Secondary Structure State Research Articles

Mechanism of flavonols for binding protein and inhibiting cell activity: Regulation by B-ring hydroxyl groups and Cu(II) coordination

In this study, we aimed to elucidate the mechanism of flavonol-Cu(II) coordination binding to human serum albumin (HSA) and its effect on cellular activity, focusing on the regulation by the number of B-ring hydroxyl groups and Cu(II) coordination. The number of B-ring hydroxyl groups was positively associated with fluorescence quenching ability, secondary structure modification, and protein affinity. The coordination of Cu(II) altered the binding capacity, interaction, binding site, and binding kinetics between flavonols and proteins, as well as the secondary structure, fluorescence lifetime, and microscopic state of proteins. The number of B-ring hydroxyl groups was positively correlated with the ability of flavonols to inhibit HepG2 cell activity, and this correlation was not affected by Cu(II) coordination. However, Cu(II) binding weakened the ability of flavonols to inhibit HepG2 cell activity. This study contributes to a comprehensive understanding of how the number of hydroxyl groups in the B-ring of flavonols and the presence of copper ions affect their transport in the blood, providing new insights into the bioavailability of flavonols.

Purification and Biochemical Characterization of the DNA Binding Domain of the Nitrogenase Transcriptional Activator NifA from Gluconacetobacter diazotrophicus.

NifA is a σ54 activator that turns on bacterial nitrogen fixation under reducing conditions and when fixed cellular nitrogen levels are low. The redox sensing mechanism in NifA is poorly understood. In α- and β-proteobacteria, redox sensing involves two pairs of Cys residues within and immediately following the protein's central AAA+ domain. In this work, we examine if an additional Cys pair that is part of a C(X)5C motif and located immediately upstream of the DNA binding domain of NifA from the α-proteobacterium Gluconacetobacter diazotrophicus (Gd) is involved in redox sensing. We hypothesize that the Cys residues' redox state may directly influence the DNA binding domain's DNA binding affinity and/or alter the protein's oligomeric sate. Two DNA binding domain constructs were generated, a longer construct (2C-DBD), consisting of the DNA binding domain with the upstream Cys pair, and a shorter construct (NC-DBD) that lacks the Cys pair. The Kd of NC-DBD for its cognate DNA sequence (nifH-UAS) is equal to 20.0 µM. The Kd of 2C-DBD for nifH-UAS when the Cys pair is oxidized is 34.5 µM. Reduction of the disulfide bond does not change the DNA binding affinity. Additional experiments indicate that the redox state of the Cys residues does not influence the secondary structure or oligomerization state of the NifA DNA binding domain. Together, these results demonstrate that the Cys pair upstream of the DNA binding domain of Gd-NifA does not regulate DNA binding or domain dimerization in a redox dependent manner.

Biohybrid membranes for organophosphate pesticides degradation: Hyperactivation of immobilized phosphotriesterase by surfactants

The aim of this work is to develop a hyperactivated biocatalytic membrane (BM) for the degradation of organophosphate pesticides (OPs), combining an enzyme-loaded membrane with cationic (cetyl trimethyl ammonium bromide, CTAB) or anionic (sodium dodecyl sulfate, SDS) surfactants. To analyze the effect of surfactants on the catalytic performance of BM, a systematic study of enzyme activity, stability, change in the secondary structure, protein aggregate formation, and kinetic parameters ( KM, Kcat, Kcat/KM) was performed. The results showed that the activity of the immobilized phosphotriesterase toward the pesticide paraoxon was greatly enhanced in the presence of SDS and CTAB (90% and 80%, respectively). Circular dichroism, dynamic light scattering, electrophoresis, and kinetics studies made it clear that surfactants affect catalytic performance by either changing the secondary structure and aggregation state of the enzyme (CTAB) or increasing the affinity of the enzyme for the substrate (SDS). Despite the strong improvement in free enzyme activity (2.6μmol min−1mg−1 versus 4.9 and 4.6μmol min−1mg−1 with SDS and CTAB, respectively), a smaller enhancement effect was observed with the immobilized enzyme, which may be attributed to the increased stiffness of the enzyme due to immobilization. Nevertheless, stable BMs with doubled specific activity were prepared that remained almost constant across various reaction cycles. Moreover, tests in a biocatalytic membrane reactor showed a 96% conversion at one-third of the residence time compared to the literature. The proposed strategy is a real advance in improving the activity/stability of BMs, which is one of the main drawbacks of this technology.

