Charged Amino Acid Side Chains Research Articles

Thermodynamics of Metal-Acetate Interactions.

Metal ions play crucial roles in protein- and ligand-mediated interactions. They not only act as catalysts to facilitate biological processes but are also important as protein structural elements. Accurately predicting metal ion interactions in computational studies has always been a challenge, and various methods have been suggested to improve these interactions. One such method is the 12-6-4 Lennard-Jones (LJ)-type nonbonded model. Using this model, it has been possible to successfully reproduce the experimental properties of metal ions in aqueous solution. The model includes induced dipole interactions typically ignored in the standard 12-6 LJ nonbonded model. In this we expand the applicability of this model to metal ion-carboxylate interactions. Using 12-6-4 parameters that reproduce the solvation free energies of the metal ions leads to an overestimation of metal ion-acetate interactions, thus, prompting us to fine-tune the model to specifically handle the latter. We also show that the standard 12-6 LJ model significantly falls short in reproducing the experimental binding free energy between acetate and 11 metal ions (Ni(II), Mg(II), Cu(II), Zn(II), Co(II), Cu(I), Fe(II), Mn(II), Cd(II), Ca(II), and Ag(I)). In this study, we describe optimized C4 parameters for the 12-6-4 LJ nonbonded model to be used with three widely employed water models (Transferable Intermolecular Potential with 3 Points (TIP3P), Simple Point Charge Extended (SPC/E), and Optimal Point Charge (OPC) water models). These parameters can accurately match the experimental binding free energy between 11 metal ions and acetate. These parameters can be applied to the study of metalloproteins and transition metal ion channels and transporters, as acetate serves as a representative of the negatively charged amino acid side chains from aspartate and glutamate.

Ion binding with charge inversion combined with screening modulates DEAD box helicase phase transitions

Membraneless organelles, or biomolecular condensates, enable cells to compartmentalize material and processes into unique biochemical environments. While specific, attractive molecular interactions are known to stabilize biomolecular condensates, repulsive interactions, and the balance between these opposing forces, are largely unexplored. Here, we demonstrate that repulsive and attractive electrostatic interactions regulate condensate stability, internal mobility, interfaces, and selective partitioning of molecules both invitro and in cells. We find that signaling ions, such as calcium, alter repulsions between model Ddx3 and Ddx4 condensate proteins by directly binding to negatively charged amino acid sidechains and effectively inverting their charge, in a manner fundamentally dissimilar to electrostatic screening. Using a polymerization model combined with generalized stickers and spacers, we accurately quantify and predict condensate stability over a wide range of pH, salt concentrations, and amino acid sequences. Our model provides a general quantitative treatment for understanding how charge and ions reversibly control condensate stability.

Disruption of Spatial Proximities among Charged Groups in Equilibrium-Denatured States of Proteins Tracked Using Protein Charge Transfer Spectra.

The absorption and luminescence originating from protein charge transfer spectra (ProCharTS) depend on the proximity between multiple charged groups in a protein. This makes ProCharTS absorbance/luminescence intensity a sensitive probe for detecting changes in the protein structure, which alter the proximity among charged groups in the protein. In this work, ProCharTS absorbance of charge-rich proteins like human serum albumin (HSA), α3C, and α3W was used to monitor structural changes upon chemical denaturant-induced protein unfolding under equilibrium conditions. The denaturation midpoints were estimated using nonlinear regression analysis. For HSA, absorbance at 325 and 340 nm estimated the GdnHCl-induced denaturation midpoints to be 0.80 and 0.61 M, respectively. A similar analysis of α3C and α3W ProCharTS absorbance yielded denaturation midpoints of 0.88 and 0.86 M at 325 nm and 0.96 and 0.66 M at 340 nm, respectively. A previously reported molten globule-like state in the GdnHCl-induced HSA unfolding pathway was detected by the increase in HSA ProCharTS absorbance at 0.5 M GdnHCl. To validate the above results, protein unfolding was additionally monitored using conventional methods like circular dichroism (CD), Trp, and dansyl fluorescence. Our results suggest that disruption of charged amino acid sidechain contacts as revealed by ProCharTS occurs at lower denaturant concentrations compared to the loss of secondary/folded structure monitored by CD and fluorescence. Further, HSA ProCharTS absorbance at 315-340 nm revealed that tertiary contacts among charged residues were disrupted at lower GdnHCl concentrations compared to sequence adjacent contacts. Our data underscore the utility of ProCharTS as a novel label-free tool to track unfolding in charge-rich proteins.

