Hydrophobic Protein Research Articles

Unique Behavior of Bacterially Expressed Rat Carnitine Palmitoyltransferase 2 and Its Catalytic Activity.

Mammalian type 2 carnitine parmitoyltransferase (EC 2.3.1.21), abbreviated as CPT2, is an enzyme involved in the translocation of fatty acid into the mitochondrial matrix space, and catalyzes the reaction acylcarnitine + CoA = acyl-CoA + carnitine. When rat CPT2 was expressed in Escherichia coli, its behavior was dependent on the presence or absence of i) its mitochondrial localization sequence and ii) a short amino acid sequence thought to anchor it to the mitochondrial inner membrane: CPT2 containing both sequences behaved as a hydrophobic protein, while recombinant CPT2 lacking both regions behaved as a water soluble protein; if only one region was present, the resultant proteins were observed in both fractions. Because relatively few protein species could be obtained from bacterial lysates as insoluble pellets under the experimental conditions used, selective enrichment of recombinant CPT2 protein containing both hydrophobic sequences was easily achieved. Furthermore, when CPT2 enriched in insoluble fraction was resuspended in an appropriate medium, it showed catalytic activity typical of CPT2: it was completely suppressed by the CPT2 inhibitor, ST1326, but not by the CPT1 inhibitor, malonyl-CoA. Therefore, we conclude that the bacterial expression system is an effective tool for characterization studies of mammalian CPT2.

Protein aggregation monitoring in cells under oxidative stress: a novel fluorescent probe based on a 7-azaindole-BODIPY derivative.

The development of new fluorescent probes as molecular sensors is a critical step for the understanding of molecular mechanisms. BODIPY-based probes offer versatility due to their high fluorescence quantum yields, photostability, and tunable absorption/emission wavelengths. Here, we report the synthesis and evaluation of a novel 7-azaindole-BODIPY derivative to probe hydrophobic proteins as well as protein misfolding and aggregation. In organic solvents, this compound shows two efficiently interconverting emissive excited states. In aqueous environments, it forms molecular aggregates with unique photophysical properties. The complex photophysics of the 7-azaindole-BODIPY derivative was explored for sensing applications. In the presence of albumin, the compound is stabilized in hydrophobic protein regions, significantly increasing its fluorescence emission intensity and lifetime. Similar effects occur in the presence of protein aggregates but not with other macromolecules like pepsin, DNA, Ficoll 40, and coconut oil. Fluorescence lifetime imaging microscopy (FLIM) and two-photon fluorescence microscopy on breast (MCF-7) and lung (A549) cancer cells incubated with this compound display longer fluorescence lifetimes and higher emission intensity under oxidative stress. Synchrotron FTIR micro spectroscopy confirmed that the photophysical changes observed were due to protein misfolding and aggregation caused by the oxidative stress. These findings demonstrate that this compound can serve as a fluorescent probe to monitor protein misfolding and aggregation triggered by oxidative stress.

Surface-segregating zwitterionic copolymers to control poly(dimethylsiloxane) surface chemistry.

The use of microfluidic devices in biomedicine is growing rapidly in applications such as organs-on-chip and separations. Polydimethylsiloxane (PDMS) is the most popular material for microfluidics due to its ability to replicate features down to the nanoscale, flexibility, gas permeability, and low cost. However, the inherent hydrophobicity of PDMS leads to the adsorption of macromolecules and small molecules on device surfaces. This curtails its use in "organs-on-chip" and other applications. Current technologies to improve PDMS surface hydrophilicity and fouling resistance involve added processing steps or do not create surfaces that remain hydrophilic for long periods. This work describes a novel, simple, fast, and scalable method for improving surface hydrophilicity and preventing the nonspecific adsorption of proteins and small molecules on PDMS through the use of a surface-segregating zwitterionic copolymer as an additive that is blended in during manufacture. These highly branched copolymers spontaneously segregate to surfaces and rearrange in contact with aqueous solutions to resist nonspecific adsorption. We report that mixing a minute amount (0.025 wt%) of the zwitterionic copolymer in PDMS considerably reduces hydrophobicity and nonspecific adsorption of proteins (albumin and lysozyme) and small molecules (vitamin B12 and reactive red). PDMS blended with these zwitterionic copolymers retains its mechanical and physical properties for at least six months. Moreover, this approach is fully compatible with existing PDMS device manufacture protocols without additional processing steps and thus provides a low-cost and user-friendly approach to fabricating reliable biomicrofluidics.