Investigation into the distinct structural features of the internal fusion loop within the LASV fusion domain.

Infection with Lassa virus (LASV), an Old-World arenavirus that is endemic to West Africa, causes Lassa fever – a lethal hemorrhagic fever. Delivery of LASV's genetic material into the host cell is an integral component of its lifecycle. This is accomplished via membrane fusion – a process widely accepted to be initiated by a hydrophobic sequence known as the fusion domain (FD).The LASV FD (G260–N295) consists of two structurally distinct regions: an N-terminal fusion peptide (FP: G260–P275) and an internal fusion loop (FL: G276–N295). Here, we identified a conserved disulfide bond (C279 and C292) and salt bridge (R282 and E289) within the FL that are pertinent to fusion. Through a FRET-based fusion assay, we witnessed a significant reduction in fusion when the disulfide bond was broken. Additionally, we found that fusion is reduced in the presence of high salt concentrations. When single point mutations were made such that each residue was the same charge (i.e., R282E and E289R) to ablate the salt bridge, we observed reduced fusion, which was partially returned when a double mutation was introduced (R282E/E289R). This was corroborated by ITC where the binding affinity to the lipid bilayer was decreased in comparison to wildtype for each salt bridge mutant and without the disulfide bond. While it could be suggested aggregation is causing this loss in function, CD spectroscopy and gel filtration revealed that both the global secondary structure and oligomeric state, respectively, of the FD remains unchanged, even after mutagenesis or disruption of the disulfide bond. In conclusion, our findings suggest that both the salt bridge and disulfide bond within the FL are critical for the proper association of the complete FD with the membrane to elicit efficient fusion.

Epitopedia: identifying molecular mimicry between pathogens and known immune epitopes

Upon infection, foreign antigenic proteins stimulate the host's immune system to produce antibodies targeting the pathogen. These antibodies bind to regions on the antigen called epitopes. Structural similarity (molecular mimicry) of epitopes between an infecting pathogen and host proteins or other pathogenic proteins the host has previously encountered can impact the host immune response to the pathogen and may lead to cross-reactive antibodies. The ability to identify potential regions of molecular mimicry in a pathogen can illuminate immune effects which are especially important to pathogen treatment and vaccine design. Here we present Epitopedia, a software pipeline that facilitates the identification of regions that may exhibit potential three-dimensional molecular mimicry between an antigenic pathogen protein and known immune epitopes as catalogued by the Immune Epitope Database (IEDB). Epitopedia is open-source software released under the MIT license and is freely available on GitHub, including a Docker container with all other software dependencies preinstalled. We performed an analysis describing how various secondary structure states, identity between pentapeptide pairs, and identity between the parent sequences of pentapeptide pairs affects RMSD. We found that pentapeptides pairs in a helical conformation had considerably lower RMSD values than those in extended or coil conformations. We also found that RMSD is significantly increased when pentapeptide pairs are from non-homologous sequences.