Decomposing Hofmeister effects on amino acid residues with symmetry adapted perturbation theory

Hofmeister effects, and more generally specific ion effects, are observed broadly in biological systems. However, there are many cases where the Hofmeister series might not be followed in complex biological systems, such as ion channels which can be highly specific to a particular ion. An understanding of how ions from the Hofmeister series interact with the proteinogenic amino acids will assist elucidation of why some binding interactions may be favoured over others. Using symmetry adapted perturbation theory (SAPT2 + 3), the interaction energies between a selection of anions and each amino acid have been investigated. The interaction strengths become more favourable in accordance with the Hofmeister series, and also with increasing polarity of the amino acids (with the exception of the negatively charged amino acid side chains). Furthermore, the interactions are generally most favourable when they simultaneously involve the side chain and both protic moieties of the backbone. The total interaction energy in these anion–amino acid complexes is also primarily determined by its electrostatic component, in a manner proportional to the þ (‘sho’) value of the anion.

Effects of Salts and Surface Charge on the Biophysical Stability of a Low pI Monoclonal Antibody

The impact of five representative Hofmeister salts (NaCl, KCl, MgCl2, Na2SO4, and NaSCN) on the thermal stability and aggregation kinetics of a slightly acidic monoclonal antibody (mAb) were investigated under different pH conditions. The thermal stability of the mAb was assessed by measuring the lowest unfolding transition temperature, Tm, with differential scanning fluorimetry. MgCl2 and NaSCN significantly decreased Tm at all three charged states of the mAb, but to the greatest extent when the mAb surface charge was net positive. Non-native aggregation kinetics was monitored by measuring Rayleigh light scattering. When the mAb surface charge was net positive or net neutral, the nucleation rate increased non-monotonically with MgCl2 and NaSCN but decreased monotonically with NaCl, KCl, and Na2SO4. By contrast, when the mAb surface was negatively charged, there were only minor changes in the nucleation rate with all salts tested. Furthermore, there was less structural perturbation and slower aggregation rates when the mAb was net negatively charged than when it was net neutrally or positively charged. The observed salt effects on thermal unfolding are consistent with ion-specific mechanisms dominated by short-range amide backbone binding. On the other hand, the salt effects on nucleation rates appear to be influenced by both amide backbone binding and long-range electrostatic binding of ions to charged amino acid side chains.

The Effects of Charged Amino Acid Side-Chain Length on Diagonal Cross-Strand Interactions between Carboxylate- and Ammonium-Containing Residues in a β-Hairpin.

The β-sheet is one of the common protein secondary structures, and the aberrant aggregation of β-sheets is implicated in various neurodegenerative diseases. Cross-strand interactions are an important determinant of β-sheet stability. Accordingly, both diagonal and lateral cross-strand interactions have been studied. Surprisingly, diagonal cross-strand ion-pairing interactions have yet to be investigated. Herein, we present a systematic study on the effects of charged amino acid side-chain length on a diagonal ion-pairing interaction between carboxylate- and ammonium-containing residues in a β-hairpin. To this end, 2D-NMR was used to investigate the conformation of the peptides. The fraction folded population and the folding free energy were derived from the chemical shift data. The fraction folded population for these peptides with potential diagonal ion pairs was mostly lower compared to the corresponding peptide with a potential lateral ion pair. The diagonal ion-pairing interaction energy was derived using double mutant cycle analysis. The Asp2-Dab9 (Asp: one methylene; Dab: two methylenes) interaction was the most stabilizing (−0.79 ± 0.14 kcal/mol), most likely representing an optimal balance between the entropic penalty to enable the ion-pairing interaction and the number of side-chain conformations that can accommodate the interaction. These results should be useful for designing β-sheet containing molecular entities for various applications.