Prosaposin is a novel coenzyme Q10-binding protein.

Coenzyme Q10 (CoQ10) is essential for mitochondrial ATP production and functions as an important antioxidant in every biomembrane and lipoprotein. Due to its hydrophobicity, a binding and transfer protein for CoQ10 is plausible, and we previously described saposin B as a CoQ10-binding and transfer protein. Here, we report that prosaposin, the precursor of saposin B, also binds CoQ10. As prosaposin is both a secretory protein and integral membrane protein, it is ubiquitous in the body. Prosaposin was isolated from human seminal plasma, and CoQ10 was extracted from hexane solution into the water phase. It was additionally found that immunoprecipitates of mouse brain cytosol generated using two different anti-prosaposin antibodies contained coenzyme Q9. Furthermore, mouse liver cytosol and mouse kidney cytosol also contained prosaposin-coenzyme Q9 complex. These results suggest that prosaposin binds CoQ10 in human cells and body fluids. The significance and role of the Psap-CoQ10 complex in vivo is also discussed.

Surfactant proteins as protective factors in bronchial asthma

Bronchial asthma remains a serious global health problem affecting all age groups. This disease causes significant damage to the health care system and society. The problem of late diagnosis of bronchial asthma in children remains relevant today. The article presents data on surfactant proteins as protective factors of the respiratory tract. The main function – the reduction of surface tension in the alveoli at the air-water phase boundary – is carried out due to the lipid fraction and hydrophobic surfactant proteins B and C. Hydrophilic proteins A and D are involved in the implementation of the immune response. They act as immunomodulatory factors, actively contribute to phagocytosis, alternatively affect the activity of mast, dendritic cells, lymphocytes and alveolar macrophages. These apoproteins regulate the processes of apoptosis, play an important role in the pathogenesis of allergic reactions, preventing the development of IgE-dependent inflammation. Both proteins are alternatively able to influence the type of immune response, being the so-called regulators of the immunological environment. The described pneumoproteins can probably be used as biomarkers of the severity of the course of bronchial asthma.

Visualizing Orientation and Topology of ER Membrane Proteins In Planta.

The orientation of membrane proteins within the lipid bilayer is key to understanding their molecular function. Similarly, the proper topology of multispanning membrane proteins is crucial for their function. Although bioinformatics tools can predict these parameters assessing the presence of hydrophobic protein domains sufficiently long to span the membrane and other structural features, the predictions from different algorithms are often inconsistent. Therefore, experimental analysis becomes mandatory. Redox-based topology analysis exploits the steep gradient in the glutathione redox potential (EGSH) across the ER membrane of about 80mV to visualize the orientation of ER membrane proteins by fusing the EGSH biosensor roGFP2 to either the N- or the C-termini of the investigated protein sequence. Transient expression of these fusion proteins in tobacco leaves allows direct visualization of orientation and topology of ER membrane proteins in planta. The protocol outlined here is based on either a simple merge of the two excitation channels of roGFP2 or a colocalization analysis of the two channels and thus avoids ratiometric analysis of roGFP2 fluorescence.

Development of Yellow Discoloration in Sawai (Pangasianodon hypophthalmus) Muscle due to Lipid Oxidation.

