IPRO+/−: Computational Protein Design Tool Allowing for Insertions and Deletions

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Reversible colour change in Arthropoda

The mechanisms and functions of reversible colour change in arthropods are highly diverse despite, or perhaps due to, the presence of an exoskeleton. Physiological colour changes, which have been recorded in 90 arthropod species, are rapid and are the result of changes in the positioning of microstructures or pigments, or in the refractive index of layers in the integument. By contrast, morphological colour changes, documented in 31 species, involve the anabolism or catabolism of components (e.g. pigments) directly related to the observable colour. In this review we highlight the diversity of mechanisms by which reversible colour change occurs and the evolutionary context and diversity of arthropod taxa in which it has been observed. Further, we discuss the functions of reversible colour change so far proposed, review the limited behavioural and ecological data, and argue that the field requires phylogenetically controlled approaches to understanding the evolution of reversible colour change. Finally, we encourage biologists to explore new model systems for colour change and to engage scientists from other disciplines; continued cross‐disciplinary collaboration is the most promising approach to this nexus of biology, physics, and chemistry.     

Rapid assessment of early biophysical changes in K562 cells during apoptosis determined using dielectrophoresis

Apoptosis, or programmed cell death, is a vital cellular process responsible for causing cells to self-terminate at the end of their useful life. Abrogation of this process is commonly linked to cancer, and rapid detection of apoptosis in vitro is vital to the discovery of new anti-cancer drugs. In this paper, we describe the application of the electrical phenomenon dielectrophoresis for detecting apoptosis at very early stages after drug induction, on the basis of changes in electrophysiological properties. Our studies have revealed that K562 (human myelogenous leukemia) cells show a persistent elevation in the cytoplasmic conductivity occurring as early as 30 minutes following exposure to staurosporine. This method therefore allows a far more rapid detection method than existing biochemical marker methods.     

Resolving the kinetics of lipid,protein and peptide diffusion in membranes

Abstract

Recent developments in the understanding of molecular diffusion phenomena in membranes are reviewed. Both model bilayers and biological membranes are considered in respect of lateral diffusion, rotational diffusion and transverse diffusion (flip-flop). For model systems, particular attention is paid to recent data obtained using surface-specific techniques such as sum frequency generation vibrational spectroscopy on supported lipid bilayers, and fluorescence correlation spectroscopy on giant unilamellar vesicles, both of which have yielded new insights into the intrinsic rates of diffusion and the energetic barriers to processes such as lipid flip-flop. Advances in single-molecule and many-molecule fluorescence methodologies have enabled the observation of processes such as anomalous diffusion for some membrane species in biological membranes. These are discussed in terms of new models for the role of membrane interactions with the cytoskeleton, the effects of molecular crowding in membranes, and the formation of lipid rafts. The diffusion of peptides, proteins and lipids is considered, particularly in relation to the means by which antimicrobial peptide activity may be rationalized in terms of membrane poration and lipid flip-flop.     

Computation and Functional Studies Provide a Model for the Structure of the Zinc Transporter hZIP4

Members of the Zrt and Irt protein (ZIP) family are a central participant in transition metal homeostasis as they function to increase the cytosolic concentration of zinc and/or iron. However, the lack of a crystal structure hinders elucidation of the molecular mechanism of ZIP proteins. Here, we employed GREMLIN, a co-evolution-based contact prediction approach in conjunction with the Rosetta structure prediction program to construct a structural model of the human (h) ZIP4 transporter. The predicted contact data are best fit by modeling hZIP4 as a dimer. Mutagenesis of residues that comprise a central putative hZIP4 transmembrane transition metal coordination site in the structural model alter the kinetics and specificity of hZIP4. Comparison of the hZIP4 dimer model to all known membrane protein structures identifies the 12-transmembrane monomeric Piriformospora indica phosphate transporter (PiPT), a member of the major facilitator superfamily (MFS), as a likely structural homolog.     

Chitoporin from the Marine Bacterium Vibrio harveyi: PROBING THE ESSENTIAL ROLES OF TRP136 AT THE SURFACE OF THE CONSTRICTION ZONE*

VhChiP is a sugar-specific porin present in the outer membrane of the marine bacterium Vibrio harveyi. VhChiP is responsible for the uptake of chitin oligosaccharides, with particular selectivity for chitohexaose. In this study, we employed electrophysiological and biochemical approaches to demonstrate that Trp¹³⁶, located at the mouth of the VhChiP pore, plays an essential role in controlling the channel''s ion conductivity, chitin affinity, and permeability. Kinetic analysis of sugar translocation obtained from single channel recordings indicated that the Trp¹³⁶ mutations W136A, W136D, W136R, and W136F considerably reduce the binding affinity of the protein channel for its best substrate, chitohexaose. Liposome swelling assays confirmed that the Trp¹³⁶ mutations decreased the rate of bulk chitohexaose permeation through the VhChiP channel. Notably, all of the mutants show increases in the off-rate for chitohexaose of up to 20-fold compared with that of the native channel. Furthermore, the cation/anion permeability ratio P_c/P_a is decreased in the W136R mutant and increased in the W136D mutant. This demonstrates that the negatively charged surface at the interior of the protein lumen preferentially attracts cationic species, leading to the cation selectivity of this trimeric channel.     

