broken, making the side chain of Arg188 be free to move and form tighter contacts with negatively charged LPS. These findings suggest that residue 190 combined with surrounding positively charged residues largely contribute to bactericidal and endotoxin-neutralizing activities of mBPI5.
Sujet(s)
Peptides antimicrobiens cationiques/génétique , Peptides antimicrobiens cationiques/pharmacologie , Acide aspartique/génétique , Protéines du sang/génétique , Protéines du sang/pharmacologie , Mutation faux-sens , Animaux , Peptides antimicrobiens cationiques/composition chimique , Protéines du sang/composition chimique , Relation dose-effet des médicaments , Escherichia coli/effets des médicaments et des substances chimiques , Escherichia coli/croissance et développement , Souris , Viabilité microbienne/effets des médicaments et des substances chimiques , Modèles moléculaires , Conformation des protéines , Pseudomonas aeruginosa/effets des médicaments et des substances chimiques , Pseudomonas aeruginosa/croissance et développement , Protéines recombinantes/composition chimique , Protéines recombinantes/pharmacologieRÉSUMÉ
Unraveling the mechanistic workings of membrane transporters has remained a challenging task. We describe a novel strategy that involves subjecting the residues of the hydrophobic face of a transmembrane helix to a charged/polar scanning mutagenesis. TMD9 of the yeast glutathione transporter, Hgt1p, has been identified as being important in substrate binding, and two residues, F523 and Q526, are expected to line the substrate translocation channel while the other face is hydrophobic. The hydrophobic face of TMD9 helix consists of residues A509, V513, L517, L520, I524, and I528, and these were mutated to lysine, glutamine, and glutamic acid. Among the 16 charged mutants created, six were nonfunctional, revealing a surprising tolerance of charged residues in the hydrophobic part of TM helices. Furthermore, the only position that did not tolerate any charged residue was I524, proximal to the substrate binding residues. However, P525, also proximal to the substrate binding residues, did tolerate charged/polar residues, suggesting that mere proximity to the substrate binding residues was not the only factor. The I524K/E/Q mutants expressed well and localized correctly despite lacking any glutathione uptake capability. Isolation of suppressors for all nonfunctional mutants yielded second-site suppressors only for I524K and I524Q, and suppressors for these mutations appeared at G202K/I and G202K/Q, respectively. G202 is in the hydrophilic loop between TMD3 and TMD4. The results suggest that I524 in the hydrophobic face interacts with this region and is also in a conformationally critical region for substrate translocation.
Sujet(s)
Acides aminés , Protéines de transport membranaire/composition chimique , Protéines de transport membranaire/métabolisme , Conformation des protéines , Motifs et domaines d'intéraction protéique , Levures/métabolisme , Transport biologique , Expression des gènes , Test de complémentation , Interactions hydrophobes et hydrophiles , Cinétique , Protéines de transport membranaire/génétique , Mutation , Levures/génétiqueRÉSUMÉ
Lantibiotics are antimicrobial peptides which can have a broad spectrum activity against many Gram positive pathogens. Many of these peptides contain charged amino acids which may be of critical importance with respect to antimicrobial activity. We have recently carried out an in-depth bioengineering based investigation of the importance of charged residues in a representative two peptide lantibiotic, lacticin 3147, and here we discuss the significance of these findings in the context of other lantibiotics and cationic antimicrobial peptides.