In this study, we investigated the impact of lipid oxidation on the discoloration of Sawai (Pangasianodon hypophthalmus) lipids and proteins. Sawai microsomes, liposomes, and salt-soluble myofibrillar proteins were prepared and subjected to lipid oxidation process. The results revealed that the levels of thiobarbituric acid-reactive substances, yellowness (as indicated by b* values), and pyrrole compounds increased when Sawai liposomes and microsomes were oxidized using iron and ascorbate. Meanwhile, the levels of free amines decreased, particularly as the iron content (25∼100 μM) and incubation time (0∼20 h) increased. The impact of oxidized liposomes at different levels (1, 2, and 5%) on the salt-soluble Sawai myofibrillar proteins was also evaluated. The findings revealed that lipid oxidation products reduced the sulfhydryl content and increased the surface hydrophobicity and carbonyl content of the salt-soluble Sawai myofibrillar proteins. These results imply that the formation of yellow discoloration in Sawai muscle could be due to nonenzymatic browning reactions occurring between lipid oxidation products and amines in the muscle protein.

In silico Docking Analysis of the FDA-Approved Drugs on Envelope Protein of SARS CoV-2 Omicron Variant

The current pandemic situation is created by the highly evolving SARS coronavirus 2 which is having several mutations in its structural proteins. The structural proteins of SARS CoV-2 include spike (S), Envelope (E), Membrane (M), and Nucleocapsid (N) which are primarily responsible for the infection, transmission, and pathogenesis of the virus. Envelope protein is the smallest of the four proteins containing 75 amino acids with a molecular weight of about 8 kDa. The major functions of the hydrophobic envelope protein include envelope formation, budding, replication, and release of the virion. The presence of mutation on the envelope protein results in improper formation of the pentameric structure and also hinders other functional properties. Our computational analysis majorly focuses on several FDA-approved inhibitory compounds that bind to SARS CoV-2 envelope protein that help in the inhibition of virion formation. The percentage of similarity of the envelope protein between SARS CoV and SARS CoV-2 is approximately 96 percent. The homology-modeled structure of the SARS CoV E protein was downloaded from Protein Model Database (PMDB) and the mutation which was found to be consistent among most of the SARS CoV-2 variants was selected as T9I which is present in the N-terminal region. This mutation was introduced into the SARS CoV Envelope protein and was remodeled. The ligands which were approved by FDA were selected for docking analysis to understand their binding capabilities with the envelope protein. Ligands such as Beta-D-Fucose, Mycophenolic Acid, Castanospermine, 1-Deoxynojirimycin, Nafcillin, Guaifenesin, Nabumetone, Cinametic Acid, Lauric acid were used in our study. The docking simulations revealed that Lauric acid, Nafcillin, Nabumetone, and Mycophenolic acid have high binding energy with the SARS CoV-2 wild type (Wuhan) and mutant E protein of the SARS CoV-2 (Omicron) variant. This Insilico data gives insights to test these high binding compounds in invitro studies to prove their efficacy and the protein-protein interactions of envelope protein with its other partnering proteins. These pharmaceutical compounds are a potential alternative in the future for a novel drug development to treat several emerging variants of SARS CoV-2.

Mitochondrial Protein SLIRP Affects Biosynthesis of Cytochrome c Oxidase Subunits in HEK293T Cells.

Mitochondria carry out various vital roles in eukaryotic cells, including ATP energy synthesis, the regulation of apoptosis, Fe-S cluster formation, and the metabolism of fatty acids, amino acids, and nucleotides. Throughout evolution, mitochondria lost most of their ancestor's genome but kept the replication, transcription, and translation machinery. Protein biosynthesis in mitochondria is specialized in the production of highly hydrophobic proteins encoded by mitochondria. These proteins are components of oxidative phosphorylation chain complexes. The coordination of protein synthesis must be precise to ensure the correct assembly of nuclear-encoded subunits for these complexes. However, the regulatory mechanisms of mitochondrial translation in human cells are not yet fully understood. In this study, we examined the contribution of the SLIRP protein in regulating protein biosynthesis in mitochondria. Using a click-chemistry approach, we discovered that deletion of the SLIRP gene disturbs mitochondrial translation, leading to the dysfunction of complexes I and IV, but it has no significant effect on complexes III and V. We have shown that this protein interacts only with the small subunit of the mitochondrial ribosome, which may indicate its involvement in the regulation of the mitochondrial translation initiation stage.

