Formation Of Hydrophobic Cluster Research Articles

Hydrophobic Cluster Formation in Aqueous Ethanol Solutions Probed by Soft X-ray Absorption Spectroscopy.

Hydrophobic cluster structures in aqueous ethanol solutions at different concentrations have been investigated by soft X-ray absorption spectroscopy (XAS). In the O K-edge XAS, we have found that hydrogen bond structures among water molecules are enhanced in the middle-concentration region by the hydrophobic interaction of the ethyl groups in ethanol. In the C K-edge XAS, the lower energy features arise from a transition from the terminal methyl C 1s electron to an unoccupied orbital of 3s Rydberg character, which is sensitive to the nearest-neighbor intermolecular interactions. From the comparison of C K-edge XAS with the inner-shell calculations, we have found that ethanol clusters are easily formed in the middle-concentration region due to the hydrophobic interaction of the ethyl group in ethanol, resulting in the enhancement of the hydrogen bond structures among water molecules. This behavior is different from aqueous methanol solutions, where the methanol-water mixed clusters are more predominant in the middle-concentration region due to the relatively weak hydrophobic interactions of the methyl group in methanol.

Hydrogen Bonding Sequence Directed Coil-Globule Transition in Water Soluble Thermoresponsive Polymers.

The origin of the coil-globule transition for water-soluble thermoresponsive polymers frequently used in nanomaterials remains elusive. Using polypropylene oxide as an example we demonstrate by means of atomistic molecular dynamics simulations that temperature-induced increase in the sequence length of monomers that are not hydrogen bonded to water drives the coil-globule transition. Longer chains statistically exhibit longer sequences which serve as nucleation sites for hydrophobic cluster formation, facilitating chain collapse at lower temperature in agreement with experimental data.

Why are ACE2 binding coronavirus strains SARS-CoV/SARS-CoV-2 wild and NL63 mild?

Coronaviruses are responsible for several epidemics, including the 2002 SARS, 2012 MERS, and COVID‐19. The emergence of recent COVID‐19 pandemic due to SARS‐CoV‐2 virus in December 2019 has resulted in considerable research efforts to design antiviral drugs and other therapeutics against coronaviruses. In this context, it is crucial to understand the biophysical and structural features of the major proteins that are involved in virus‐host interactions. In the current study, we have compared spike proteins from three strains of coronaviruses NL63, SARS‐CoV, and SARS‐CoV, known to bind human angiotensin‐converting enzyme 2 (ACE2), in terms of sequence/structure conservation, hydrophobic cluster formation and importance of binding site residues. The study reveals that the severity of coronavirus strains correlates positively with the interaction area, surrounding hydrophobicity and interaction energy and inversely correlate with the flexibility of the binding interface. Also, we identify the conserved residues in the binding interface of spike proteins in all three strains. The systematic point mutations show that these conserved residues in the respective strains are evolutionarily favored at their respective positions. The similarities and differences in the spike proteins of the three viruses indicated in this study may help researchers to deeply understand the structural behavior, binding site properties and etiology of ACE2 binding, accelerating the screening of potential lead molecules and the development/repurposing of therapeutic drugs.

Immobilization of Small‐Molecule Ligands Containing Secondary or Tertiary Amine Groups onto TiO2‐Supported Ru Catalysts Driven by the Hydrophobic Effect

AbstractA strategy for effectively immobilizing small‐molecule ligands onto TiO2‐supported Ru catalysts is described. The immobilization was based on a simple two‐phase centrifugation technique and driven by the hydrophobic effect, likely leading to the formation of hydrophobic clusters of small‐molecule ligands. By using this strategy, a library of ligands containing secondary or tertiary amine groups and hydrophobic chains were successfully immobilized onto TiO2‐supported Ru catalysts. Of these ligands, ligands containing 2,2,6,6‐tetramethyl‐1‐piperidine‐N‐oxyl (TEMPO) moieties significantly improved the selectivity for aldehyde at high conversion in aerobic oxidation of alcohols, resulting from the inhibition of auto‐oxidation of aldehydes. This strategy can generate diversity in preparation of organic/inorganic hybrid catalysts.

In situ formation of hydrophobic clusters to enhance mechanical performance of biodegradable poly(l-glutamic acid)/poly(ε-caprolactone) hydrogel towards meniscus tissue engineering.