Longer charged amino acids favor β-strand formation in hairpin peptides.

Interactions between charged amino acids significantly influence the structure and function of proteins. The encoded charged amino acids Asp, Glu, Arg, and Lys have different number of hydrophobic methylenes linking the backbone to the charged functionality. It remains to be fully understood how does this difference in the number of methylenes affect protein structure stability. Protein secondary structures are the fundamental three-dimensional building blocks of protein structures. β-Sheet structures are particularly interesting, because these structures have been associated with a number of protein misfolding diseases. Herein, we report the effect of charged amino acid side chain length at two β-strand positions individually on the stability of a β-hairpin. The charged amino acids include side chains with a carboxylate, an ammonium, or a guanidinium group. The experimental peptides, fully folded reference peptides, and fully unfolded reference peptides were synthesized by solid phase peptide synthesis and analyzed by 2D NMR methods including TOCSY, DQF-COSY, and ROESY. Sequence specific assignments were performed for all peptides. The chemical shift data were used to derive the fraction folded population and the folding free energy for the experimental peptides. Results showed that the fraction folded population increased with increasing charged amino acid side chain length. These results should be useful for developing functional peptides that adopt the β-conformation.

Two positively charged amino acid side-chains in the inner vestibule of the CFTR channel pore play analogous roles in controlling anion binding and anion conductance.

Positively charged amino acid side-chains play important roles in anion binding and permeation through the CFTR chloride channel. One pore-lining lysine residue in particular (K95) has been shown to be indispensable for anion binding, conductance, and selectivity. Here, we use functional investigation of CFTR to show that a nearby arginine (R134) plays a functionally analogous role. Removal of this positive charge (in the R134Q mutant) drastically reduces single-channel conductance, weakens binding of both permeant and blocking anions, and abolishes the normal anion conductance selectivity pattern. Each of these functional effects was reversed by a second-site mutation (S1141K) that introduces an ectopic positive charge to a nearby pore-lining residue. Substituted cysteine accessibility experiments confirm that R134-but not nearby residues in the same transmembrane helix-is accessible within the pore lumen. These results suggest that K95 and R134, which are very close together within the inner vestibule of the pore, play analogous, important roles, and that both are required for the normal anion binding and anion conductance properties of the pore. Nevertheless, that fact that both positive charges can be "transplanted" to other sites in the inner vestibule with little effect on channel permeation properties indicates that it is the overall number of charges-rather than their exact locations-that controls pore function.

Electronic and Structural Properties of the Double Cubane Iron-Sulfur Cluster

The double-cubane cluster (DCC) refers to an [Fe8S9] iron-sulfur complex that is otherwise only known to exist in nitrogenases. Containing a bridging µ2-S ligand, the DCC in the DCC-containing protein (DCCP) is covalently linked to the protein scaffold via six coordinating cysteine residues. In this study, the nature of spin coupling and the effect of spin states on the cluster’s geometry are investigated computationally. Using density functional theory (DFT) and a broken symmetry (BS) approach to study the electronic ground state of the system, we computed the exchange interaction between the spin-coupled spins of the four FeFe dimers contained in the DCC. This treatment yields results that are in excellent agreement with both computed and experimentally determined exchange parameters for analogously coupled di-iron complexes. Hybrid quantum mechanical (QM)/molecular mechanical (MM) geometry optimizations show that cubane cluster A closest to charged amino acid side chains (Arg312, Glu140, Lys146) is less compact than cluster B, indicating that electrons of the same spin in a charged environment seek maximum separation. Overall, this study provides the community with a fundamental reference for subsequent studies of DCCP, as well as for investigations of other [Fe8S9]-containing enzymes.