Evolution and ecology of Jeilongvirus among wild rodents and shrews in Singapore

BackgroundJeilongvirus was proposed as a new genus within the Paramyxoviridae in 2018. The advancement in metagenomic approaches has encouraged multiple reports of Jeilongvirus detection following the initial species discovery, enriching species diversity and host range within the genus. However, Jeilongvirus remains understudied in Singapore, where interfaces between humans and small mammals are plentiful.MethodsHere, we utilized metagenomic sequencing for the exploration of viral diversity in small mammal tissues. Upon discovery of Jeilongvirus, molecular screening and full genome sequencing was conducted, with the data used to conduct statistical modelling and phylogenetic analysis.ResultsWe report the presence of Jeilongvirus in four species of Singapore wild small mammals, detected in their spleen and kidney. We show that full genomes of three Singapore Jeilongvirus encode for eight ORFs including the small hydrophobic and transmembrane proteins. All generated genomes cluster phylogenetically within the small mammal subclade, but share low genetic similarity with representative Jeilongvirus species. Statistical modelling showed no spatial or temporal patterns and differences among species, life history traits and habitat types.ConclusionsThis study serves as a basis for understanding dynamics between Jeilongvirus and small mammal hosts in Singapore by displaying the virus generalist nature. In addition, the initial detection can help to invoke improved routine surveillance and detection of circulating pathogens in synanthropic hosts.

Effect of atmospheric cold plasma treatment on structural, thermal, and mechanical properties of pea protein isolate edible films

The current study has been undertaken to examine the influence of atmospheric cold plasma (ACP) treatment on the thermo-mechanical, structural, and barrier properties of pea protein isolate (PPI) edible films. ACP treatment was applied to the PPI films for selected exposure times (30, 60, 90, and 120 s) at a fixed voltage of 20 kV. The ACP treatment increased the surface hydrophobicity of the films considerably, from 38.43 ± 3.32° to 61.67 ± 2.06°, demonstrating the unfolding of hydrophobic protein groups due to surface etching. The effect was further pronounced with the reduced protein aggregates and improved compactness in the surface microstructure. FTIR and XRD results demonstrated ACP-induced modifications in the secondary protein structures, including the transformation of α-helix into β-sheet structures. The DSC protein denaturation temperature increased from 61.16 °C to 72.56 °C after 30 s of ACP treatment, suggesting cross-linking on the PPI surface. After 120 s of ACP treatment, the tensile strength increased from 2.40 ± 0.08 to 3.98 ± 0.15 MPa, and elongation at break increased from 77.53 ± 1.26 to 106.95 ± 2.70%. Additionally, the water vapor transmission rate decreased from 2.32 ± 0.13 to 1.80 ± 0.03 g/h.m2. Furthermore, the treatment also enhanced the biodegradation process of PPI films. Overall, the ACP treatment appears to be a successful structural modification technique for plant-based edible films with improved packaging properties.

Diversity and evolution of mitochondrial translation apparatus

The evolution of mitochondria proceeded independently in different eukaryotic lines, reflected in the diversity of mitochondrial genomes and mechanisms of their expression observed in different eukaryotic species. Mitochondria lost most of the genes of their bacterial ancestor, partially transferred them to the nucleus or completely eliminated. However, the mitochondria of almost all eukaryotic cells retain relatively small genomes, as well as their replication, transcription, and translation apparatuses. Dependence on the nuclear genome, features of mitochondrial transcripts, and the necessity to synthesize highly hydrophobic membrane proteins led to a number of significant changes in translation apparatus derived from the bacterial ancestor. It retained the basic structure necessary for protein synthesis but became more specialized and labile. In this review, we consider what changes take place during the initiation of mitochondrial translation, and how the evolution of mitochondria affected the functions of the main factors initiating protein biosynthesis in these organelles.

