Role Of His Residues Research Articles

Unraveling the implications of multiple histidine residues in the potent antimicrobial peptide Gaduscidin-1

The development of antimicrobial peptides (AMPs) as potential therapeutics requires resolving the foundational principles behind their structure-activity relationships. The role of histidine residues within AMPs remains a mystery despite the fact that several potent peptides containing this amino acid are being considered for further clinical development. Gaduscidin-1 (Gad-1) is a potent AMP from Atlantic cod fish that has a total of five His residues. Herein, the role of His residues and metal-potentiated activity of Gad-1 was studied. The five His residues contribute to the broad-spectrum activity of Gad-1. We demonstrated that Gad-1 can coordinate two Cu2+ ions, one at the N-terminus and one at the C-terminus, where the C-terminal binding site is a novel Cu2+ binding motif. High affinity Cu2+ binding at both sites was observed using mass spectrometry and isothermal titration calorimetry. Electron paramagnetic resonance was used to determine the coordination environment of the Cu2+ ions. Cu2+ binding was shown to be responsible for an increase in antimicrobial activity and a new mode of action. Along with the traditional AMP mode of action of pore formation, Gad-1 in the presence of Cu2+ (per)oxidizes lipids. Importantly, His3, His11, His17, and His21 were found to be important to lipid (per)oxidation. This insight will help further understand the inclusion and role of His residues in AMPs, the role of the novel C-terminal binding site, and can contribute to the field of designing potent AMPs that bind metal ions to potentiate activity.

Copper(II) partially protects three histidine residues and the N‐terminus of amyloid‐β peptide from diethyl pyrocarbonate (DEPC) modification

Diethyl pyrocarbonate (DEPC) has been primarily used as a residue‐specific modifying agent to study the role of His residues in peptide/protein and enzyme function; however, its action is not specific, and several other residues can also be modified. In the current study, we monitored the reaction of DEPC with amyloid‐beta (Aβ) peptides and insulin by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) and determined the modification sites by electrospray ionization quadrupole time‐of‐flight tandem MS (ESI Q‐TOF MS/MS). Our results indicate that five residues in Aβ1–42 are modified in the presence of 30‐fold molar excess of DEPC. After hydroxylamine treatment (which removes modifications from three His residues), two labels remain bound at the peptide N terminus and Lys16. DEPC treatment of Aβ1–42 promotes peptide aggregation, as monitored through the loss of soluble Aβ42 in a semi‐quantitative MALDI‐TOF MS assay. It has been previously proposed that Cu(II) ions protect Aβ1–16 from DEPC modification through binding to His6. We confirmed that Cu(II) ions decrease the stoichiometry of Aβ1–16 modification with the excess of DEPC being lower as compared to the control, which indicates that Cu(II) protects Aβ from DEPC modification. Sequencing of obtained Cu(II)‐protected Aβ1–16 samples showed that Cu(II) does not protect any residues completely, but partially protects all three His residues and the N terminus. Thus, the protection by Cu(II) ions is not related to specific metal binding to a particular residue (e.g. His6), but rather all His residues and the N terminus are involved in binding of Cu(II) ions. These results allow us to elucidate the effect of DEPC modification on amyloidogenity of human Aβ and to speculate about the role of His residues in these processes.

Antimicrobial and anti-inflammatory activities of three chensinin-1 peptides containing mutation of glycine and histidine residues

The natural peptide chensinin-1 doesnot exhibit its desired biological properties. In this study, the mutant MC1-1 was designed by replacing Gly in the chensinin-1 sequence with Trp. Mutants MC1-2 and MC1-3 were designed based on the MC1-1 sequence to investigate the specific role of His residues. The mutated peptides presented α-helicity in a membrane-mimetic environment and exhibited broad-spectrum antimicrobial activities; in contrast to Trp residues, His residues were dispensable for interacting with the cell membrane. The interactions between the mutant peptides and lipopolysaccharide (LPS) facilitated the ingestion of peptides by Gram-negative bacteria. The binding affinities of the peptides were similar, at approximately 10 μM, but ΔH for MC1-2 was −7.3 kcal.mol−1, which was 6-9 folds higher than those of MC1-1 and MC1-3, probably due to the conformational changes. All mutant peptides demonstrated the ability to inhibit LPS-induced tumour-necrosis factor-α (TNF-α) and interleukin-6 (IL-6) release from murine RAW264.7 cells. In addition, the representative peptide MC1-1showed better inhibition of serum TNF-α and IL-6 levels compared to polymyxin B (PMB), a potent binder and neutralizer of LPS as positive control in LPS-challenged mice model. These data suggest that the mutant peptides could be promising molecules for development as chensinin-based therapeutic agents against sepsis.