Effects of citrus fibre and soybean protein isolate on heat‐induced pork myofibrillar protein gel properties under low‐sodium salt conditions

SummaryThe aim of this study was to investigate the effect of the combination of citrus fibre (CF) and soy protein isolate (SPI) on pork myofibrillar protein (MP) gel properties under low‐sodium salt conditions during the heating process. The composite (with CF and SPI) and single (with 1% and 2% NaCl) MP gels properties were evaluated by colour, water holding capacity (WHC), gel strength, rheological properties, microstructure, secondary structure, and water state. The addition of 0.45% CF and 1.5% SPI significantly improved WHC and gel strength, as well as increased the values of G′ and G′′ (P < 0.05). The microstructure indicated that the CF/SPI could decrease the water channels and form a denser structure through filling effects, which was also proved by the fastT21relaxation time. However, the addition of 0.9% CF and 3.0% SPI could inhibit the protein aggregation and disrupt the stable and homogeneous structure. The analysis of the secondary structure showed that the CF/SPI resulted in a lower α‐helix content and a higher β‐sheet content compared with MP gels. This study may provide some guidance for the use of CF/SPI to improve MP gel properties in low‐salt meat products.

Effect of ultrasound on pH-shift to improve thermal stability of coconut milk by modifying physicochemical properties of coconut milk protein

In this study, ultrasound was subjected to pH-shift treatment to explore their synergistic effects on thermal stability of coconut milk and the underlying mechanism was explored by investigating the changes in functional properties and structures of coconut milk protein. The results showed that ultrasound combined with alkaline pH-shift treatment improved the viscosity (from 239.5 to 309.2 mPa), increased zeta potential (from −22.3 to −24.8 mV) and decreased the size distribution curve right shift distance (from 4.07 to 2.15 μm) of coconut milk compared to alkaline pH-shift treatment alone. This was future elucidated by the enhance effect of ultrasound assisted alkaline pH-shift to improve physicochemical properties of coconut milk protein, including higher solubility (from 43.4% to 51.8%), higher surface hydrophobicity (from 5877.2 to 13372.7) and fewer free sulfhydryl group (from 441.8 to 352.5 μmol/g). The improved properties were consistent with changes in the secondary structure, tertiary structure and microscopic state of coconut globin reflected by the fourier transform infrared spectroscopy, fluorescence spectroscopy and scanning electron microscopy, respectively. In conclusion, ultrasound can amplify the effect of pH-shift on increasing the thermal stability of coconut milk by modifying functional properties and structures of coconut milk protein, especially under alkaline pH-shift treatment.

Changes in the Secondary Structure and Assembly of Proteins on Fluoride Ceramic (CeF3) Nanoparticle Surfaces.

Fluoride nanoparticles (NPs) are materials utilized in the biomedical field for applications including imaging of the brain. Their interactions with biological systems and molecules are being investigated, but the mechanism underlying these interactions remains unclear. We focused on possible changes in the secondary structure and aggregation state of proteins on the surface of NPs and investigated the principle underlying the changes using the amyloid β peptide (Aβ16–20) based on infrared spectrometry. CeF3 NPs (diameter 80 nm) were synthesized via thermal decomposition. Infrared spectrometry showed that the presence of CeF3 NPs promotes the formation of the β-sheet structure of Aβ16–20. This phenomenon was attributed to the hydrophobic interaction between NPs and Aβ peptides in aqueous environments, which causes the Aβ peptides to approach each other on the NP surface and form ordered hydrogen bonds. Because of the coexisting salts on the secondary structure and assembly of Aβ peptides, the formation of the β-sheet structure of Aβ peptides on the NP surface was suppressed in the presence of NH4+ and NO3– ions, suggesting the possibility that Aβ peptides were adsorbed and bound to the NP surface. The formation of the β-sheet structure of Aβ peptides was promoted in the presence of NH4+, whereas it was suppressed in the presence of NO3– because of the electrostatic interaction between the lysine residue of the Aβ peptide and the ions. Our findings will contribute to comparative studies on the effect of different NPs with different physicochemical properties on the molecular state of proteins.

Zinc-Dependent Oligomerization of Thermus thermophilus Trigger Factor Chaperone.