(Invited) Single-Molecule Electronics for DNA Sequencing

Nanoscale materials provide new opportunities to interface solid-state electronics with biomolecules and biochemical activity. For example, single-walled carbon nanotubes (SWNTs) have the special property of electronic resistance that is sensitive to single electrons. We have exploited this sensitivity to build nanoelectronic biosensors that monitor the biochemical activity of individual proteins [1]. As an attached protein moves, binds, or performs catalysis, its charged amino acid sidechains induce resistance fluctuations in the SWNT device that may be monitored with microsecond resolution [2].Recently, we have used this measurement platform for single-molecule measurements of DNA polymerases [3]. Polymerases are the key enzymes for converting single-stranded DNA to double-stranded helices, the primary step in DNA replication, amplification, and most sequencing technologies. When a polymerase processing DNA is also attached to a SWNT device, the electrical signal provides a high-resolution readout of single-nucleotide incorporations and exciting possibilities for high-density, high-throughput electronic DNA sequencing.To investigate the feasibility of electronic DNA sequencing, we have compared single-molecule transduction by DNA polymerases from three different organisms. By working with multiple families of DNA polymerases, we have tested the applicability of the electronic technique while also generating detailed records of differences among the enzymes. For example, we observe an anomalous rate variability when measuring the polymerase from the bacillus phage φ29. Base incorporation rates average 20 s-1 for most the enzymes processing single-stranded DNA templates, but rates up to 200 and 400 s-1 occurred when φ29 encountered homopolymeric sequences of poly(dT) or poly(dC), respectively. When processing poly(dA) and poly(dG) sequences, on the other hand, φ29 had bursts of activity interrupted by pauses lasting 50 to 300 s. This sequence-dependent activity illustrates one way that single-molecule methods reveal information hidden in ensemble-based techniques.Another workhorse protein in DNA sequencing technologies is the DNA polymerase derived from the thermophilic bacteria Thermus aquaticus (Taq). Anomalous stability at high temperatures makes Taq a unique enzyme for the polymerase chain reaction (PCR) and commercial amplification of DNA. In a first for single-molecule biophysics, SWNT devices have recorded Taq activity over a wide temperature range from 22 to 94 °C, including the typical PCR operating temperature of 72 °C. Even operating at this high temperature, the technique resolved Taq testing incoming nucleotides for complementarity and incorporating correct matches in the base-by-base construction of Watson-Crick pairs. The detailed recordings reveal the similarities of Taq’s operation to other, room temperature polymerases.[1] Y. Choi, et. al., Science 335, 319 (2012). [2] M. V. Akhterov, et. al., ACS Chem. Biol. 10, 1495 (2015). [3] T. J. Olsen et. al., JACS 135, 7855 (2013); O. T. Gul et. al., Biosensors 6, 29 (2016)

Biological control of S-nitrosothiol reactivity: potential role of sigma-hole interactions.

S-Nitrosothiols (RSNOs) are ubiquitous biomolecules whose chemistry is tightly controlled in vivo, although the specific molecular mechanisms behind this biological control remain unknown. In this work, we demonstrate, using high-level ab initio and DFT calculations, the ability of RSNOs to participate in intermolecular interactions with electron pair donors/Lewis bases (LBs) via a σ-hole, a region of positive electrostatic potential on the molecular surface at the extension of the N-S bond. Importantly, σ-hole binding is able to modulate the properties of RSNOs by changing the balance between two chemically opposite (antagonistic) resonance components, R-S+[double bond, length as m-dash]N-O- (D) and R-S-/NO+ (I), which are, in addition to the main resonance structure R-S-N[double bond, length as m-dash]O, necessary to describe the unusual electronic structure of RSNOs. σ-Hole binding at the sulfur atom of RSNO promotes the resonance structure D and reduces the resonance structure I, thereby stabilizing the weak N-S bond and making the sulfur atom more electrophilic. On the other hand, increasing the D-character of RSNO by other means (e.g. via N- or O-coordination of a Lewis acid) in turn enhances the σ-hole bonding. Our calculations suggest that in the protein environment a combination of σ-hole bonding of a negatively charged amino acid sidechain at the sulfur atom and N- or O-coordination of a positively charged amino acid sidechain is expected to have a profound effect on the RSNO electronic structure and reactivity.