Antigen‐specific mRNA lipid nanoparticle platforms for the prevention and treatment of allergy and autoimmune diseases

AbstractWhile most nanomedicines primarily aim to stimulate the immune system against infections or tumors, there is a growing demand for inducing immune tolerance under certain conditions, such as allergic and autoimmune diseases. Researchers have explored nanotechnology‐based strategies to induce immune tolerance in a targeted and specific manner. One approach involves the use of nanoparticles (NPs) to encapsulate immunosuppressive drugs and/or antigens to educate naïve T cells and promote the generation of antigen‐specific regulatory T cells that inhibit immune responses. However, this approach has certain limitations. The hydrophobicity of proteins or peptides restricts the degree to which they can be encapsulated in NPs, which in turn, affects their loading efficiency and treatment efficacy. With the emergence of mRNA lipid nanoparticle (LNP) platforms, there is the possibility of overcoming the limitations of protein and peptide encapsulation. To date, mRNA LNP systems have been shown to provide organ, cellular, and subcellular targeting for the induction of immune tolerance. This method of drug delivery is flexible and scalable that can be customized for a specific patient, resulting in an effective means of administering relevant proteins or epitopes to induce antigen‐specific immune tolerance. With continued research and development, this technology could offer a safer and more effective alternative to current therapies, ultimately improving the quality of life of patients worldwide.

Physicochemical and functional properties of fermented pea and navy bean protein isolates

AbstractBackground and ObjectivesThere has been a significant increase in the use of fermentation for protein modification by the food industry. This research aimed to investigate the use of solid‐state fermentation (SSF) by Aspergillus oryzae NRRL 5590 on pea and navy bean protein isolates (PPI and NBPI, respectively) to enhance their physicochemical and functional properties.FindingsThe impact of fermentation was more profound on PPI than NBPI with a higher degree of hydrolysis achieved for the former (9.3% vs. 4.4%). Fermented PPI had significantly increased protein content, surface charge and hydrophobicity, solubility, and foaming properties, but decreased emulsion stability. For NBPI, modifications were only observed for surface hydrophobicity and water hydration capacity (WHC), which both increased after fermentation. Overall, navy bean proteins were less susceptible to protein hydrolysis than pea proteins upon fermentation, possibly due to the phaseolin protein in navy bean.ConclusionsIn summary, fermentation may be used to enhance the solubility and foaming properties of PPI and WHC of NBPI for their use as ingredients in applications where such higher functionalities are favorable.Significance and NoveltyThe results provide insights into pulse protein modification by bioprocessing, specifically fermentation, and opportunities for potential value‐added applications for pea and navy bean proteins.

The Gβ-like protein Bcgbl1 regulates development and pathogenicity of the gray mold Botrytis cinerea via modulating two MAP kinase signaling pathways.

The fungal Gβ-like protein has been reported to be involved in a variety of biological processes, such as mycelial growth, differentiation, conidiation, stress responses and infection. However, molecular mechanisms of the Gβ-like protein in regulating fungal development and pathogenicity are largely unknown. Here, we show that the Gβ-like protein gene Bcgbl1 in the gray mold fungus Botrytis cinerea plays a pivotal role in development and pathogenicity by regulating the mitogen-activated protein (MAP) kinases signaling pathways. The Bcgbl1 deletion mutants were defective in mycelial growth, sclerotial formation, conidiation, macroconidial morphogenesis, plant adhesion, and formation of infection cushions and appressorium-like structures, resulting in a complete loss of pathogenicity. Bcgbl1 interacted with BcSte50, the adapter protein of the cascade of MAP kinase (MAPK). Bcgbl1 mutants had reduced phosphorylation levels of two MAPKs, namely Bmp1 and Bmp3, thereby reducing infection. However, deletion of Bcgbl1 did not affect the intracellular cAMP level, and exogenous cAMP could not restore the defects. Moreover, Bcgbl1 mutants exhibited defects in cell wall integrity and oxidative stress tolerance. Transcriptional profiling revealed that Bcgbl1 plays a global role in regulation of gene expression upon hydrophobic surface induction. We further uncovered that three target genes encoding the hydrophobic surface binding proteins (HsbAs) contributed to the adhesion and virulence of B. cinerea. Overall, these findings suggest that Bcgbl1 had multiple functions and provided new insights for deciphering the Bcgbl1-mediated network for regulating development and pathogenicity of B. cinerea.