Extended and dynamic linker histone-DNA Interactions control chromatosome compaction

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Regulation of Oligomeric Organization of the Serotonin 5-Hydroxytryptamine 2C (5-HT2C) Receptor Observed by Spatial Intensity Distribution Analysis

The questions of whether G protein-coupled receptors exist as monomers, dimers, and/or oligomers and if these species interconvert in a ligand-dependent manner are among the most contentious current issues in biology. When employing spatial intensity distribution analysis to laser scanning confocal microscope images of cells stably expressing either a plasma membrane-associated form of monomeric enhanced green fluorescent protein (eGFP) or a tandem version of this fluorophore, the eGFP tandem was identified as a dimer. Similar studies on cells stably expressing an eGFP-tagged form of the epidermal growth factor receptor demonstrated that, although largely a monomer in the basal state, this receptor rapidly became predominantly dimeric upon the addition of its ligand epidermal growth factor. In cells induced to express an eGFP-tagged form of the serotonin 5-hydroxytryptamine 2C (5-HT_2C) receptor, global analysis of construct quantal brightness was consistent with the predominant form of the receptor being dimeric. However, detailed spatial intensity distribution analysis demonstrated the presence of multiple forms ranging from monomers to higher-order oligomers. Furthermore, treatment with chemically distinct 5-HT_2C receptor antagonists resulted in a time-dependent change in the quaternary organization to one in which there was a preponderance of receptor monomers. This antagonist-mediated effect was reversible, because washout of the ligand resulted in the regeneration of many of the oligomeric forms of the receptor.     

Molecular organization of the lipid matrix in intact Stratum corneum using ATR-FTIR spectroscopy

ATR-FTIR spectroscopy is useful in investigating the lateral organization of Stratum corneum (SC) lipids in full-thickness skin. Based on studies of the thermotropic phase transitions in n-tricosane and in excised human skin, the temperature dependence of the CH₂ scissoring bandwidth emerged as a measure of the extent of orthorhombic and hexagonal phases. This dependence provides a simpler measure of the lateral order in lipid assemblies than the common spectroscopic approaches based on difference spectra, curve fitting of the CH₂ scissoring region, and the position of the CH₂ stretching vibrations. It has the advantages of ease of determination, relatively low variability, and high discriminative power for the type of lateral intermolecular chain packing. A comparison of the lateral organization of the lipids at the SC surface of mammalian skin using the scissoring bandwidth revealed considerable differences between human abdominal skin (containing mostly orthorhombic phases), porcine ear skin (containing mostly hexagonal phases), and reconstructed human epidermis (containing mostly disordered phases). This parameter also correctly described the different effects of propylene glycol (minimally disturbing) and oleic acid (formation of a highly disordered phase) on the SC lipids in excised human skin. The procedure described here is applicable to in vivo studies in the areas of dermatology, transdermal drug delivery, and skin biophysics.     

A tale of two tails: The importance of unstructured termini in the aggregation pathway of β2‐microglobulin

The identification of intermediate states for folding and aggregation is important from a fundamental standpoint and for the design of novel therapeutic strategies targeted at conformational disorders. Protein human β2‐microglobulin (HB2m) is classically associated with dialysis‐related amyloidosis, but the single point mutant D76N was recently identified as the causative agent of a hereditary systemic amyloidosis affecting visceral organs. Here, we use D76N as a model system to explore the early stage of the aggregation mechanism of HB2m by means of an integrative approach framed on molecular simulations. Discrete molecular dynamics simulations of a structured‐based model predict the existence of two intermediate states populating the folding landscape. The intermediate I₁ features an unstructured C‐terminus, while I₂, which is exclusively populated by the mutant, exhibits two unstructured termini. Docking simulations indicate that I₂ is the key species for aggregation at acidic and physiological pH contributing to rationalize the higher amyloidogenic potential of D76N relative to the wild‐type protein and the ΔN6 variant. The analysis carried out here recapitulates the importance of the DE‐loop in HB2m self‐association at a neutral pH and predicts a leading role of the C‐terminus and the adjacent G‐strand in the dimerization process under acidic conditions. The identification of aggregation hot‐spots is in line with experimental results that support the importance of Phe56, Asp59, Trp60, Phe62, Tyr63, and Tyr66 in HB2m amyloidogenesis. We further predict the involvement of new residues such as Lys94 and Trp95 in the aggregation process.