Electrostatic Tuning of Anion Attraction from the Cytoplasm to the Pore of the CFTR Chloride Channel.

Anions enter from the cytoplasm into the channel pore of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel not via a central pathway but via a single lateral portal or fenestration. High Cl- conductance is dependent on electrostatic attraction of cytoplasmic Cl- ions by four positively charged amino acid side-chains located within this portal. Here we use a mutagenic approach to investigate the functional effects of transplanting or supplementing these positive charges at nearby portal-lining sites. Using patch clamp recording, we find that the functionally important positive charges at K190 and R303 can be transplanted to four nearby sites (N186, L197, W356, and A367) with little loss of Cl- conductance. Introduction of additional positive charge at these sites had almost no effect on Cl- conductance, but did increase the sensitivity to channel block by intracellular suramin and Pt(NO2)42- anions. We suggest that it is the number of positive charges within the portal, rather than their exact location, that is the most important factor influencing Cl- conductance. The portal appears well optimized in terms of charge distribution to maximize Cl- conductance.

Dynamic ion pair behavior stabilizes single α-helices in proteins

Ion pairs are key stabilizing interactions between oppositely charged amino acid side chains in proteins. They are often depicted as single conformer salt bridges (hydrogen-bonded ion pairs) in crystal structures, but it is unclear how dynamic they are in solution. Ion pairs are thought to be particularly important in stabilizing single α-helix (SAH) domains in solution. These highly stable domains are rich in charged residues (such as Arg, Lys, and Glu) with potential ion pairs across adjacent turns of the helix. They provide a good model system to investigate how ion pairs can contribute to protein stability. Using NMR spectroscopy, small-angle X-ray light scattering (SAXS), and molecular dynamics simulations, we provide here experimental evidence that ion pairs exist in a SAH in murine myosin 7a (residues 858-935), but that they are not fixed or long lasting. In silico modeling revealed that the ion pairs within this α-helix exhibit dynamic behavior, rapidly forming and breaking and alternating between different partner residues. The low-energy helical state was compatible with a great variety of ion pair combinations. Flexible ion pair formation utilizing a subset of those available at any one time avoided the entropic penalty of fixing side chain conformations, which likely contributed to helix stability overall. These results indicate the dynamic nature of ion pairs in SAHs. More broadly, thermodynamic stability in other proteins is likely to benefit from the dynamic behavior of multi-option solvent-exposed ion pairs.

The Leishmania PABP1-eIF4E4 interface: a novel 5'-3' interaction architecture for trans-spliced mRNAs.

Trans-splicing of trypanosomatid polycistronic transcripts produces polyadenylated monocistronic mRNAs modified to form the 5′ cap4 structure (m7Gpppm36,6,2′Apm2′Apm2′Cpm23,2′U). NMR and X-ray crystallography reveal that Leishmania has a unique type of N-terminally-extended cap-binding protein (eIF4E4) that binds via a PAM2 motif to PABP1. This relies on the interactions of a combination of polar and charged amino acid side-chains together with multiple hydrophobic interactions, and underpins a novel architecture in the Leishmania cap4-binding translation factor complex. Measurements using microscale thermophoresis, fluorescence anisotropy and surface plasmon resonance characterize the key interactions driving assembly of the Leishmania translation initiation complex. We demonstrate that this complex can accommodate Leishmania eIF4G3 which, unlike the standard eukaryotic initiation complex paradigm, binds tightly to eIF4E4, but not to PABP1. Thus, in Leishmania, the chain of interactions 5′cap4-eIF4E4–PABP1-poly(A) bridges the mRNA 5′ and 3′ ends. Exceptionally, therefore, by binding tightly to two protein ligands and to the mRNA 5′ cap4 structure, the trypanosomatid N-terminally extended form of eIF4E acts as the core molecular scaffold for the mRNA-cap-binding complex. Finally, the eIF4E4 N-terminal extension is an intrinsically disordered region that transitions to a partly folded form upon binding to PABP1, whereby this interaction is not modulated by poly(A) binding to PABP1.

