Protein and Peptide Letters - Volume 17, Issue 11, 2010

Antimicrobial peptides (AMPs) are a class of defence peptides that selectively target micro-organisms in order to protect the host. AMPs form part of the innate immune system of organisms such as plants (thormatin), insects (ceratoxin), amphibians (magainins, brevins, aureins) and mammals (indolicidin, LL-37, defensins, dermaseptin) [1-4]. Many of these peptides are cationic, composed of 10 to 45 amino acids, and adopt amphiphilic structures at the membrane interface. These peptides have a potent ability to target and kill a wide range of Gram-negative and Gram-positive bacteria [5-6], protozoa [7] parasites [7], fungi [8], viruses [9] and some tumour cells [10]. Based on this ability, AMPs are attractive propositions for development as therapeutically useful antimicrobial and anticancer agents [11]. It is generally accepted that the killing mechanisms of defence peptides involve the invasion of target cell membranes, although there remain many questions around the precise structure / function relationships involved. Membrane binding is thought to provide a key rate limiting step in these mechanisms, which can then lead to passage of the peptide through the membrane in order to attack intracellular targets or, as in most cases, membrane disruption and cell death [12]. In the opening review by Peter Bond and Syma Khalid, molecular dynamic simulations are applied to investigate the role of conformation in the activity of AMPs. More recently, course grain simulations have been used to investigate the impact of factors such as peptide concentration, lipid composition and membrane curvature and also the effect these factors have on the lytic mechanism employed by AMPs. Whilst primary sequence data can provide key information regarding activity, a range of biophysical and molecular analysis have shown that the overall molecular architecture of AMPs is important for peptide-membrane interaction. Over 1500 AMPs have so far been identified and the majority of these peptides adopt β-sheet or α-helical structure. The paper by Kowlaski et al. characterises the antimicrobial properties of two synthetic β-sheet peptides derived from Limulus anti-lipopolysaccharides. Isothermal titration calorimetry is used to investigate the binding of the peptide to lipopolysaccharides and to investigate its potential antiseptic activity. The review paper by Christopher Dempsey and co-workers summarises a membrane model system used to investigate the thermodynamics of interfacial binding and subsequent membrane disruption by α-helical antimicrobial peptides. RTA3 and magainin analogues are used to investigate how the balance of hydrophobicity, amphiphilicity and positive charge affected binding and disruption of membranes. Once bound, many AMPs exert their activity by disruption of bilayer structure via one of a number of mechanisms. To further understand this activity, it is therefore important to investigate the impact of the peptide on the phospholipid. The paper by Farid Sa'adedin and Jeremy Bradshaw adds an interesting aspect to the literature about the effect of the antimicrobial peptide LS3 on the phase transition temperature of model lipid membranes. Differential scanning calorimetry studies were used to investigate the effect of the peptide on the phase transition of mixtures of cis and trans isomers of phosphatidylethanolamine and the data was used to discuss the orientation of the peptide in the membrane. The work undertaken by Angeliki Damianoglou et al. uses a well characterised AMP, melittin, and the enzyme, phospholipase A2 (PLA2), to analyse peptidemembrane interactions and insertion behaviour using linear dichroism (LD) to study the orientation of the peptide in the membrane. Although LD improves our understanding of peptide-membrane interactions, this paper emphasises that such work needs to be complemented by a range of other biophysical techniques such as dynamic light scattering, circular dichroism and mass spectrometry...........

Antimicrobial peptides (AMPs) are short, cationic, membrane-interacting proteins that exhibit broad-spectrum antimicrobial activity, and are hence of significant biomedical interest. They exert their activity by selectively binding to and lysing target cell membranes, but the precise molecular details of their mechanism are not known. This is further complicated by the fact that their structural characteristics are dependent upon the local lipid environment. As a result, molecular dynamics (MD) simulations have been applied to understand the conformation and mechanism of AMPs, as well as related viral and cell-penetrating peptides. In particular, atomically detailed MD simulation studies on the timescale of tens to hundreds of nanoseconds have successfully helped to: (i) model or refine the conformation of AMPs and their aggregates in the presence of membrane-mimicking solvent mixtures, detergent micelles, and lipid bilayers; (ii) follow the process of adsorption of individual AMPs to membrane surfaces; and (iii) observe the spontaneous assembly of multiple peptides and subsequent cooperative membrane lysis. More recently, coarse-grained (CG) models have been developed to extend the time and length scales accessible to simulations of membrane/peptide systems. CG simulations on the order of microseconds have provided insight into AMP lytic mechanisms, and how they depend upon such factors as peptide concentration, lipid composition, and bilayer curvature. These studies have been supplemented by combined atomistic/ CG and integrated multiscale models. Together, simulations have deepened our understanding of the interactions between AMPs and biological membranes, and will help to design new synthetic peptides with enhanced biomedical potential.