The preparation of scaffolds represents a huge challenge for meniscus tissue engineering, as there are particular biomechanical and biodegradability requirements. Here, an amphiphilic polymer was prepared by grafting hydrophobic poly(ε-caprolactone) (PCL) with short chains onto hydrophilic biodegradable poly(l-glutamic acid) (PLGA). PLGA-g-PCL was then crosslinked with triethylene glycol (TEG) to form a chemically crosslinked network (CCN), followed by the in situ formation of stable hydrophobic PCL clusters inside the preformed CCN to increase the strength and elasticity of the hydrogel. The spindle-like PCL clusters in the hydrogel were observed to possess a mean size of 5-10 μm on the long axis and 3-4 μm on the minor axis, with no crystallization according to WAXS. Owing to the hydrophobic association of PCL, the swollen CCN was secondarily crosslinked and stiffened. Dynamic mechanical analysis (DMA) demonstrated excellent rebound resilience, and the maximum compressive strength of all the PLGA-g-PCL hydrogels was greater than 0.60 MPa with a maximum strain of about 70%, which represented significant increases in comparison with a PLGA hydrogel. Owing to the short chains of PCL, degradation of the PLGA-g-PCL hydrogel was accelerated within 3 months in vivo. A hydrogel carrying adipose-derived stem cells (ASCs) effectively regenerated meniscus-like tissue in vivo and preserved the corresponding articular cartilage from degeneration over a 16 week period.

PNIPAm-based microgels with a UCST response

Thermoresponsive microgels with an upper critical solution temperature (UCST) response (i.e., showing volume expansion at higher temperature) are highly desirable for a variety of applications, but quite uncommon, in particular if they are to work in physiological conditions. Here we present a novel UCST-type microgel showing a remarkable transition in physiological conditions, with a diameter increase larger than 150% from 34 °C to 40 °C. This novel material is based in poly(N-isopropyl acrylamide), pNIPAm, a polymer well known for its opposite temperature response (featuring a lower critical solution temperature, LCST). The unusual properties of our material are due to a small fraction of copolymerized methacrylic acid and nitrocatechol monomers. The collapsed conformation in PBS buffer below 34 °C is stabilized by hydrogen bonding, with repulsion by partially ionized nitrocatechol and methacrylic units being screened by the ionic strength and the formation of hydrophobic clusters by the methyl groups in methacrylic acid. The increase in temperature leads to the collapse of pNIPAm segments, which disrupts the balance of supramolecular interactions, leading to the large volume expansion of the gel.

Characterization of human small heat shock protein HspB1 that carries C-terminal domain mutations associated with hereditary motor neuron diseases

Physico-chemical properties of four mutants (T164A, T180I, P182S and R188W) of human small heat shock protein HspB1 (Hsp27) associated with neurodegenerative diseases were analyzed by means of fluorescence spectroscopy, dynamic light scattering, size-exclusion chromatography and measurement of chaperone-like activity. Mutation T164A was accompanied by destabilization of the quaternary structure and decrease of thermal stability without any significant changes of chaperone-like activity. Mutations T180I and P182S are adjacent or within the conserved C-terminal motif IPI/V. Replacement T180⇒I leading to the formation of hydrophobic cluster consisting of three Ile produced small increase of thermal stability without changes of chaperone-like activity. Mutation P182S induced the formation of metastable large oligomers of HspB1 with apparent molecular weight of more than 1000kDa. Oligomers of P182S have very low thermal stability and undergo irreversible aggregation at low temperature. The P182S mutant forms mixed oligomers with the wild type HspB1 and the properties of these mixed oligomers are intermediate between those of the wild type HspB1 and its mutant. Mutation R188W did not significantly affect quaternary structure or thermal stability of HspB1, but was accompanied by a pronounced decrease of its chaperone-like activity. All mutations analyzed are associated with hereditary motor neuropathies or Charcot–Marie–Tooth disease type 2; however, molecular mechanisms underlying pathological effects are specific for each of these mutants.

Atomic-level structure characterization of an ultrafast folding mini-protein denatured state.