Simple SummaryMetal ions often play important roles in biological processes. Thermus thermophilus trigger factor (TtTF) is a zinc-dependent molecular chaperone where Zn2+ has been shown to enhance its folding-arrest activity. However, the mechanisms of how Zn2+ binds to TtTF and how Zn2+ affects the activity of TtTF are yet to be elucidated. As a first step in understanding the mechanism, we performed in vitro biophysical experiments on TtTF to investigate the zinc-binding site on TtTF and unveil how Zn2+ alters the physical properties of TtTF, including secondary structure, thermal stability, and oligomeric state. Our results showed that TtTF binds Zn2+ in a 1:1 ratio, and all three domains of TtTF are involved in zinc-binding. We found that Zn2+ does not affect the thermal stability of TtTF, whereas it does induce partial structural change and promote the oligomerization of TtTF. Given that the folding-arrest activity of Escherichia coli TF (EcTF) is regulated by its oligomerization, our results imply that TtTF exploits Zn2+ to modulate its oligomeric state to regulate the activity.Thermus thermophilus trigger factor (TtTF) is a zinc-dependent molecular chaperone whose folding-arrest activity is regulated by Zn2+. However, little is known about the mechanism of zinc-dependent regulation of the TtTF activity. Here we exploit in vitro biophysical experiments to investigate zinc-binding, the oligomeric state, the secondary structure, and the thermal stability of TtTF in the absence and presence of Zn2+. The data show that full-length TtTF binds Zn2+, but the isolated domains and tandem domains of TtTF do not bind to Zn2+. Furthermore, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectra suggested that Zn2+-binding induces the partial structural changes of TtTF, and size exclusion chromatography-multi-angle light scattering (SEC-MALS) showed that Zn2+ promotes TtTF oligomerization. Given the previous work showing that the activity regulation of E. coli trigger factor is accompanied by oligomerization, the data suggest that TtTF exploits zinc ions to induce the structural change coupled with the oligomerization to assemble the client-binding site, thereby effectively preventing proteins from misfolding in the thermal environment.

Molecular Insights into the Copper-Sensitive Operon Repressor in Acidithiobacillus caldus.

The copper-sensitive operon repressor (CsoR) family, which is the main Cu(I)-sensing family, is widely distributed and regulates regulons involved in detoxification in response to extreme copper stress (a general range of ≥3 g/liter copper ions). Here, we identified CsoR in hyper-copper-resistant Acidithiobacillus caldus (CsoRAc), an organism used in the bioleaching process of copper ores. CsoRAc possesses highly conserved Cu(I) ligands and structures within the CsoR family members. Transcriptional analysis assays indicated that the promoter (PIII) of csoR was active but weakly responsive to copper in Escherichia coli. Copper titration assays gave a stoichiometry of 0.8 mol Cu(I) per apo-CsoRAc monomer in vitro combined with atomic absorption spectroscopy analysis. CuI-CsoRAc and apo-CsoRAc share essentially identical secondary structures and assembly states, as demonstrated by circular dichroism spectra and size exclusion chromatography profiles. The average dissociation constants (KD = 2.26 × 10-18 M and 0.53 × 10-15 M) and Cu(I) binding affinity of apo-CsoRAc were estimated by bathocuproine disulfonate (BCS) and bicinchoninic acid (BCA) competition assays, respectively. Site-directed mutations of conserved Cu(I) ligands in CsoRAc did not significantly alter the secondary structure or assembly state. Competition assays showed that mutants had the same order of magnitude of Cu(I) binding affinity as apo-CsoRAc. Moreover, apo-CsoRAc could bind to the DNA fragment P08430 in vitro, although with low affinity. Finally, a working model was developed to illustrate putative copper resistance mechanisms in A. caldus. IMPORTANCE Research on copper resistance among various species has attracted considerable interest. However, due to the lack of effective and reproducible genetic tools, few studies regarding copper resistance have been reported for A. caldus. Here, we characterized a major Cu(I)-sensing family protein, CsoRAc, which binds Cu(I) with an attomolar affinity higher than that of the Cu(I)-specific chelator bathocuproine disulfonate. In particular, CsoR family proteins were identified only in A. caldus, rather than A. ferrooxidans and A. thiooxidans, which are both used for bioleaching. Meanwhile, A. caldus harbored more copper resistance determinants and a relatively full-scale regulatory system involved in copper homeostasis. These observations suggested that A. caldus may play an essential role in the application of engineered strains with higher copper resistance in the near future.

