Internal segregation and side chain ordering in hairy-rod polypeptide monolayers at the gas/water interface: An x-ray scattering study

We report studies of the structure and packing of Langmuirmonolayers (LMs) of polypeptide poly(γ-4-(n-hexadecyloxy)benzyl α,L-glutamate) (C16–O–PBLG) on the surface of water. The molecule is a “hairy rod” and consists of side attachments of hexadecyloxy chains (–O–C16) to the rigid rod-like core made up of α-helical poly(γ-benzyl L-glutamate) (PBLG). Measurements include surface pressure (Π) versus area/monomer (A) isotherms, x-ray specular reflectivity (XR), and grazing incidence diffraction(GID). In contrast to the LM of bare PBLG on water, which undergoes a monolayer/bilayer transition with increasing Π, monolayers of C16–O–PBLG remain stable up to the highest densities. On the basis of XR and GID results, the structure of the C16–O–PBLG monolayer is characterized by the following main features. First, hydrophobicity causes the –O–C16 chains to segregate towards the film/gas interface and away from water and the PBLG cores, which sit parallel to and near the water/film interface. Since the attachment position of some of the side chains is at the core/water interface, the segregation forces these chains into the space between neighboring core rods. Compression associated with increasing Π thickens the film but the internally segregated structure is maintained for all Π (i.e., >∼30 dyne/cm). Second, the C16–O–PBLG rods form domains in which the rods are aligned parallel to each other and to the interface. The correlation length for the interhelix positional order of the rods is short and typically comparable to or less than the length of the rods. With increasing Π the spacing d between nearest-neighbor rods decreases linearly with A at high Π, indicating a direct correspondence between the macroscopic compressibility and the microscopic interhelix compressibility. Third, as Π increases past ∼5 dyne/cm, the local packing of tethered –O–C16 chains displays the same herringbone (HB) order that is common for high-density bulk and monolayer phases of alkyl chains. Various features of the observed GID peaks also imply that the HB order of –O–C16 chains is oriented with respect to the helical axes of aligned PBLG cores. We propose that the HB order is established initially by one-dimensionally confined chains between aligned rods at low Π and grows laterally with compression

Internal Segregation and Side Chain Ordering in Hairy-Rod Polypeptide Monolayers at the Gas/Water Interface: An X-Ray Scattering Study

We report studies of the structure and packing of Langmuir monolayers (LMs) of polypeptide poly($\gamma$-4-(n-hexadecyloxy)benzyl $\alpha$,L-glutamate) (C16–O–PBLG) on the surface of water. The molecule is a “hairy rod” and consists of side attachments of hexadecyloxy chains (–O–C16) to the rigid rod-like core made up of $\alpha$-helical poly($\gamma$-benzyl L-glutamate) (PBLG). Measurements include surface pressure ($\Pi$) versus area/monomer (A) isotherms, x-ray specular reflectivity (XR), and grazing incidence diffraction (GID). In contrast to the LM of bare PBLG on water, which undergoes a monolayer/bilayer transition with increasing $\Pi$, monolayers of C16–O–PBLG remain stable up to the highest densities. On the basis of XR and GID results, the structure of the C16–O–PBLG monolayer is characterized by the following main features. First, hydrophobicity causes the –O–C16 chains to segregate towards the film/gas interface and away from water and the PBLG cores, which sit parallel to and near the water/film interface. Since the attachment position of some of the side chains is at the core/water interface, the segregation forces these chains into the space between neighboring core rods. Compression associated with increasing $\Pi$ thickens the film but the internally segregated structure is maintained for all $\Pi$ (i.e., >$\sim$30 dyne/cm). Second, the C16–O–PBLG rods form domains in which the rods are aligned parallel to each other and to the interface. The correlation length for the interhelix positional order of the rods is short and typically comparable to or less than the length of the rods. With increasing $\Pi$ the spacing d between nearest-neighbor rods decreases linearly with A at high $\Pi$, indicating a direct correspondence between the macroscopic compressibility and the microscopic interhelix compressibility. Third, as $\Pi$ increases past $\sim$5 dyne/cm, the local packing of tethered –O–C16 chains displays the same herringbone (HB) order that is common for high-density bulk and monolayer phases of alkyl chains. Various features of the observed GID peaks also imply that the HB order of –O–C16 chains is oriented with respect to the helical axes of aligned PBLG cores. We propose that the HB order is established initially by one-dimensionally confined chains between aligned rods at low Π and grows laterally with compression.Engineering and Applied Science

Function and Regulation of Vibrio campbellii Proteorhodopsin: Acquired Phototrophy in a Classical Organoheterotroph