We have synthesized a series of short peptides (17 to 20 amino acids), originally derived from Limulus antilipopolysaccharide factor LALF, which were primarily designed to act as antimicrobial agents as well as neutralizers of bacterial endotoxin (lipopolysaccharide, LPS), Here, two selected peptides, a 17- and a 19-mer, were characterized physicochemically and in biological test systems. The secondary structure of the peptides indicates essentially a β-sheet including antiparallel strands, the latter being reduced when the peptides bind to LPS. A very strong exothermic binding due to attractive Coulomb interactions governs the LPS-peptide reaction, which additionally leads to a fluidization of the acyl chains of LPS. A comparison of the interaction of the peptide with negatively charged phosphatidylserine shows in contrast a rigidification of the acyl chains of the lipid. Finally, the biological assays reveal a diverging behaviour of the two peptides, with higher antibacterial activity of the 17-mer, but a much higher activity of the 19-mer in its ability to inhibit the LPS-induced cytokine production in human mononuclear cells.

Amphipathic peptides are accommodated within the diffuse gradient of polarity that characterizes the interfacial regions of phospholipid bilayer membranes. Interfacial membrane interactions are key to the diverse biological functions and activities of these peptides, which encompass a large class of antimicrobial peptides including the helical peptides magainin, melittin, and RTA3 derived from the commensal bacterium Streptococcus mitis. For these peptides in vitro efficacy (high antimicrobial activity with minimal mammalian cell toxicity, equivalent to high potential therapeutic index; PTI), can be broadly understood in relation to the thermodynamics of interfacial binding and membrane disruption in membranes having surface charges that correspond to bacterial and mammalian cell membranes, respectively. Peptides with disrupted amphipathicity resulting from a positively charged amino acid residue on the non-polar helix face, can have greatly enhanced PTI, although a balance of amphipathicity, hydrophobicity and positive charge is required for retention of high antimicrobial activity. These observations are illustrated with recent examples from the literature, and studies on RTA3 and magainin analogues from our laboratories. Despite the identification and optimisation of peptides with very good PTI, a focus on addressing toxicity upon systemic administration and poor in vivo efficacy is likely to be required to translate growing understanding of the relationships between peptide interfacial activity and effects on cells, into novel systemic therapeutics.

Differential Scanning Calorimetry studies of a synthetic peptide revealed the peptide decreased the temperature of the lamellar-hexagonal phase transition of cis-trans mixtures of phosphatidylethanolamine. The transition enthalpy varied significantly with lipid composition. The findings are discussed with reference to peptide saturation on the bilayer surface, bilayer thinning and peptide orientation.

Here we present data on the kinetics of insertion of melittin, a peptide from bee venom, into lipid membranes of different composition. Another component of bee venom is the enzyme phospholipase A2 (PLA2). We have examined the interaction of melittin and PLA2 with liposomes both separately and combined and demonstrate that they work synergistically to disrupt the membranes. A dramatic difference in the action of melittin and PLA2 is observed when the composition of the membrane is altered. Temperature also has a large effect on the kinetics of insertion and membrane disruption. We use a combination of techniques to measure liposome size (dynamic light scattering), peptide secondary structure (circular dichroism spectroscopy), peptide orientation relative to the membrane (linear dichroism spectroscopy) and enzymatic digestion of the lipids (mass spectrometry).

The Langmuir Blodgett apparatus provides a versatile system for studying the interfacial properties of peptides and peptide-membrane interactions under controlled conditions. Using amphiphilic α-helical peptides to highlight studies undertaken, here we discuss the use of this system to provide information on the surface activity of peptides and describe the insights these studies give into biological function.

Tyrosine hydroxylase is studied in terms of adsorption behaviour on gold surfaces and various passivating layers. Results reveal differences in layer formation, where mercaptoundecanoic acid-coated gold shows the best potential in terms of adsorbed mass. Nanoparticles with this coating are subsequently tested for enzymatic activity, which remains at attenuated levels.

