Tryptophan- and arginine-rich antimicrobial peptides: Structures and mechanisms of action

Antimicrobial peptides encompass a number of different classes, including those that are rich in a particular amino acid. An important subset are peptides rich in Arg and Trp residues, such as indolicidin and tritrpticin, that have broad and potent antimicrobial activity. The importance of these two amino acids for antimicrobial activity was highlighted through the screening of a complete combinatorial library of hexapeptides. These residues possess some crucial chemical properties that make them suitable components of antimicrobial peptides. Trp has a distinct preference for the interfacial region of lipid bilayers, while Arg residues endow the peptides with cationic charges and hydrogen bonding properties necessary for interaction with the abundant anionic components of bacterial membranes. In combination, these two residues are capable of participating in cation-π interactions, thereby facilitating enhanced peptide-membrane interactions. Trp sidechains are also implicated in peptide and protein folding in aqueous solution, where they contribute by maintaining native and nonnative hydrophobic contacts. This has been observed for the antimicrobial peptide from human lactoferrin, possibly restraining the peptide structure in a suitable conformation to interact with the bacterial membrane. These unique properties make the Arg- and Trp-rich antimicrobial peptides highly active even at very short peptide lengths. Moreover, they lead to structures for membrane-mimetic bound peptides that go far beyond regular α-helices and β-sheet structures. In this review, the structures of a number of different Trp- and Arg-rich antimicrobial peptides are examined and some of the major mechanistic studies are presented.     

Molecular dynamics simulation of thionated hen egg white lysozyme

Understanding of the driving forces of protein folding is a complex challenge because different types of interactions play a varying role. To investigate the role of hydrogen bonding involving the backbone, the effect of thio substitutions in a protein, hen egg white lysozyme (HEWL), was investigated through molecular dynamics simulations of native as well as partly (only residues in loops) and fully thionated HEWL using the GROMOS 54A7 force field. The results of the three simulations show that the structural properties of fully thionated HEWL clearly differ from those of the native protein, while for partly thionated HEWL they only changed slightly compared with native HEWL. The analysis of the torsional-angle distributions and hydrogen bonds in the backbone suggests that the α-helical segments of native HEWL tend to show a propensity to convert to 3(10)-helical geometry in fully thionated HEWL. A comparison of the simulated quantities with experimental NMR data such as nuclear overhauser effect (NOE) atom-atom distance bounds and (3)J((H)(N)(H)(α))-couplings measured for native HEWL illustrates that the information content of these quantities with respect to the structural changes induced by thionation of the protein backbone is rather limited.     

Direct observation of basic protein HEWL as an oxoanions receptor by electrospray ionization mass spectrometry

Positive ion mode of electrospray ionization mass spectrometry (ESI-MS) has been used for the detection and study of protein interaction. From the measurement of molecular mass of the intact complex and individual binding partners, the binding stoichiometry can be derived. In our work, one basic protein, hen egg white lysozyme (HEWL) as an anion receptor shows high sensitivity and selectivity responses to most oxoanions detected by ESI-MS. But neutral protein, such as insulin, does not response to anions. It was found that HSO₄ ^-, IO₄ ^-, ClO₄ ^-, H₂PO₄ ^-, HCO₃ ^- and AcO^- have strong affinity to interact with HEWL under present condition, butHSO₃ ^-, NO₃ ^-, Cl^- and F^- could not be trapped by HEWL. ESI-MS condition and concentration of anions areconsidered. This is an important evidence obtained by mass spectrometryfor the distribution of anion recognition with a native protein.     

Nonnative Electrostatic Interactions Can Modulate Protein Folding: Molecular Dynamics with a Grain of Salt