A New Raman Spectroscopic Probe of Both the Protonation State and Noncovalent Interactions of Histidine Residues

The amino acid histidine (His) has a number of unique roles that can dictate function in proteins, and these roles are typically conferred through noncovalent interactions that depend on the protonation state of His's 4-substituted imidazole ring. His's protonation state can vary near physiological pH, and a probe of His's variable protonation state and its resulting noncovalent interactions that has both high time resolution and no sample limitations could find wide use in determining the role of particular His residues in proteins. Here we use a classic deuterium exchange reaction to replace the C2-H hydrogen atom of the His imidazole ring with deuterium, leading to a unique aromatic C2-D stretching vibration whose frequency is sensitive to environmental changes across the entire imidazole ring. Using nonresonant Raman spectroscopy, we demonstrate using model compounds that the frequency of this C2-D vibration shifts by 35 cm(-1) upon changes in the His protonation state. The C2-D band is a very weak infrared absorber, so this vibration is not expected to be useful in infrared transmission experiments for proteins. Solvent-dependent Raman experiments indicate that the C2-D band of the neutral imidazole ring is sensitive to H-bonding interaction with donors and acceptors of varying strengths, suggesting that the C2-D frequency can be used to identify H-bonding partners of specific His residues. Raman spectra at varying concentrations of Cu(2+) also show the C2-D band's sensitivity to metal coordination, with differences due to changes in the coordination environment. The strong Raman signal of this band and the sampling flexibility of Raman spectroscopy suggest that this vibration could be very useful in documenting the local role of His residues in many His-containing proteins and protein assemblies.

Influence of metal ions on thermal aggregation of bovine serum albumin: Aggregation kinetics and structural changes

Metal ions are implicated in protein aggregation processes of several neurodegenerative pathologies. In this work the effects of Cu(II) and Zn(II) ions on heat-induced structural modifications of bovine serum albumin (BSA) were studied, with the aim of delineating the role of these ions in the early stages of proteins aggregation kinetics. A joint application of different techniques was used. The aggregate growth was followed by dynamic light scattering measurements, whereas the conformational changes occurring in the protein structure were monitored by Raman and IR spectroscopy. Both in absence and in presence of metal ions, heating treatment gave rise to beta-structures to the detriment of alpha-helix conformation of BSA. The temperature of protein unfolding was not sensitively affected by the presence of Zn(II) or Cu(II) ions; on the contrary, only Zn(II) ions slightly promoted the heat-induced aggregation of the protein, since bigger aggregates were formed in their presence. The different efficacy of the Cu(II) and Zn(II) ions in promoting the BSA aggregation were highlighted by Raman measurements, assessing the role of His residues in metal binding. A distinct polypeptide folding of the two metal-BSA systems takes place, since the predominant mode of metal binding depends on metal. In particular, in Zn-BSA the metal coordination involves the imidazole N(tau) atom of His which can promote inter-molecular cross-linking.

The Functional Roles of the His247 and His281 Residues in Folate and Proton Translocation Mediated by the Human Proton-coupled Folate Transporter SLC46A1

This report addresses the functional role of His residues in the proton-coupled folate transporter (PCFT; SLC46A1), which mediates intestinal folate absorption. Of ten His residues, only H247A and H281A mutations altered function. The folic acid influx Kt at pH 5.5 for H247A was downward arrow 8.4-fold. Although wild type (WT)-PCFT Ki values varied among the folates, Ki values were much lower and comparable for H247-A, -R, -Q, or -E mutants. Homology modeling localized His247 to the large loop separating transmembrane domains 6 and 7 at the cytoplasmic entrance of the translocation pathway in hydrogen-bond distance to Ser172. The folic acid influx Kt for S172A-PCFT was decreased similar to H247A. His281 faces the extracellular region in the seventh transmembrane domain. H281A-PCFT results in loss-of-function due to approximately 12-fold upward arrow in the folic acid influx Kt. When the pH was decreased from 5.5 to 4.5, the WT-PCFT folic acid influx Kt was unchanged, but the Kt decreased 4-fold for H281A. In electrophysiological studies in Xenopus oocytes, both WT-PCFT- and H281A-PCFT-mediated folic acid uptake produced current and acidification, and both exhibited a low level of folate-independent proton transport (slippage). Slippage was markedly increased for the H247A-PCFT mutant. The data suggest that disruption of the His247 to Ser172 interaction results in a PCFT conformational alteration causing a loss of selectivity, increased substrate access to a high affinity binding pocket, and proton transport in the absence of a folate gradient. The His281 residue is not essential for proton coupling but plays an important role in PCFT protonation, which, in turn, augments folate binding to the carrier.

Role of His residues in Bacillus subtilis cytochrome b558 for haem binding and assembly of succinate: quinone oxidoreductase (complex II)

Cytochrome b558 in the cytoplasmic membrane of Bacillus subtilis constitutes the anchor and electron acceptor to the flavoprotein (Fp) and iron-sulphur protein (Ip) in succinate:quinone oxidoreductase, and seemingly contains two haem groups. EPR and MCD spectroscopic data indicate bis-imidazole ligation of the haem. Apo-cytochrome was found in the membrane fraction of haem-deficient B. subtilis, suggesting that during biogenesis of the oxidoreductase the cytochrome b558 polypeptide is embedded into the membrane prior to the incorporation of haem and subsequent binding of Fp and Ip. The six His residues in cytochrome b558 were individually changed to Tyr to attempt identification of residues serving as haem axial ligands and to analyse the role of His residues for assembly and function of the oxidoreductase. From the properties of the mutants, His-47 can be excluded as a haem ligand. The remaining His residues (at positions 13, 28, 70, 113 and 155) are located in or close to four predicted transmembrane segments. The Tyr-28 and Tyr-70 mutant proteins appeared to lack one of the two haems. Only the Tyr-13 and Tyr-47 mutant cytochromes were found to function as anchors for Fp and Ip, but the Tyr-13 mutant cytochrome assembles into an enzymatically defective succinate:quinone oxidoreductase. It is concluded from a combination of the experimental findings, sequence comparisons and membrane topology data that His-28, His-70 and His-155 are probably haem axial ligands in a dihaem cytochrome b558. His-70 and His-155 may be ligands to the same haem.