Efficiency aspects of regioselective testosterone hydroxylation with highly active CYP450-based whole-cell biocatalysts.

Steroid hydroxylations belong to the industrially most relevant reactions catalysed by cytochrome P450 monooxygenases (CYP450s) due to the pharmacological relevance of hydroxylated derivatives. The implementation of respective bioprocesses at an industrial scale still suffers from several limitations commonly found in CYP450 catalysis, that is low turnover rates, enzyme instability, inhibition and toxicity related to the substrate(s) and/or product(s). Recently, we achieved a new level of steroid hydroxylation rates by introducing highly active testosterone-hydroxylating CYP450 BM3 variants together with the hydrophobic outer membrane protein AlkL into Escherichia coli-based whole-cell biocatalysts. However, the activity tended to decrease, which possibly impedes overall productivities and final product titres. In this study, a considerable instability was confirmed and subject to a systematic investigation regarding possible causes. In-depth evaluation of whole-cell biocatalyst kinetics and stability revealed a limitation in substrate availability due to poor testosterone solubility as well as inhibition by the main product 15β-hydroxytestosterone. Instability of CYP450 BM3 variants was disclosed as another critical factor, which is of general significance for CYP450-based biocatalysis. Presented results reveal biocatalyst, reaction and process engineering strategies auguring well for industrial implementation of the developed steroid hydroxylation platform.

Ultrasound-assisted alkaline hydrolysis of fish(Cyprinus carpio L.) scale gelatin: A possible curcumin delivery system with increased water solubility and sustained release

This study investigated the effect of ultrasound-assisted alkaline treatment on the structural characteristics and curcumin loading efficiency in fish scale gelatin (FSG). The results showed that FSG treated with a combination of ultrasound and alkaline hydrolysis outperformed non-treated FSG, as well as alkaline- or ultrasound-treated FSG in augmenting curcumin loading efficiency (from 3.02 to 18.36 μg/mL). In contrast to FSG exposed to single treatments (alkaline or ultrasound), the FSG subjected to combined treatment exhibited a notably higher ratio of hydrophobic amino acids (47.51 ± 2.27%) and protein elution amount (180.87 μg/mL). Meanwhile, single and combined treatments significantly modified the molecular conformation as well as the microstructure of FSG, transforming the α-helical conformation to the β-sheet structure and presenting an ordered structure with increased voids. The curcumin-loaded FSG subjected to ultrasound-assisted alkaline treatment revealed a sustained release profile under simulated gastrointestinal conditions. The elucidated outcomes underscore the significant potential of FSG subjected to ultrasound-assisted alkaline treatment as a promising avenue for facilitating the controlled delivery of bioactive hydrophobic nutrients.

Edible electrospun zein nanofibrous scaffolds close the gaps in biofilm formation ability between microorganisms

Biofilm is an important survival mode of microorganisms in nature. Biofilm culture is supposed to be a way of learning from nature to endow microorganisms with a strong tolerance. However, microorganisms possess different biofilm formation abilities, and universal scaffolds, especially edible scaffolds, suitable for fast biofilm formation are not found yet. Zein is a hydrophobic and biocompatible prolamine-rich protein from corn and is one of the most used biomacromolecules now. In this work, natural zein was utilized to produce electrospun zein nanofibrous biomaterial. The results demonstrate that edible electrospun zein nanofibrous scaffolds significantly enhance biofilm formation, where bacterial probiotics and yeasts are 0.9–1.4 Log cfu/cm2 (8–25 × ) and 1.7–3.3 Log cfu/cm2 (50–2000 × ) higher than the values on polystyrene plates and zein films, respectively. Gaps in biofilm formation abilities between bacteria and yeasts are narrowed from 1.7–1.8 Log cfu/cm2 (50–63 × ) to 0.5–0.6 Log cfu/cm2 (3–4 × ), respectively. Biofilms quickly formed on electrospun zein nanofibrous scaffolds before the occurrence of serious swelling deformation of the scaffolds. This is the first attempt to use edible electrospun fibrous scaffolds based on natural biomacromolecules for enhancing biofilm formation, crucially important for the application of probiotics and yeast in food and medical industries. Electrospun zein nanofibrous scaffolds can serve as powerful tools to enhance the biofilm formation of valuable food microorganisms and close the huge gaps in biofilm formation abilities between microorganisms, providing a convenient and universal strategy to produce biofilm-phenotyped probiotics and yeasts.