Humanin Inactivates Pro‐Apoptotic BCL‐2 Proteins Through a Common Fibril‐Forming Mechanism

Neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's are associated with accumulation of amyloid plaques or amyloid-like complexes that foster cellular dysregulation. For example, neurons with amyloid inclusions often undergo premature apoptosis resulting from excessive expression of p38 mitogen-activated protein kinase. The increased abundance of this enzyme leads to mass phosphorylation and activation of a protein from the B-cell lymphoma 2 (BCL-2) family, BAX. BAX is the central regulatory protein for mitochondrial outer membrane permeabilization (MOMP), a poration process that commits cells to apoptosis by releasing death-propagating factors from the mitochondria. Thus, it may be possible to prevent premature neuronal apoptosis by identifying endogenous factors that decrease the MOMP capacity of BAX and other pro-apoptotic BCL-2 proteins. Humanin (HN) is a mitochondria-derived peptide that is released by the organelle in response to several stress stimuli. In the cytosol or on the mitochondrial membrane HN can counteract the death signal by forming specific complexes with some of the BCL-2 proteins. We recently reported the in vitro amyloid-like fibrillation of BAX and a second pro-apoptotic family member, BID, with HN. We proposed this as a novel anti-apoptotic mechanism that inhibits pro-apoptotic BCL-2 proteins from initiating MOMP by sequestering them into fibrils, a heretofore unprecedented phenomenon that involves refolding globular BCL-2 proteins into fibrils where they undergo significant alpha-helix to beta-sheet fold-switching. These fibrils represent a new direction for the field of cell death and elucidating mechanisms of the BCL-2 family. The existence of this fold-switching mechanism and fibrillation insists that at least some pro-apoptotic members of the BCL-2 family have unappreciated, functional beta-sheet conformations. We hypothesize that in the membrane-bound conformations these pro-apoptotic BCL-2 proteins might adopt beta-sheet structures. Perhaps their fold-switching dynamics may play a role in the pore-forming process in the mitochondrial membrane, altering its membrane potential and leading up to the release of death factors into the cytosol. Therapeutics that can affect BCL-2 protein propensity for either secondary structure state may prove to be fruitful toward ameliorating disease states where BCL-2 family activity has become misregulated.

A Chimeric Variant of Two Gentisate 1,2‐Dioxygenases with Improved Catalytic Activity, Altered Structural Conformation and Substrate Specificity

Gentisate 1,2-dioxygenases catalyze the critical ring-opening step in the metabolism of gentisate (2,5-dihydroxybenzoate) by microorganisms under aerobic conditions. Gentisate 1,2-dioxgenases from bacterial species that have been characterized to date are strictly Fe(II)-dependent and they are mostly homotetrameric in solution. In bacteria, genes coding for gentisate 1,2-dioxygenase and other enzymes in the rest of the pathway for gentisate degradation are often organized in operons. While genes and enzymes in the gentisate pathway have been relatively well-studied in bacteria, reports about their fungal counterparts remain scarce in the literature. Here, we report the biochemical characterization of two gentisate 1,2-dioxygenases from Aspergillus niger with different specific activities that appear to be encoded within two split gene clusters in the A. niger genome. These differences in activities seem to be accompanied by differences in secondary structure and oligomerization states between these two proteins in solution. Furthermore, we report the engineering of a chimeric variant consisting of two domains derived from these two A. niger gentisate 1,2-dioxgenases that shows improved activity with gentisate, altered structural conformation and substrate specificity able to cleave a substrate that neither of the two wild type gentisate 1,2-dioxygenases is able to attack.