In recent years, a growing number of protein folding studies have focused on the unfolded state, which is now recognized as playing a major role in the folding process. Some of these studies show that interactions occurring in the unfolded state can significantly affect the stability and kinetics of the protein folding reaction. In this study, we modeled the effect of electrostatic interactions, both native and nonnative, on the folding of three protein systems that underwent selective charge neutralization or reversal or complete charge suppression. In the case of the N-terminal L9 protein domain, our results directly attribute the increase in thermodynamic stability to destabilization of the unfolded ensemble, reaffirming the experimental observations. These results provide a deeper structural insight into the ensemble of the unfolded state and predict a new mutation site for increased protein stability. In the second case, charge reversal mutations of RNase Sa affected protein stability, with the destabilizing mutations being less destabilizing at higher salt concentrations, indicating the formation of charge-charge interactions in the unfolded state. In the N-terminal L9 and RNase Sa systems, changes in electrostatic interactions in the unfolded state that cause an increase in free energy had an overall compaction effect that suggests a decrease in entropy. In the third case, in which we compared the β-lactalbumin and hen egg-white lysozyme protein homologues, we successfully eliminated differences between the folding kinetics of the two systems by suppressing electrostatic interactions, supporting previously reported findings. Our coarse-grained molecular dynamics study not only reproduces experimentally reported findings but also provides a detailed molecular understanding of the elusive unfolded-state ensemble and how charge-charge interactions can modulate the biophysical characteristics of folding.     

Thermal Response with Exothermic Effects of β2-Microglobulin Amyloid Fibrils and Fibrillation

Calorimetric measurements were carried out using a differential scanning calorimeter to characterize the thermal response of β₂-microglobulin amyloid fibrils, the deposition of which results in dialysis-related amyloidosis. The fibril solution showed a large decrease in heat capacity (exothermic effect) before the temperature-induced depolymerization of the fibrils, which was characterized by a definite dependence on heating rate. To understand the factors that determine the heating-rate-dependent thermal response, the concentration dependence of polyethylene glycol, which inhibits the association of amyloid fibrils with heating, on exothermic effect was examined in detail and showed a causal link between the exothermic effect and fibril association. The results suggest that the transient association driven by a spatial approach and the concomitant dehydration of hydrophobic areas of amyloid fibrils may be significant factors determining the thermal response with exothermic effect, which has not been observed in calorimetric studies of monomolecular globular proteins. The heating-rate-dependent thermal response with the exothermic effect was observed not only for other amyloid fibrils formed from amyloid β-peptides but also during the processes of the temperature-induced conversion of β₂-microglobulin protofibrils and hen egg-white lysozyme into amyloid fibrils. These results highlight the physics related to the heating-rate-dependent behaviors of heat capacity in terms of interactions between the specific structures of amyloid fibrils and water molecules.     

Temperature derivative fluorescence spectroscopy as a tool to study dynamical changes in protein crystals

Motions through the energy landscape of proteins lead to biological function. At temperatures below a dynamical transition (150-250 K), some of these motions are arrested and the activity of some proteins ceases. Here, we introduce the technique of temperature-derivative fluorescence microspectrophotometry to investigate the dynamical behavior of single protein crystals. The observation of glass transitions in thin films of water/glycerol mixtures allowed us to demonstrate the potential of the technique. Then, protein crystals were investigated, after soaking the samples in a small amount of fluorescein. If the fluorophore resides within the crystal channels, temperature-dependent changes in solvent dynamics can be monitored. Alternatively, if the fluorophore binds to the protein, local dynamical transitions within the biomolecule can be probed directly. A clear dynamical transition was observed at 175 K in the active site of crystalline human butyrylcholinesterase. The results suggest that the dynamics of crystalline proteins is strongly dependent on solvent composition and confinement in the crystal channels. Beyond applications in the field of kinetic crystallography, the highly sensitive temperature-derivative fluorescence microspectrophotometry technique opens the way to many studies on the dynamics of biological nanosamples.     

Membrane integrity and amyloid cytotoxicity: a model study involving mitochondria and lysozyme fibrillation products