Characterization of translocon proteins in the type III secretion system of Lawsonia intracellularis

Lawsonia intracellularis, the etiologic agent of proliferative enteropathy (PE), is an obligate intracellular Gram-negative bacterium possessing a type III secretion system (T3SS), which enables the pathogen to translocate effector proteins into targeted host cells to modulate their functions. T3SS is a syringe-like apparatus consisting of a base, an extracellular needle, a tip, and a translocon. The translocon proteins assembled by two hydrophobic membrane proteins can form pores in the host-cell membrane, and therefore play an essential role in the function of T3SS. To date, little is known about the T3SS and translocon proteins of L. intracellularis. In this study, we first analyzed the conservation of the T3S apparatus between L. intracellularis and Yersinia, and characterized the putative T3S hydrophobic major translocon protein LI1158 and minor translocon protein LI1159 in the L. intracellularis genome. Then, by using Yersinia pseudotuberculosis as a surrogate system, we found that the full-length LI1158 and LI1159 proteins, but not the putative class II chaperone LI1157, were secreted in a − Ca2+ and T3SS-dependent manner and the secretion signal was located at the N terminus (aa 1–40). Furthermore, yeast-two hybrid experiments revealed that LI1158 and LI1159 could self-interact, and LI1159 could interact with LI1157. However, unlike CPn0809 and YopB, which are the major hydrophobic translocon proteins of the T3SS of C. pneumoniae and Yersinia, respectively, full-length LI1158 was non-toxic to both yeast and Escherichia coli cells, but full-length LI1159 showed certain toxicity to E. coli cells. Taken together, despite some differences from the findings in other bacteria, our results demonstrate that LI1158 and LI1159 may be the translocon proteins of L. intracellularis T3SS, and probably play important roles in the translocation of effector proteins at the early pathogen infection stage.

Comparative Analysis of Protein Surface Hydrophobicity Maps Determined by Sparse Sampling INDUS and Spatial Aggregation Propensity.

Protein surface hydrophobicity plays a central role in various biological processes such as protein folding and aggregation, as well as in the design and manufacturing of biotherapeutics. While the hydrophobicity of protein surface patches has been linked to their constituent residue hydropathies, recent research has shown that protein surface hydrophobicity is more complex and characterized by the response of water to these surfaces. In this work, we employ water density perturbations to map the surface hydrophobicity of a set of model proteins using sparse indirect umbrella sampling simulations (SSI). This technique is used to identify hydrophobic surface patches for the set of model proteins, and the results are compared to those obtained from the widely adopted spatial aggregation propensity (SAP) technique. While SAP-based calculations show agreement with SSI in some cases, there are several examples of disagreement. We identify four general classes of difference in behavior and study factors that contribute to these differences. We find that the SAP method can sometimes mask the effect of weakly nonpolar or isolated nonpolar residues that can lead to strong hydrophobic patches on the protein surface. In addition, hydrophobic patches identified by SAP can exhibit shifts in both position and strength on the SSI map. Our results demonstrate that the combination of topography and chemical context controls the hydrophobicity of a given patch above and beyond the intrinsic polarity of the residues present on the patch surface. The availability of more accurate protein hydrophobicity maps in concert with new classes of hydrophobic molecular descriptors may create significant opportunities for in silico prediction of protein behavior for a range of applications, such as protein design, biomanufacturability, and downstream bioprocessing.