Atomic-level analyses of non-native protein ensembles constitute an important aspect of protein folding studies to reach a more complete understanding of how proteins attain their native form exhibiting biological activity. Previously, formation of hydrophobic clusters in the 6 M urea-denatured state of an ultrafast folding mini-protein known as TC5b from both photo-CIDNP NOE transfer studies and FCS measurements was observed. Here, we elucidate the structural properties of this mini-protein denatured in 6 M urea performing 15N NMR relaxation studies together with a thorough NOE analysis. Even though our results demonstrate that no elements of secondary structure persist in the denatured state, the heterogeneous distribution of R 2 rate constants together with observing pronounced heteronuclear NOEs along the peptide backbone reveals specific regions of urea-denatured TC5b exhibiting a high degree of structural rigidity more frequently observed for native proteins. The data are complemented with studies on two TC5b point mutants to verify the importance of hydrophobic interactions for fast folding. Our results corroborate earlier findings of a hydrophobic cluster present in urea-denatured TC5b comprising both native and non-native contacts underscoring their importance for ultra rapid folding. The data assist in finding ways of interpreting the effects of pre-existing native and/or non-native interactions on the ultrafast folding of proteins; a fact, which might have to be considered when defining the starting conditions for molecular dynamics simulation studies of protein folding.

Interfacial Viscoelasticity of Myoglobin at Air/Water and Air/Solution Interfaces: Role of Folding and Clustering

This study describes the folding and organization of myoglobin (Mb) at the solution/air interface at different pH values of 2.5, 3.5, 5.5, 7.5, and 8.5. Dynamic surface tension and the associated dilational and shear viscoelasticity for Mb at these pH's have been studied using a sinusoidal surface compression and expansion for frequencies ranging from 0.01 to 0.4 Hz. The changes in dilational viscosity, elasticity, and fluorescence lifetime measurements have been related to the conformational changes of the protein films at the interface. It is observed that while acid-induced denaturation of the protein does not lead to large changes in dilational properties, the shear properties on the other hand are strongly influenced by it, and the protein behaves like a shear-thickening fluid. At higher pH, particularly at the isoelectric point, Mb is pseudoplastic indicating an increase in the shear viscosity. These results are strongly suggestive of formation of hydrophobic clusters at the protein-buffer interface because of the change in the overall charge distributions.

Structural characterization and unfolding mechanism of human 4F2hc ectodomain

4F2hc (CD98hc) is a multifunctional type II membrane glycoprotein involved in several functions as amino acid transport, cell fusion, β1-integrin-signaling and transformation. 4F2hc ectodomain has been crystallized and its three-dimensional structure determined. We have carried out a spectroscopical/structural characterization of the recombinant ectodomain in order to obtain information on its dynamic structure in solution and on its ability to form homodimers by itself in the absence of the transmembrane helix and of the potential interactions with the plasma membrane. Analytical ultracentrifugation and crosslinking experiments showed that the ectodomain is monomeric in solution. The secondary structure determined by far-UV circular dichroism (CD) spectroscopy (around 30% α-helix and 20% β-sheets, 12% antiparallel and 8% parallel) reveals a compact and thermally stable structure with a high melting temperature (57–59°C). Tryptophan residues are mainly buried and immobilized in the hydrophobic core of the protein as suggested by near-UV CD spectrum, the position of the Trp maximum fluorescence emission (323nm) and from the acrylamide quenching constant (2.6M−1). Urea unfolding equilibrium has been studied by far-UV CD and fluorescence spectroscopy to gain information on the folding/unfolding process of the ectodomain. The analyses suggest the existence of two intermediate states as reported for other TIM barrel-containing proteins rather than an independent unfolding of each domain [A, (βα)8 barrel; C, antiparallel β8 sandwich]. Folding seems to be directed by the initial formation of hydrophobic clusters within the first strands of the β-barrel of domain A followed by additional hydrophobic interactions in domain C.