A new protein nucleic-acid coarse-grained force field based on the UNRES and NARES-2P force fields.

Based on the coarse-grained UNRES and NARES-2P models of proteins and nucleic acids, respectively, developed in our laboratory, in this work we have developed a coarse-grained model of systems containing proteins and nucleic acids. The UNRES and NARES-2P effective energy functions have been applied to the protein and nucleic-acid components of a system, respectively, while protein-nucleic-acid interactions have been described by the respective coarse-grained potentials developed in our recent work (Yin et al., J. Chem Theory Comput. 2015, 11, 1792). The Debye-Hückel screening has been applied to the electrostatic-interaction energy between the phosphate groups and charged amino-acid side chains. The model has been integrated into the UNRES package for coarse-grained molecular dynamics simulations of proteins and the implementation has been tested for energy conservation in microcanonical molecular dynamics runs and for temperature conservation in canonical molecular dynamics runs. Two case studies were performed: (i) the dynamics of the Ku protein heterodimer bound to DNA, for which it was found that the Ku70/Ku80 protein complex plays an active role in DNA repairing and (ii) conformational changes of the multiple antibiotic resistance (MarA) protein occurring during DNA binding, for which the functionally important motions occurring during this process were identified. © 2018 Wiley Periodicals, Inc.

Ionic Polypeptide Polymers with Unusual β-Sheet Stability.

Incorporating charged amino acid side chains in polypeptide polymer backbones to improve solubility usually leads to reduced secondary structuring. Here we show that highly water soluble (>15 mg.mL-1) β-sheetscan be obtained via nucleotide monophosphate grafting onto simple poly(γ-propargyl- L-glutamate) backbone. This synthetic methodology has been applied to the synthesis of thymidine-based nucleopolypeptides presenting stable β-sheet conformation in aqueous solutions with pH values comprised between 4 and 8. These polymeric analogues of nucleoproteins exhibited selective interaction with simple DNA sequences displaying adenine.

CpAMs induce assembly of HBV capsids with altered electrophoresis mobility: Implications for mechanism of inhibiting pgRNA packaging

Native agarose gel electrophoresis-based particle gel assay has been commonly used for examination of hepatitis B virus (HBV) capsid assembly and pregenomic RNA encapsidation in HBV replicating cells. Interestingly, treatment of cells with several chemotypes of HBV core protein allosteric modulators (CpAMs) induced the assembly of both empty and DNA-containing capsids with faster electrophoresis mobility. In an effort to determine the physical basis of CpAM-induced capsid mobility shift, we found that the surface charge, but not the size, of capsids is the primary determinant of electrophoresis mobility. Specifically, through alanine scanning mutagenesis analysis of twenty-seven charged amino acids in core protein assembly domain and hinge region, we showed that except for K7 and E8, substitution of glutamine acid (E) or aspartic acid (D) on the surface of capsids reduced their mobility, but substitution of lysine (K) or arginine (R) on the surface of capsids increased their mobility in variable degrees. However, alanine substitution of the charged amino acids that are not exposed on the surface of capsid did not apparently alter capsid mobility. Hence, CpAM-induced electrophoresis mobility shift of capsids may reflect the global alteration of capsid structure that changes the exposure and/or ionization of charged amino acid side chains of core protein. Our findings imply that CpAM inhibition of pgRNA encapsidation is possibly due to the assembly of structurally altered nucleocapsids. Practically, capsid electrophoresis mobility shift is a diagnostic marker of compounds that target core protein assembly and predicts sensitivity of HBV strains to specific CpAMs.