QseC is a histidine kinase (HK) receptor involved in quorum sensing, a mechanism by which bacteria respond to fluctuations in cell population. We conducted a structural study of the cytoplasmic domain of QseC (QseC-CD) using X-ray crystallography. The 2.5 Å structure of the apo-enzyme revealed that the kinase domain of QseC retains the overall fold of the typical HK kinase domain. The construct that we used is inactive in the autokinase reaction and its inactivity is most likely caused by its atypical dimerization interface, as compared to that observed in the T.maritima HK cytoplasmic domain structure. Restoration of the activity may require that the entire dimerization domain be present in the protein construct. QseC, which plays an important role in bacterial pathogenesis, is a promising drug target and the structure of QseCCD provides a platform for developing more potent inhibitors of pathogen virulence.

The paper deals with investigation of silver ion interaction with sulfur-bearing amino acids and cysteinebearing peptides using an electrospray ionisation orthogonal ion introduction time-of-flight (ESI-o-TOF) mass spectrometer. It has been shown that Cys and Hcy demonstrate the largest affinity for silver, both in relation to methionine and cysteine residues in peptides. The studies have for the first time revealed the effect of predominant forming of ions containing two silver atoms per a sulfhydryl group of cysteine-bearing peptides if the nearest microenvironment does not hinder this.

We evaluated the i-peptides occurrence frequency in the protein sequences, belonging to two reference datasets containing structured and disordered protein domains. Moreover we estimated the most frequent i-peptides (with i= 2, 3, 4) into these sequences in order to select specific i-peptides for each structural classification. According to these specific ipeptides, a new binary classification method was developed for predicting if a given protein sequence can be classified as “disordered” or “structured”. The best results were obtained using the tri-peptides, much more able to gain structural information from sequences compared to the di-peptides.

Cyclodextrin glycosyltransferase (EC 2.4.1.19, CGTase) is an important industrial enzyme in the production of cyclodextrins. Thermal stability is of great importance for this enzyme. Rational design of thermostable variants of mesophilic proteins is well motivated. In this work, molecular dynamics simulations have been performed to study thermal stabilization of CGTase protein via electrostatic interactions of salt bridges. To predict behaviors of the salt bridges engineered into a mesophilic protein to increase stability, in silico mutant of CGTase from the mesophilic Bacillus macerans is generated. Dynamic motions of salt bridges in thermal unstable regions are monitored during the simulations. Among the five salt bridges, Lys88-Glu91, Asp296-Arg335 and Arg336-Asp370 are found to be more important for stability than the others. Especially, the region C is stabilized by a well-organized strong multiple salt bridge interactions. The results reveal that salt bridges involved in thermal unstable regions are relatively strong and prone to be tightened at elevated temperature, which can hold the stable conformation of the spatial neighborhood. Meanwhile, we use the heat capacity and total energy as the measure of stability difference between the original and its mutant variant, and then, quantify the contribution of salt bridges in thermal unstable regions for the mutant protein. Therefore, the viable computational strategy has been demonstrated to improve thermal stability of the mesophilic CGTase by introducing stable salt bridge interactions into its thermal unstable regions and it can be universally applied to other enzymes.

In this work, we have compared the stability and activity of immobilized lipase and free enzyme of same specific activity. The immobilization was carried out on (3Å x 1.5 mm) molecular sieve (a porous support) derivatized with glutaraldehyde as the functional group. Immobilization of the enzyme allowed the maintenance of 85% of the enzyme activity even after 8th cycle. In fact, only 12% of the enzyme activity was lost whereas the soluble enzyme lost 90% of its initial activity when incubated at 55°C for 2 hrs. Additionally, the enzyme was stable between pH 7.5-9.0 unlike free enzyme. Kinetic parameters Km and Vmax for free and molecular sieve-immobilized lipase were found to be 0.3 mM and 1 μmole/min/ml, 3.7 mM and 8 μmole/min/ml, respectively. Moreover, the immobilized enzyme, on the porous support, cannot be denatured with detergents, and, therefore, it maintained the stability achieved by means of the multipoint covalent attachment on the molecular sieve support.

Microarray technologies have begun to feature widely in biomedical science. These techniques, allow for high throughput and quantitative analysis of protein-carbohydrate interactions. Lectin and antibody have been evaluated with these new techniques which extend to the detection of viruses and bacteria. This review outlines some of the basic principles of ‘glycoarrays’ and illustrates their recent applications. Moreover, the review also gives an overview about a recently launched powerful detection platform using lectin microarrays with a potential to revolutionize the use of lectins in biomedical diagnosis and glycomics in general. In addition, two analytical techniques including mass spectrometry and glycan microarrays expected to play important role to characterize binding profile of new lectins are described in brief. Finally, strong and weak points of lectins as biorecognition molecules currently used in biomedical diagnosis are shown with conclusions drawn from molecular modelling of biorecognition events.