Impact of protein dynamics on secondary structure prediction

Protein 3D structures support their biological functions. As the number of protein structures is negligible in regards to the number of available protein sequences, prediction methodologies relying only on protein sequences are essential tools. In this field, protein secondary structure prediction (PSSPs) is a mature area, and is considered to have reached a plateau.Nonetheless, proteins are highly dynamical macromolecules, a property that could impact the PSSP methods. Indeed, in a previous study, the stability of local protein conformations was evaluated demonstrating that some regions easily changed to another type of secondary structure.The protein sequences of this dataset were used by PSSPs and their results compared to molecular dynamics to investigate their potential impact on the quality of the secondary structure prediction. Interestingly, a direct link is observed between the quality of the prediction and the stability of the assignment to the secondary structure state. The more stable a local protein conformation is, the better the prediction will be. The secondary structure assignment not taken from the crystallized structures but from the conformations observed during the dynamics slightly increase the quality of the secondary structure prediction. These results show that evaluation of PSSPs can be done differently, but also that the notion of dynamics can be included in development of PSSPs and other approaches such as de novo approaches.

Evolutionary model of protein secondary structure capable of revealing new biological relationships.

Ancestral sequence reconstruction has had recent success in decoding the origins and the determinants of complex protein functions. However, phylogenetic analyses of remote homologues must handle extreme amino acid sequence diversity resulting from extended periods of evolutionary change. We exploited the wealth of protein structures to develop an evolutionary model based on protein secondary structure. The approach follows the differences between discrete secondary structure states observed in modern proteins and those hypothesized in their immediate ancestors. We implemented maximum likelihood-based phylogenetic inference to reconstruct ancestral secondary structure. The predictive accuracy from the use of the evolutionary model surpasses that of comparative modeling and sequence-based prediction; the reconstruction extracts information not available from modern structures or the ancestral sequences alone. Based on a phylogenetic analysis of a sequence-diverse protein family, we showed that the model can highlight relationships that are evolutionarily rooted in structure and not evident in amino acid-based analysis.

Effects of rice bran rancidity on the oxidation and structural characteristics of rice bran protein

Rice bran rancidity may affect rice bran protein through protein oxidation. However, little is known about the relationship between rice bran rancidity and rice bran protein oxidation. The effects of rice bran rancidity on the oxidation extent and structural characteristics of rice bran protein were investigated. As storage time of rice bran increased, the acid value, peroxide value, and value of thiobarbituric acid reactive substances in crude rice bran oil increased from 4.31 mg KOH/g, 2.84 Meq/kg, and 6.22 μg MDA/g to 38.72 mg KOH/g, 15.58 Meq/kg, and 28.99 μg MDA/g, respectively, which indicated that hydrolytic rancidity and oxidative rancidity of rice bran occurred simultaneously. The gradual increase in protein carbonyl and dityrosine content from 2.12 nmol/mg and 88.61 A.U. to 13.8 nmol/mg and 159.37 A.U. was accompanied by a steady decrease in free sulfhydryl content of rice bran protein from 22.6 to 9.6 nmol/mg, which implied that the products of rice bran rancidity induced rice bran protein oxidation. The rice bran protein oxidation subsequently resulted in a loss of the ordered state of secondary structure and the formation of aggregates as well as cross-link, when both disulfide bonds and non-disulfide covalent bonds participated in cross-link formation.

Automated High-Throughput Capillary Circular Dichroism and Intrinsic Fluorescence Spectroscopy for Rapid Determination of Protein Structure.

Assessing the physical stability of proteins is one of the most important challenges in the development, manufacture, and formulation of biotherapeutics. Here, we describe a method for combining and automating circular dichroism and intrinsic protein fluorescence spectroscopy. By robotically injecting samples from a 96-well plate into an optically compliant capillary flow cell, complementary information about the secondary and tertiary structural state of a protein can be collected in an unattended manner from considerably reduced volumes of sample compared to conventional techniques. We demonstrate the accuracy and reproducibility of this method. Furthermore, we show how structural screening can be used to monitor unfolding of proteins in two case studies using (i) a chaotropic denaturant (urea) and (ii) low-pH buffers used for monoclonal antibody (mAb) purification during Protein A chromatography.