Proteorhodopsins (PRs) are retinal-binding photoproteins that mediate light-driven proton translocation across prokaryotic cell membranes. Despite their abundance, wide distribution and contribution to the bioenergy budget of the marine photic zone, an understanding of PR function and physiological significance in situ has been hampered as the vast majority of PRs studied to date are from unculturable bacteria or culturable species that lack the tools for genetic manipulation. In this study, we describe the presence and function of a horizontally acquired PR and retinal biosynthesis gene cluster in the culturable and genetically tractable bioluminescent marine bacterium Vibrio campbellii. Pigmentation analysis, absorption spectroscopy and photoinduction assays using a heterologous over-expression system established the V. campbellii PR as a functional green light absorbing proton pump. In situ analyses comparing PR expression and function in wild type (WT) V. campbellii with an isogenic ΔpR deletion mutant revealed a marked absence of PR membrane localization, pigmentation and light-induced proton pumping in the ΔpR mutant. Comparative photoinduction assays demonstrated the distinct upregulation of pR expression in the presence of light and PR-mediated photophosphorylation in WT cells that resulted in the enhancement of cellular survival during respiratory stress. In addition, we demonstrate that the master regulator of adaptive stress response and stationary phase, RpoS1, positively regulates pR expression and PR holoprotein pigmentation. Taken together, the results demonstrate facultative phototrophy in a classical marine organoheterotrophic Vibrio species and provide a salient example of how this organism has exploited lateral gene transfer to further its adaptation to the photic zone

Monodisperse benzyl -ester derivatives of poly(α, L-glutamate) and poly(α, L-aspartate) modeling nature\u27s delicate state of matter

Biosynthesis of monodisperse poly(α,L-aspartic acid) and optimization of poly(α,L-glutamic acid) production have been accomplished. Protein design having as ultimate goal the synthesis of monodisperse benzyl-ester derivatives of α-helical polypeptides by genetic engineering has been performed. This comprises design of a monomeric DNA cassette, cloning, multimerization, ligation into an expression vector, and expression of the target gene followed by purification of the resultant protein. Advantages of such techniques include control of molecular size, composition, sequence and stereochemistry of the synthesized polymers. 1H NMR (nuclear magnetic resonance), amino acid analysis, and MALDI-MS (matrix-assisted laser desorption mass spectrometry) analytical techniques were employed for characterization of monodisperse poly(α, L-amino acid)s. Modification of conventional purification and cyanogen bromide cleavage reactions are included as part of the strategy to obtain monodisperse poly(α, L-aspartic acid). Optimization of polymer production was achieved by means of utilization of a variety of Escherichia coli strains and plasmids, modification of large scale fermentation conditions, and alterations of purification procedures. Monodisperse poly(α,L-amino acid)s were benzylated by treating the acid forms of the polymers with phenyldiazomethane. Polarized light optical microscopy was used to observe textures from birefringent samples of poly(β-benzyl α,L-glutamate) and poly(β-benzyl α,L-aspartate). Small-angle X-ray diffraction data shows evidence of smectic ordering of monodisperse poly(β-benzyl α,L-glutamate) (DP = 76). poly(γ-4-(hexadecyloxy)benzyl α,L-glutamate) DP = 300, was synthesized by methods developed in our laboratories. Differential scanning calorimetry (DSC) and polarized light optical microscopy (POM) data is presented as evidence for thermotropic liquid crystal behavior

Protein engineering and other bio-synthetic routes for bio-based materials: Current uses and potential applications

In the past 20 years protein engineering has been used for the production of proteins mostly for biological applications. The incorporation of artificial amino acids and chemical handles into proteins had made possible the design and production of protein-based materials like hybrid inorganic-organic materials, smart/ responsive materials, monodisperse polymers, and nanoscale assemblies. In the current topic, we cover current uses and envision future applications of materials generated using protein engineering and biosynthesis techniques

Nanometer-Scale Smectic Ordering of Genetically Engineered Rodlike Polymers: Synthesis and Characterization of Monodisperse Derivatives of Poly(γ-benzyl α,L-glutamate)

Macromolecules with precisely defined architectures on the nanometer scale are ideal building blocks for self-assembled materials and nanometer-scale devices. By utilizing recombinant DNA technology and bacterial protein expression, we have prepared a set of rodlike artificial proteins, Glu(OBzl)Asp(OBzl)[Glu(OBzl)_(17)Asp(OBzl)]_X-Glu(OBzl)Glu(OBzl) (PBLG-X, X = 3−6, 1), which serve as monodisperse analogues of poly(γ-benzyl α,l-glutamate) (PBLG), with variation of rod length from 8.7 to 17 nm. Synthesis was accomplished by (i) bacterial production of a precursor polypeptide GluAsp(Glu_(17)Asp)_XGluGlu (PLGA-X, X = 3−6), as a fusion to mouse dihydrofolate reductase (DHFR), (ii) CNBr digestion of the expressed protein to liberate PLGA-X, and (iii) side chain benzylation of PLGA-X via treatment with phenyldiazomethane. The growth rates of cultures transformed with the recombinant genes, and the yields of protein expressed therein, depended on the number of Glu_(17)Asp repeats appended to the fusion partner, decreasing as the number of repeats increased. PBLG-3, with the lowest aspect ratio (6.9), did not exhibit liquid crystalline (LC) behavior while each of the other polymers formed lyotropic LC phases. The phase transition behavior of PBLG-X was distinctly different from that of conventional polydisperse PBLG; when the concentration of the polymer solution was gradually increased, the transition from the isotropic to the liquid crystalline state occurred uniformly throughout the sample without formation of distinct birefringent droplets. Solution-cast films of PBLG-4 and PBLG-5 formed smectic supramolecular architectures with layer spacings (11.4 and 14.0 nm, respectively) precisely determined by the length of the monodisperse rods. Smectic ordering was observed only in films cast from mixtures of chloroform (97%) and trifluoroacetic acid (3%). In films prepared from dioxane, both polydisperse and monodisperse PBLGs adopted columnar order without formation of smectic layers