Heterotrophic production of Chlorella sp. TISTR 8990—biomass growth and composition under various production conditions

The green microalga Chlorella sp. TISTR 8990 was grown heterotrophically in the dark using various concentrations of a basal glucose medium with a carbon‐to‐nitrogen mass ratio of 29:1. The final biomass concentration and the rate of growth were highest in the fivefold concentrated basal glucose medium (25 g L^?1 glucose, 2.5 g L^?1 KNO₃) in batch operations. Improving oxygen transfer in the culture by increasing the agitation rate and decreasing the culture volume in 500‐mL shake flasks improved growth and glucose utilization. A maximum biomass concentration of nearly 12 g L^?1 was obtained within 4 days at 300 rpm, 30°C, with a glucose utilization of nearly 76% in batch culture. The total fatty acid (TFA) content of the biomass and the TFA productivity were 102 mg g^?1 and 305 mg L^?1 day^?1, respectively. A repeated fed‐batch culture with four cycles of feeding with the fivefold concentrated medium in a 3‐L bioreactor was evaluated for biomass production. The total culture period was 11 days. A maximum biomass concentration of nearly 26 g L^?1 was obtained with a TFA productivity of 223 mg L^?1 day^?1. The final biomass contained (w/w) 13.5% lipids, 20.8% protein and 17.2% starch. Of the fatty acids produced, 52% (w/w) were saturated, 41% were monounsaturated and 7% were polyunsaturated (PUFA). A low content of PUFA in TFA feedstock is required for producing high quality biodiesel. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1589–1600, 2017     

Denatured State Structural Property Determines Protein Stabilization by Macromolecular Crowding: A Thermodynamic and Structural Approach

Understanding of protein structure and stability gained to date has been acquired through investigations made under dilute conditions where total macromolecular concentration never surpasses 10 g l⁻¹. However, biological macromolecules are known to evolve and function under crowded intracellular environments that comprises of proteins, nucleic acids, ribosomes and carbohydrates etc. Crowded environment is known to result in altered biological properties including thermodynamic, structural and functional aspect of macromolecules as compared to the macromolecules present in our commonly used experimental dilute buffers (for example, Tris HCl or phosphate buffer). In this study, we have investigated the thermodynamic and structural consequences of synthetic crowding agent (Ficoll 70) on three different proteins (Ribonuclease-A, lysozyme and holo α-lactalbumin) at different pH values. We report here that the effect of crowding is protein dependent in terms of protein thermal stability and structure. We also observed that the structural characteristics of the denatured state determines if crowding will have an effect or not on the protein stability.     

Butanol production from sweet sorghum bagasse with high solids content: Part I—comparison of liquid hot water pretreatment with dilute sulfuric acid

In these studies, we pretreated sweet sorghum bagasse (SSB) using liquid hot water (LHW) or dilute H₂SO₄ (2 g L^?1) at 190°C for zero min (as soon as temperature reached 190°C, cooling was started) to reduce generation of sugar degradation fermentation inhibiting products such as furfural and hydroxymethyl furfural (HMF). The solids loading were 250–300 g L^?1. This was followed by enzymatic hydrolysis. After hydrolysis, 89.0 g L^?1 sugars, 7.60 g L^?1 acetic acid, 0.33 g L^?1 furfural, and 0.07 g L^?1 HMF were released. This pretreatment and hydrolysis resulted in the release of 57.9% sugars. This was followed by second hydrolysis of the fibrous biomass which resulted in the release of 43.64 g L^?1 additional sugars, 2.40 g L^?1 acetic acid, zero g L^?1 furfural, and zero g L^?1 HMF. In both the hydrolyzates, 86.3% sugars present in SSB were released. Fermentation of the hydrolyzate I resulted in poor acetone‐butanol‐ethanol (ABE) fermentation. However, fermentation of the hydrolyzate II was successful and produced 13.43 g L^?1 ABE of which butanol was the main product. Use of 2 g L^?1 H₂SO₄ as a pretreatment medium followed by enzymatic hydrolysis resulted in the release of 100.6–93.8% (w/w) sugars from 250 to 300 g L^?1 SSB, respectively. LHW or dilute H₂SO₄ were used to economize production of cellulosic sugars from SSB. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:960–966, 2018     