Synthesis and Study of Bimetallic Pd-Rh System Supported on Zirconia-Doped Alumina as a Component of Three-way Catalysts

The present work is aimed at preparation and detailed characterization of the Pd-Rh/γ-Al2O3-ZrO2 composition used as a component of three-way catalysts. Physicochemical properties, such as specific surface area, morphology, secondary structure, dispersity and chemical state of active components, and thermal stability along with catalytic activity were studied within this research. Textural parameters of the samples were examined using low-temperature nitrogen adsorption. Analysis of the morphology and secondary structure was performed by means of electron microscopy. Concentration of metals located on the surface and accessible for the reagents was estimated using test reaction of ethane hydrogenolysis. Method of X-ray photoelectron spectroscopy was applied to investigate the chemical state of active components. Catalytic performance and thermal stability of the samples were tested in a prompt thermal aging regime using CO oxidation reaction as a criterion for in situ characterization of the catalyst state. It was found that both these characteristics strongly depend on the nature and composition of the precursors used.

Solubilization and recovery of heat-aggregated cytochrome c using alkylammonium nitrate

We investigated the solubilization and recovery of heat-aggregated cytochrome c using alkylammonium nitrates (RAN-ILs), which are a group of protic ionic liquids (ILs), including methylammonium nitrate (MAN), ethylammonium nitrate (EAN), and propylammonium nitrate (PAN), in a wide range of concentrations (X = 0–40 mol%IL for MAN and 0–100 mol%IL for EAN and PAN) using absorption and circular dichroism spectroscopy to obtain basic information on solubilization/recovery solvents for heat-aggregated proteins using ILs. On the whole, the increase of absorbance at the soret band (~405 nm) of cytochrome c indicated that solubilization ability was observed in aqueous solutions with MAN and EAN (X > 10) and PAN (X > 5). These results indicate that the three RAN-ILs studied have solubilization ability for heat-aggregated cytochrome c, and increasing the alkyl-chain length of the cation causes an enhancement of the solubilization ability for heat-aggregated cytochrome c. The activity, secondary structure and heme coordination state (Fe–His18/Met80) of the solubilized cytochrome c in aqueous PAN solutions, which have the highest solubilization ability among the three RAN-ILs, were recovered up to X = 30 after removing PAN by dialysis. The present findings indicate that aqueous solutions with RAN-ILs have a high potential as recovery agents for heat-aggregated proteins.

Abstract 2357: Utilizing biological and protein structure-guided features to improve driver mutation discovery

Abstract Distinguishing between driver and passenger somatic mutations to pinpoint genetic alterations leading to oncogenesis still presents significant challenges. To meet these challenges, computational tools have been developed as effective filters, pruning most of the somatic mutations to a shortlist of high-priority, functional candidates for experimental validation. Most tools include searching for genes or pathways having mutation rates higher than explained by chance, mutations in conserved regions, or genes with neighboring mutations on the linear DNA or protein sequence. Recently, there has been a shift to utilize tertiary/quaternary protein structures to identify mutations clustering proximal to each other in 3D space. Such enrichment of mutations can indicate specific domains critical to normal protein function and when mutated, can drive tumor initiation and progression. HotSpot3D, a protein structure-based tool, identifies clusters enriched with proximal mutations within proteins. Though HotSpot3D has been valuable in identifying clusters of residues that are important to cancer, it does not distinguish the driving potential or structural impact of different mutations within a cluster nor does it consider the physical impact of different amino acid substitutions at the same site. The prediction power of HotSpot3D in distinguishing driver mutations from passenger mutations can be improved if spatial clustering considers physical/biological features proximal to mutations in significant clusters as well as the specific amino acid substitutions of mutations. We have created a machine learning algorithm that further prioritizes putative driver mutations found in HotSpot3D clusters by incorporating structural/biological features such as proximity of mutations to functional sites (active sites, phosphorylation sites, disulfide bonds, etc.), solvent accessibility, physiochemical property change of mutations, free energy change of mutations, conservation of residue sites, secondary structure state of residue sites, and expression/phosphorylation changes of samples containing mutations. We have curated experimentally validated mutations identified as neutral or oncogenic from various databases to serve as our training sets. This algorithm can be trained on the curated mutations in various protein subclasses such as homologous proteins, oncogenes, tumor suppressors, etc. to identify distinct structural feature signatures per subclass specific to driver mutations. This tool will aid in revealing putative driver mutations in genes not previously linked with cancer and help pinpoint mutations in known cancer genes that are driving cancer. Specifically, we are interested in applying the algorithm to druggable protein families such as G-Protein Coupled Receptors, Kinases, and Nuclear Hormone Receptors to better understand their role in tumor initiation and progression. Citation Format: Sohini Sengupta, Adam Scott, Amila Weerasinghe, Dan C. Zhou, Matthew A. Wyczalkowski, Reyka G. Jayasinghe, Ken Chen, Gordon Mills, Mike C. Wendl, John Dipersio, Li Ding. Utilizing biological and protein structure-guided features to improve driver mutation discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2357.