The effects of mixed crowding agents containing both sucrose and dextran 70 on refolding process of human muscle creatine kinase (HCK) were studied by enzyme activity assay and aggregation measurements. The results showed that sucrose and dextran have opposite effect on parameters of HCK during refolding: reactivation yield, refolding rates and amount of aggregation, as they were both used in the mixed crowding agents. The exclusion volume effect of dextran and osmophobic effect of sucrose on HCK refolding can be counteracted by each other: sucrose bated the aggregation induced by dextran and increased the final reactivation yield and refolding rate of the slow track, while dextran inhibited the effect of sucrose to prevent aggregation and help correct folding. The effects of human cyclophilin 18 (hCyp18) and casein on folding of HCK were also studied in crowding conditions, and it was found that the chaperone function of hCyp18 was additive with sucrose but blocked by dextran, and that the aggregation-improving effect of casein was additive with dextran 70 but impaired by sucrose to certain extent. This study indicates osmolytes and macromolecule crowding agents play different roles in the physiological conditions and could lead to better understanding of protein folding in the intracellular environment.

The polypeptide leptin exerts a multitude of regulatory functions. It has been implicated in the pathophysiology of inflammatory, metabolic and psychiatric disorders and has been found to be differentially expressed in men and women. Although a clear increase of leptin levels with age has been repeatedly observed in men, the association of leptin levels and age in women is an issue of scientific discussion. To investigate the association of age, gender, body mass index (BMI) and selected diseases with plasma levels of leptin in 551 adults randomly chosen from the Bavarian population, we assessed subjects' characteristics, lifestyle, and medical history including life time history of frequent diseases and performed blood sampling and standardized anthropometric measurements. Leptin plasma levels were measured using a Radioimmunoassay. Leptin levels were significantly higher in women as compared to men and this difference persisted even after controlling for differences in age or BMI. Leptin levels increased across the age groups in both men and women. Controlling for differences in BMI substantially attenuated the influence of age on leptin levels. In women, age was no longer significantly associated with leptin levels after controlling for BMI. With regard to medical history, hyperuricemia and gout were significantly associated with higher leptin levels, even after controlling for BMI, whereas subjects with high blood pressure or dyslipoproteinemia showed higher leptin levels only if the BMI was not considered as control variable. The BMI and its influence on the interrelations of gender, age and leptin should be considered when interpreting leptin levels.

Predicting enzyme subfamily class is an imbalance multi-class classification problem due to the fact that the number of proteins in each subfamily makes a great difference. In this paper, we focus on developing the computational methods specially designed for the imbalance multi-class classification problem to predict enzyme subfamily class. We compare two support vector machine (SVM)-based methods for the imbalance problem, AdaBoost algorithm with RBFSVM (SVM with RBF kernel) and SVM with arithmetic mean (AM) offset (AM-SVM) in enzyme subfamily classification. As input features for our predictive model, we use the conjoint triad feature (CTF). We validate two methods on an enzyme benchmark dataset, which contains six enzyme main families with a total of thirty-four subfamily classes, and those proteins have less than 40% sequence identity to any other in a same functional class. In predicting oxidoreductases subfamilies, AM-SVM obtains the over 0.92 Matthew's correlation coefficient (MCC) and over 93% accuracy, and in predicting lyases, isomerases and ligases subfamilies, it obtains over 0.73 MCC and over 82% accuracy. The improvement in the predictive performance suggests the AM-SVM might play a complementary role to the existing function annotation methods.

Proteins perform various functions through interacting with other molecules. Analyzing the characteristics of residues on the interaction interfaces provides insights into the mechanisms of these interactions. In this study, we analyze the characteristics of five different interfaces: protein-protein interfaces, protein-DNA interfaces, protein-RNA interfaces, protein-carbohydrate interfaces, and protein-ligand interfaces. The analysis reveals that these interfaces are different in residue composition. These differences in residue composition reflect the differences in the mechanisms that facility different types of interactions. Regardless of the differences in residue composition, all of the five types of interfaces are more conservative than the non-interface protein surfaces. Additionally, our results also show that it is important to consider the effect of solvent accessibility when investigating residues' propensities for different parts of the proteins.