Functional organization of cytoplasmic portals controlling access to the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel pore

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that apparently has evolved from an ancestral active transporter. Key to the CFTR's switch from pump to channel function may have been the appearance of one or more "lateral portals." Such portals connect the cytoplasm to the transmembrane channel pore, allowing a continuous pathway for the electrodiffusional movement of Cl- ions. However, these portals remain the least well-characterized part of the Cl- transport pathway; even the number of functional portals is uncertain, and if multiple portals do exist, their relative functional contributions are unknown. Here, we used patch-clamp recording to identify the contributions of positively charged amino acid side chains located in CFTR's cytoplasmic transmembrane extensions to portal function. Mutagenesis-mediated neutralization of several charged side chains reduced single-channel Cl- conductance. However, these same mutations differentially affected channel blockade by cytoplasmic suramin and Pt(NO2)42- anions. We considered and tested several models by which the contribution of these positively charged side chains to one or more independent or non-independent portals to the pore could affect Cl- conductance and interactions with blockers. Overall, our results suggest the existence of a single portal that is lined by several positively charged side chains that interact electrostatically with both Cl- and blocking anions. We further propose that mutations at other sites indirectly alter the function of this single portal. Comparison of our functional results with recent structural information on CFTR completes our picture of the overall molecular architecture of the Cl- permeation pathway.

High-Efficiency Microflow and Nanoflow Negative Electrospray Ionization of Peptides Induced by Gas-Phase Proton Transfer Reactions.

Liquid chromatography coupled with electrospray ionization mass spectrometry (ESI-MS) is routinely used in proteomics research. Mass spectrometry-based peptide analysis is performed de facto in positive-ion mode, except for the analysis of some post-translationally modified peptides (e.g., phosphorylation and glycosylation). Collected mass spectrometry data after peptide negative ionization analysis is scarce, because of a lack of negatively charged amino acid side-chain residues that would enable efficient ionization (i.e., on average, every 10th amino acid residue is negatively charged). Also, several phenomena linked to negative ionization, such as corona discharge, arcing, and electrospray destabilization, because of the presence of polar mobile-phase solutions or acidic mobile-phase additives (e.g., formic or trifluoroacetic acid), reduce its use. Named phenomena influence microflow and nanoflow electrospray ionization (ESI) of peptides in a way that prevents the formation of negatively charged peptide ions. In this work, we have investigated the effects of post-column addition of isopropanol solutions of formaldehyde, 2,2-dimethylpropanal, ethyl methanoate, and 2-phenyl-2-oxoethanal as the negative-ion-mode mobile-phase modifiers for the analysis of peptides. According to the obtained data, all four modifiers exhibited significant enhancement of peptide negative ionization, while ethyl methanoate showed the best results. The proposed mechanism of action of the modifiers includes proton transfer reactions through oxonium ion formation. In this way, mobile phase protons are prevented from interfering with the process of negative ionization. To the best of our knowledge, this is the first study that describes the use and reaction mechanism of aforementioned modifiers for enhancement of peptide negative ionization.

The Importance of a Salt Bridge in FNR Transcription Factor Activity

Fumarate Nitrate Reductase transcription factor (FNR) regulates the expression of genes involved in anaerobic respiration within E. coli bacteria. In the absence of oxygen, the FNR protein contains a [4Fe‐4S] cluster. This [4Fe‐4S] cluster causes a change in the dimerization helix of FNR, allowing the protein to dimerize and bind to DNA. In a recent publication, the crystal structure of A. fischeri FNR, which has high homology to E. coli FNR, was reported[1]. The structure suggests that the [4Fe‐4S] cluster promotes dimerization through a salt bridge interaction between residues Asp 130 and Arg 140. This investigation focuses the effect of the salt bridge interaction on the activity of E. coli FNR. Two mutant variants of FNR (Asp130Arg and Arg140Glu) were constructed in a pET11a plasmid containing the FNR gene. In each variant, one of the amino acid residues constituting the salt bridge was replaced with an oppositely charged amino acid side chain. The activity of the FNR mutant proteins was then measured under anaerobic conditions using the narG‐lacZ reporter strain. The results show that the activity of FNR decreases significantly when the salt bridge is broken. In addition, the results of the mutations that swap the amino acids of the salt bridge will be discussed.