Structural and functional investigation of allophycocyanin trimer

Nature has been developing and optimizing photosynthesis billions years, including green plants, algae, photosynthetic bacteria and cyanobacteria. Phycobiliproteins system, as the phycobilisomes, in cyanobacteria and red algae is the one of two major light-harvesting systems, after evolution in billions years. Allophycocyanin (APC) trimer is a primary composition of phycobilisomes core for efficient lower-energy photons funneling pathway. In 2009, Liu et al. have firstly assembled the highly soluble self-assembled recombinant APC trimer with N-terminus polyhistidine, which the entire biosynthesis pathway of APC trimer was reconstituted in Escherichia coli strain with the full-length genes of apoprotein α subunit and β subunit from Synechocystis sp. PCC 6803. The recombinant APC trimer was used to be the subject in this research. An optimum protocol with immobilized metal-ion affinity chromatography with chelating transition metal ion (Ni2+) was used to purify the recombinant protein. Then, the second purification was performed to obtain APC trimer with a higher purity ( A 650/ A 620=1.346), using density gradient centrifugation. Single molecules of recombinant APC trimers were investigated with atomic force microscopy. Three-dimensional images with resolution at nanoscale indicated that the recombinant allophycocyanin trimers were approximately 1.5 nm in short axis, and 15 nm in long axis, which were in accordance with the sizes of natural allophycocyanin trimer measured by transmission electron microscopy or X-ray crystallography. Moreover, the correct conformation of recombinant allophycocyanin trimer was confirmed by fluorescence spectra (studied energy transfer ability), absorption spectra (studied the light-harvesting ability), and circular dichroism (CD) spectra (studied changes of secondary structure and aggregation state). The CD spectra of recombinant allophycocyanin induced by a wide range of pH were analyzed both at wavelengths in ultraviolet range (190–250 nm) and visible range (450–750 nm). The α -helices were the dominant secondary structure in recombinant allophycocyanin trimer by analysis of CD spectra in ultraviolet range, while the analysis of CD spectra in visible range suggested the existence of exciton-coupled pairs of phycocyanobilins, in which the energy transfer was mainly in the form of exciton splitting. By studying its spectra property variations in response to pH changes, we found that recombinant APC trimer showed good absorbance and fluorescence stability at varying pH. The conformation of chromophores in recombinant APC trimer was relatively stable, between the range of pH 4–9, which was revealed by the stable absorption and fluorescence spectra properties of recombinant APC trimer. The trimeric structure of recombinant APC was maintained while local variations of protein peptides were also shown in response to the environmental disturbance. Beyond this pH range, secondary structure as well as overall conformation of recombinant APC dramatically changed, and the energy absorption and transfer ability were also disrupted. This work demonstrated that correct function in energy transfer was retained in the recombinant allophycocyanin trimer compared with nature allophycocyanin trimer.