Evidence for Initial Non-specific Polypeptide Chain Collapse During the Refolding of the SH3 Domain of PI3 Kinase

Refolding of the SH3 domain of PI3 kinase from the guanidine hydrochloride (GdnHCl)-unfolded state has been probed with millisecond (stopped flow) and sub-millisecond (continuous flow) measurements of the change in fluorescence, circular dichroism, ANS fluorescence and three-site fluorescence resonance energy transfer (FRET) efficiency. Fluorescence measurements are unable to detect structural changes preceding the rate-limiting step of folding, whereas measurements of changes in ANS fluorescence and FRET efficiency indicate that polypeptide chain collapse precedes the major structural transition. The initial chain collapse reaction is complete within 150 μs. The collapsed form at this time possesses hydrophobic clusters to which ANS binds. Each of the three measured intra-molecular distances has contracted to an extent predicted by the dependence of the FRET signal in completely unfolded protein on denaturant concentration, indicating that contraction is non-specific. The extent of contraction of each intra-molecular distance in the collapsed product of sub-millisecond folding increases continuously with a decrease in [GdnHCl]. The gradual contraction is continuous with the gradual contraction seen in completely unfolded protein, and its dependence on [GdnHCl] is not indicative of an all-or-none collapse reaction. The dependence of the extent of contraction on [GdnHCl] was similar for the three distances, indicating that chain collapse occurs in a synchronous manner across different segments of the polypeptide chain. The sub-millisecond measurements of folding in GdnHCl were unable to determine whether hydrophobic cluster formation, probed by ANS fluorescence measurement, precedes chain contraction probed by FRET. To determine whether hydrogen bonding plays a role in initial chain collapse, folding was initiated by dilution of the urea-unfolded state. The extent of contraction of at least one intra-molecular distance in the collapsed product of sub-millisecond folding in urea is similar to that seen in GdnHCl, and the initial contraction in urea too appears to be gradual.

Correlation of protein-protein interactions as assessed by affinity chromatography with colloidal protein stability: A case study with lysozyme

Lysozyme-lysozyme interactions were assessed in the native state at 25°C as well in the denatured state at 80°C by affinity chromatography in order to measure the osmotic second virial coefficient (B). This parameter allows us to better understand protein aggregation pathways and colloidal protein stability. Repulsive interactions (B > 0) were weakened for both protein states by increasing salt concentration and by increasing the pH value toward lysozyme pI. This decrease was more pronounced in the denatured state, most likely caused by changes in electrostatic interactions and the formation of hydrophobic clusters. The lysozyme formulations presenting the more repulsive conditions (B > 0), as derived from the osmotic second virial coefficient, showed better colloidal stability under mechanical and thermal stresses. As expected, B values are much more negative for the interactions in the denatured state compared to the data obtained for the native state, reflecting a strong tendency of denatured lysozyme to aggregate. Thus, measurement of protein interactions by affinity chromatography allows us to gain information on protein interactions in both native and denatured states as well as to predict solution conditions prone for improving protein colloidal stability.

Fluctuating partially native-like topologies in the acid denatured ensemble of autolysis resistant HIV-1 protease

Folding, in-vivo, starts from a denatured state and thus the nature of the denatured state would play an important role in directing the folding of a protein. We report here NMR characterization of the acid-denatured state of a mutant of HIV-1 protease, designed to prevent autolysis (Q7K, L33I, L63I) and to prevent cysteine oxidation (C67A and C95A). Secondary chemical shifts, TALOS analysis of chemical shifts and 15N relaxation data ( R 1, R 2, NOE) coupled with AABUF and hydrophobicity calculations, suggest formation of hydrophobic clusters and possibility of some partially native-like topologies in the acid denatured state of the protease. The structural and dynamics characteristics of the acid denatured PR seem to be considerably different from those of the guanidine or urea denatured states of some variants of PR. These would have implications for the folding and auto-processing of the enzyme in-vivo.

Barrierless evolution of structure during the submillisecond refolding reaction of a small protein.

To determine whether a protein folding reaction can occur in the absence of a dominant barrier is crucial for understanding its complexity. Here direct ultrafast kinetic measurements have been used to study the initial submillisecond (sub-ms) folding reaction of the small protein barstar. The cooperativity of the initial folding reaction has been explored by using two probes: fluorescence resonance energy transfer, through which the contraction of two intramolecular distances is measured, and the binding of 8-anilino-1-naphthalene sulfonic acid, through which the formation of hydrophobic clusters is monitored. A fast chain contraction is shown to precede the formation of hydrophobic clusters, indicating that the sub-ms folding reaction is not cooperative. The observed rate constant of the sub-ms folding reaction monitored by 8-anilino-1-naphthalene sulfonic acid fluorescence has been found to be the same in stabilizing conditions (low urea concentrations), in which specific structure is formed, and in marginally stabilizing conditions (higher urea concentrations), where virtually no structure is formed in the product of the sub-ms folding reaction. The observation that the folding rate is independent of the folding conditions suggests that the initial folding reaction occurs in the absence of a dominant free energy barrier. These results provide kinetic evidence that the formation of specific structure need not be slowed down by any significant free energy barrier during the course of a very fast protein folding reaction.