Recent findings implicate that fibrillation products, the protein aggregates formed during the various steps leading to formation of mature fibrils, induce neurotoxicity predominantly in their intermediate oligomeric state. This has been shown to occur by increasing membrane permeability, eventually leading to cell death. Despite accumulating reports describing mechanisms of membrane permeabilization by oligomers in model membranes, studies directly targeted at characterizing the events occurring in biological membranes are rare. In the present report, we describe interaction of the original native structure, prefibrils and fibrils of hen egg white lysozyme (HEWL) with mitochondrial membranes, as an in vitro biological model, with the aim of gaining insight into possible mechanism of cytotoxicity at the membrane level. These structures were first characterized using a range of techniques, including fluorescence, size-exclusion chromatography, dynamic light scattering, transmission electron microscopy, dot blot analysis and circular dichroism. HEWL oligomers were found to be flexible/hydrophobic structures with the capacity to interact with mitochondrial membranes. Possible permeabilization of mitochondria was explored utilizing sensitive fluorometric and luminometric assays. Results presented demonstrate release of mitochondrial enzymes upon exposure to HEWL oligomers, but not native enzyme monomer or mature fibrils, in a concentration-dependent manner. Release of cytochrome c was also observed, as reported earlier, and membrane stabilization promoted by addition of calcium prevented release. Moreover, the oligomer-membrane interaction was influenced by high concentrations of NaCl and spermine. The observed release of proteins from mitochondria is suggested to occur by a nonspecific perturbation mechanism.     

Conserved folding pathways of alpha-lactalbumin and lysozyme revealed by kinetic CD, fluorescence, NMR, and interrupted refolding experiments

In this report, it is shown by a combination of stopped-flow CD, fluorescence, and time-resolved NMR studies that the Ca^2 +-induced refolding of bovine α-lactalbumin (BLA) at constant denaturant concentration (4 M urea) exhibits triple-exponential kinetics. In order to distinguish between parallel folding pathways and a strictly sequential formation of the native state, interrupted refolding experiments were conducted. We show here that the Ca^2 +-induced refolding of BLA involves parallel pathways and the transient formation of a folding intermediate on the millisecond timescale. Our data furthermore suggest that the two structurally homologous proteins BLA and hen egg white lysozyme share a common folding mechanism. We provide evidence that the guiding role of long-range interactions in the unfolded state of lysozyme in mediating intersubdomain interactions during folding is replaced in the case of BLA by the Ca^2 +-binding site. Time-resolved NMR spectroscopy, in combination with fast ion release from caged compounds, enables the measurement of complex protein folding kinetics at protein concentrations as low as 100 μM and the concomitant detection of conformational transitions with rate constants of up to 8 s^− 1.     

Direct observation of basic protein HEWL as an oxoanions receptor by electrospray ionization mass spectrometry

Summary Positive ion mode of electrospray ionization mass spectrometry (ESI-MS) has been used for the detection and study of protein interaction. From the measurement of molecular mass of the intact complex and individual binding partners, the binding stoichiometry can be derived. In our work, one basic protein, hen egg white lysozyme (HEWL) as an anion receptor shows high sensitivity and selectivity responses to most oxoanions detected by ESI-MS. But neutral protein, such as insulin, does not response to anions. It was found that HSO ₄ ⁻ , IO ₄ ⁻ , ClO ₄ ⁻ , H₂PO ₄ ⁻ , HCO ₃ ⁻ and AcO⁻ have strong affinity to interact with HEWL under present condition, but HSO ₃ ⁻ , NO ₃ ⁻ , Cl⁻ and F⁻ could not be trapped by HEWL. ESI-MS condition and concentration of anions are considered. This is an important evidence obtained by mass spectrometry for the distribution of anion recognition with a native protein.     

Hen egg white lysozyme as an inhibitor of mushroom tyrosinase

We report a kinetics study on hen egg white lysozyme's (HEWL) inhibitory effect on mushroom tyrosinase catalysis of 3-(3,4-dihydroxyphenyl)-L-alanine (L-DOPA) or L-tyrosine. For the first time, we demonstrate HEWL as a robust inhibitor against mushroom tyrosinase in catalysis of both substrates. The kinetics pattern matches a mixed (mostly non-competitive) partial inhibition. Ki and ID50 value of HEWL are more than 20-fold lower than that of kojic acid, a well-known chemical inhibitor of mushroom tyrosinase. Ki, alpha value and beta value, are almost identical in both experiments (L-DOPA and L-tyrosine as substrates, respectively), which suggests this common inhibition mechanism affects both steps. The inhibitory effect increases as both proteins were mixed and pre-incubated for less than 1 h. HEWL-depletion only removed about half of the inhibitory effect. Here we propose a novel function of HEWL, which combines the reversible inhibition and the irreversible inactivation toward mushroom tyrosinase. Discovery of HEWL as an inhibitor to mushroom tyrosinase catalysis may be commercially valuable in the food, medical and cosmetic industries.