Evolutionary Engineering a Larger Porin Using a Loop-to-Hairpin Mechanism.

In protein evolution, diversification is generally driven by genetic duplication. The hallmarks of this mechanism are visible in the repeating topology of various proteins. In outer membrane ß-barrels, duplication is visible with ß-hairpins as the repeating unit of the barrel. In contrast to the overall use of duplication in diversification, a computational study hypothesized evolutionary mechanisms other than hairpin duplications leading to increases in the number of strands in outer membrane ß-barrels. Specifically, the topology of some 16- and 18-stranded ß-barrels appear to have evolved through a loop to ß-hairpin transition. Here we test this novel evolutionary mechanism by creating a chimeric protein from an 18-stranded ß-barrel and an evolutionarily related 16-stranded ß-barrel. The chimeric combination of the two was created by replacing loop L3 of the 16-stranded barrel with the sequentially matched transmembrane ß-hairpin region of the 18-stranded barrel. We find the resulting chimeric protein is stable and has characteristics of increased strand number. This study provides the first experimental evidence supporting the evolution through a loop to ß-hairpin transition.

Evolutionary engineering a larger porin using a loop-to-hairpin mechanism.

In protein evolution, diversification is generally driven by genetic duplication. The hallmarks of this mechanism are visible in the repeating topology of various proteins. In outer membrane ß-barrels, duplication is visible with ß-hairpins as the repeating unit of the barrel. In contrast to the overall use of duplication in diversification, a computational study hypothesized evolutionary mechanisms other than hairpin duplications leading to increases in the number of strands in outer membrane ß-barrels. Specifically, the topology of some 16- and 18-stranded ß-barrels appear to have evolved through a loop to ß-hairpin transition. Here we test this novel evolutionary mechanism by creating a chimeric protein from an 18-stranded ß-barrel and an evolutionarily related 16-stranded ß-barrel. The chimeric combination of the two was created by replacing loop L3 of the 16-stranded barrel with the sequentially matched transmembrane ß-hairpin region of the 18-stranded barrel. We find the resulting chimeric protein is stable and has characteristics of increased strand number. This study provides the first experimental evidence supporting the evolution through a loop to ß-hairpin transition. Highlights: We find evidence supporting a novel diversification mechanism in membrane ß-barrelsThe mechanism is the conversion of an extracellular loop to transmembrane ß-hairpinA chimeric protein modeling this mechanism folds stably in the membraneThe chimera has more ß-structure and a larger pore, consistent with a loop-to-hairpin transition.

Detergent headgroups control TolC folding in vitro.

TolC is the trimeric outer membrane component of the efflux pump system in Escherichia coli that is responsible for antibiotic efflux from bacterial cells. Overexpression of efflux pumps has been reported to decrease susceptibility to antibiotics in a variety of bacterial pathogens. Reliable production of membrane proteins allows for the biophysical and structural characterization needed to better understand efflux and for the development of therapeutics. Preparation of recombinant protein for biochemical/structural studies often involves the production of proteins as inclusion body aggregates from which active proteins are recovered. Here, we find that the in vitro folding of TolC into its functional trimeric state from inclusion bodies is dependent on the headgroup composition of detergent micelles used. Nonionic detergent favors the formation of functional trimeric TolC, whereas zwitterionic detergents induce the formation of a non-native, oligomeric TolC fold. We also find that nonionic detergents with shorter alkyl lengths facilitate TolC folding. It remains to be seen whether the charges in lipid headgroups have similar effects on membrane insertion and folding in biological systems.

Membrane Barrels Are Taller, Fatter, Inside-Out Soluble Barrels.

Up-and-down ß-barrel topology exists in both the membrane and soluble environment. By comparing features of these structurally similar proteins, we can determine what features are particular to the environment rather than the fold. Here we compare structures of membrane ß-barrels to soluble ß-barrels and evaluate their relative size, shape, amino acid composition, hydrophobicity, and periodicity. We find that membrane ß-barrels are generally larger than soluble ß-barrels, with more strands per barrel and more amino acids per strand, making them wider and taller. We also find that membrane ß-barrels are inside-out soluble ß-barrels. The inward region of membrane ß-barrels has similar hydrophobicity to the outward region of soluble ß-barrels, and the outward region of membrane ß-barrels has similar hydrophobicity to the inward region of the soluble ß-barrels. Moreover, even though both types of ß-barrel have been assumed to have strands with amino acids that alternate in direction and hydrophobicity, we find that the membrane ß-barrels have more regular alternation than soluble ß-barrels. These features give insight into how membrane barrels maintain their fold and function in the membrane.

Outer membrane protein evolution.

Outer membrane proteins have remarkably homogeneous structure. They are all up down ß-barrels. Up down barrels themselves are composed of repeated sets of ß-hairpins. The consistency of the usage of the ß-hairpin throughout the outer membrane milieu allows for interrogation of the evolution of these repetitive structures. Here we describe recent investigations of outer membrane protein evolution and how evolutionary precepts have been used for novel outer membrane protein design.

Evaluation extracellular matrix-chitosan composite films for wound healing application.

The present study describes the preparation of extracellular matrix (ECM; from porcine omentum) based chitosan composite films for wound dressing applications. The films were prepared by varying the ECM content, whereas, the amount of chitosan was kept constant. The interactions amongst the components of the films were analyzed by FTIR and XRD studies. The films were thoroughly characterized for surface hydrophilicity, moisture retention capability, water vapor permeability, mechanical and biocompatibility. FTIR study indicated that both chitosan and ECM were present in their native form and did not lose their activity. XRD analysis suggested composition dependent change in the crystallinity of the films. The mechanical properties suggested that the composite films had sufficient properties to be used for wound dressing applications. An increase in the ECM content resulted in better hydrophilicity of the films and hence better the moisture retention capacity and retardant water vapor transmission rate property of the composite films. The films were found to be biocompatible to both blood and adipose tissue derived stem cells. In gist, the prepared films may be explored as wound dressing materials.

Triple-stranded DNA containing 8-oxo-7,8-dihydro-2'-deoxyguanosine: implication in the design of selective aptamer sensors for 8-oxo-7,8-dihydroguanine.

8-Oxo-7,8-dihydroguanine (8-oxoG, or OG) as a free base has been widely considered as a biomarker for DNA oxidative damage. Currently no fluorescence sensor has been developed to directly detect 8-oxoG less than 100 nM. In this study, two triple-stranded DNAs were selected as the scaffolds to rationally design DNA aptamer sensors for 8-oxoG. The cavity was created by deleting the 8-oxodG nucleoside in a triplex containing an A·OG-C triad or a C·OG-A triad. The results showed that the fluorescence of both sensors were completely quenched by 8-oxoG. The detection ranges of the two sensors were different, while the combined range was comparable to the detection range of an antibody-based method. This result is expected to enable a fast, low-cost, and reusable method to measure 8-oxoG concentration.