Light-responsive hydrophobic association of surfactants with azobenzene-modified polymers

We examined light-triggered formation/dissociation of complexes between azobenzene-modified polymers (AMPs) and surfactants, and we found that the origin of their response to light clearly differs from that of self-assembling of micelles of photosurfactants. We studied a set of AMPs derived from polyacrylic acids with varying integration levels of azobenzene, mixed with (non-responsive) surfactants (TX 100, SDBS) that are representative of common polymer–surfactant formulae. In dilute solutions, light scattering measurements showed that surfactant–AMP association is reversibly affected by exposure to UV/visible light. Binding isotherms of the azobenzene chromophore (into micelle-like cores) and the surfactants (into AMP chains) were determined by spectrophotometry and capillary electrophoresis, respectively. Three regimes can be distinguished with increasing surfactant concentration: i) absence of binding below the CMC, ii) cooperative binding between CMC and ca. 3 × CMC, with the formation of a hydrophobic cluster containing a significant fraction (50–20 mol%) of bound azobenzene, and iii) anticooperative association in equilibrium with excess free micelles, and formation of hydrophobic clusters containing typically 8–18 mol% azobenzene. Reversible UV-triggered dissociation is achieved over the entire anticooperative regime, irrespective of AMP structure. Both too low (a few mol%) and too high (above 30 mol%) fractions of azobenzene in the mixed micelles appear deleterious to the magnitude of response to light. Altogether, our results indicate that micelle-like clusters containing about 10 mol% photoanchors can easily swing between attachment/detachment upon photo-isomerisation. A model is proposed to interpret the responsiveness of AMPs with low integration levels of azobenzene.

A novel amphiphilic pH-sensitive hydrogel based on pullulan

Novel polyelectrolyte and amphiphilic hydrogels based on pullulan have been prepared using 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide hydrochloride and N-hydroxysuccinimide. The cross-linking reaction is fast and lead to zero length ester cross-links by the reaction of a carboxylate group with an alcohol function of the polysaccharide. The charge density and the hydrophobic rate of the precursor carboxymethylpullulan (CMP) are controlled during the carboxymethylation of pullulan and the grafting reaction of octyl chains on CMP, respectively. The grafting degree influences the conformation of the hydrophobically modified CMP (HMCMP) in solution and leads to the formation of hydrophobic clusters firstly in the HMCMP solutions and further in the HMCMP hydrogels. The swelling measurements of HMCMP hydrogels at different salt concentrations (0–0.2 M NaCl) and different pH (3–11) shows the ionic strength and pH sensitivity of the gels. The loading of a hydrophobic probe molecule can be controlled by the grafting degree of HMCMP hydrogels.

Fundamental processes of protein folding: measuring the energetic balance between helix formation and hydrophobic interactions.

Theories of protein folding often consider contributions from three fundamental elements: loops, hydrophobic interactions, and secondary structures. The pathway of protein folding, the rate of folding, and the final folded structure should be predictable if the energetic contributions to folding of these fundamental factors were properly understood. alphatalpha is a helix-turn-helix peptide that was developed by de novo design to provide a model system for the study of these important elements of protein folding. Hydrogen exchange experiments were performed on selectively 15N-labeled alphatalpha and used to calculate the stability of hydrogen bonds within the peptide. The resulting pattern of hydrogen bond stability was analyzed using a version of Lifson-Roig model that was extended to include a statistical parameter for tertiary interactions. This parameter, x, represents the additional statistical weight conferred upon a helical state by a tertiary contact. The hydrogen exchange data is most closely fit by the XHC model with an x parameter of 9.25. Thus the statistical weight of a hydrophobic tertiary contact is approximately 5.8x the statistical weight for helix formation by alanine. The value for the x parameter derived from this study should provide a basis for the understanding of the relationship between hydrophobic cluster formation and secondary structure formation during the early stages of protein folding.

Energy barriers, cooperativity, and hidden intermediates in the folding of small proteins

Current theoretical views of the folding process of small proteins (<∼100 amino acids) postulate that the landscape of potential mean force (PMF) for the formation of the native state has a funnel shape and that the free energy barrier to folding arises from the chain configurational entropy only. However, recent theoretical studies on the formation of hydrophobic clusters with explicit water suggest that a barrier should exist on the PMF of folding, consistent with the fact that protein folding generally involves a large positive activation enthalpy at room temperature. In addition, high-resolution structural studies of the hidden partially unfolded intermediates have revealed the existence of non-native interactions, suggesting that the correction of the non-native interactions during folding should also lead to barriers on PMF. To explore the effect of a PMF barrier on the folding behavior of proteins, we modified Zwanzig’s model for protein folding with an uphill landscape of PMF for the formation of transition states. We found that the modified model for short peptide segments can satisfy the thermodynamic and kinetic criteria for an apparently two-state folding. Since the Levinthal paradox can be solved by a stepwise folding of short peptide segments, a landscape of PMF with a locally uphill search for the transition state and cooperative stabilization of folding intermediates/native state is able to explain the available experimental results for small proteins. We speculate that the existence of cooperative hidden folding intermediates in small proteins could be the consequence of the highly specific structures of the native state, which are selected by evolution to perform specific functions and fold in a biologically meaningful time scale.

Effect of the Mobility of Charged Units on the Microphase Separation in Amphiphilic Polyelectrolyte Hydrogels

The effect of the migration of charged units on the structure of hydrophobically modified polyelectrolyte gels swollen by D(2)O was studied by small-angle neutron scattering on an example of gels of terpolymers of acrylic acid, n-dodecylacrylate, and 2-acrylamido-2-methyl-1-propanesulfonic acid (quenched charged units) and gels of copolymers of partially neutralized acrylic acid and n-dodecylacrylate (annealed charged units). The content of charged units did not exceed 20 mol %, so that the electrostatic repulsion was too weak to disrupt the hydrophobic domains formed by self-assembled n-dodecyl chains, which was evidenced by NMR data. It was shown that upon increasing the charge content both types of gels undergo microphase separation with the formation of hydrophobic clusters consisting of several densely packed hydrophobic domains and hydrophilic regions swollen by water, where most of the charged repeat units and counterions are located. The dimensions of the nanostructure of the gels with quenched and annealed charged groups were compared. It was shown that the size of clusters in the gels with annealed charged units is much bigger than that in the gels with the same fraction of quenched charged units. This effect was attributed to a much weaker electrostatic repulsion in the corona of the hydrophobic clusters in the gels with annealed charged groups, because the charged units repelling each other are able to move farther apart.

Antimicrobial activity of arginine- and tryptophan-rich hexapeptides: the effects of aromatic clusters, D-amino acid substitution and cyclization.

Many antimicrobial peptides bear arginine (R)- and tryptophan (W)-rich sequence motifs. Based on the sequence Ac-RRWWRF-NH2, sets of linear and cyclic peptides were generated by changes in the amino acid sequence, L-D-amino acid exchange and naphthylalanine substituted for tryptophan. Linear RW-peptides displayed moderate activity towards Gram-positive Bacillus subtilis (15 < MIC < 31 microm) and were inactive against Gram-negative Escherichia coli at peptide concentrations < 100 microm. Cyclization induced high antimicrobial activity. The effect of cyclization was most pronounced for peptides with three adjacent aromatic residues. Incorporation of d-amino acid residues had minor influence on the biological activity. The haemolytic activity of all RW-peptides at 100 microm concentration was low (< 7% lysis for linear R/W-rich peptides and < 28% for the cyclic analogues). Introduction of naphthylalanine enhanced the biological activities of both the linear and cyclic peptides. All peptides induced permeabilization of large unilamellar vesicles (LUVs) composed of lipids of the membrane of B. subtilis and erythrocytes, but surprisingly had no effect on LUVs composed of lipids of the E. coli inner membrane. The profiles of peptide activity against B. subtilis and red blood cells correlated with the permeabilizing effects on the corresponding model membranes and were related to hydrophobicity parameters as derived from reversed phase high-performance liquid chromatography (HPLC). The results underlined the importance of amphipathicity as a driving force for cell lytic activity and suggest that conformational constraints and an appropriate position of aromatic residues allowing the formation of hydrophobic clusters are highly favourable for antimicrobial activity and selectivity.