Electrodialytic separation of levulinic acid catalytically synthesized from woody biomass for use in microbial conversion

Levulinic acid (LA) is produced by the catalytic conversion of a variety of woody biomass. To investigate the potential use of desalting electrodialysis (ED) for LA purification, electrodialytic separation of levulinate from both reagent and cedar‐derived LA solution (40–160 g L^?1) was demonstrated. When using reagent LA solution with pH5.0–6.0, the recovery rates of levulinate ranged from 68 to 99%, and the energy consumption for recovery of 1 kg of levulinate ranged from 0.18 to 0.27 kWh kg^?1. With cedar‐derived LA solution (pH6.0), good agreement in levulinate recovery (88–99%), and energy consumption (0.18–0.22 kWh kg^?1) were observed in comparison to the reagent LA solutions, although a longer operation time was required due to some impurities. The application of desalting ED was favorable for promoting microbial utilization of cedar‐derived LA. From 0.5 mol L^?1 of the ED‐concentrated sodium levulinate solution, 95.6% of levulinate was recovered as LA calcium salt dihydrate by crystallization. This is the first report on ED application for LA recovery using more than 20 g L^?1 LA solutions (40–160 g L^?1). © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:448–453, 2017     

The contrasting effect of macromolecular crowding on amyloid fibril formation

Background

Amyloid fibrils associated with neurodegenerative diseases can be considered biologically relevant failures of cellular quality control mechanisms. It is known that in vivo human Tau protein, human prion protein, and human copper, zinc superoxide dismutase (SOD1) have the tendency to form fibril deposits in a variety of tissues and they are associated with different neurodegenerative diseases, while rabbit prion protein and hen egg white lysozyme do not readily form fibrils and are unlikely to cause neurodegenerative diseases. In this study, we have investigated the contrasting effect of macromolecular crowding on fibril formation of different proteins.

Methodology/Principal Findings

As revealed by assays based on thioflavin T binding and turbidity, human Tau fragments, when phosphorylated by glycogen synthase kinase-3β, do not form filaments in the absence of a crowding agent but do form fibrils in the presence of a crowding agent, and the presence of a strong crowding agent dramatically promotes amyloid fibril formation of human prion protein and its two pathogenic mutants E196K and D178N. Such an enhancing effect of macromolecular crowding on fibril formation is also observed for a pathological human SOD1 mutant A4V. On the other hand, rabbit prion protein and hen lysozyme do not form amyloid fibrils when a crowding agent at 300 g/l is used but do form fibrils in the absence of a crowding agent. Furthermore, aggregation of these two proteins is remarkably inhibited by Ficoll 70 and dextran 70 at 200 g/l.

Conclusions/Significance

We suggest that proteins associated with neurodegenerative diseases are more likely to form amyloid fibrils under crowded conditions than in dilute solutions. By contrast, some of the proteins that are not neurodegenerative disease-associated are unlikely to misfold in crowded physiological environments. A possible explanation for the contrasting effect of macromolecular crowding on these two sets of proteins (amyloidogenic proteins and non-amyloidogenic proteins) has been proposed.     

Experimental evaluation of using calcein and alizarin red S for immersion marking of bighead carp Aristichthys nobilis (Richardson, 1845) to assess growth and identification of marks in otoliths,scales and fin rays

In order to evaluate the effects of immersion marking with calcein (CAL) and alizarin red S (ARS) on growth and mortality of juvenile bighead carp Aristichthys nobilis, and assess mark quality in otoliths, scales, and fin rays, CAL from 50 to 200 mg L^?1 and ARS from 150 to 300 mg L^?1 concentrations were used. With the exception of non‐lateral line scales from 50 mg L^?1 CAL treatments, immersion for 24 h produced detectable marks in sagittae, lateral line and non‐lateral line scales, and fin rays (dorsal, pectoral, ventral, anal, and caudal) at 100 days post‐marking. Detectable fluorescent marks in sagittae were readily observed at concentrations of 150–200 mg L^?1 CAL or 150–300 mg L^?1 ARS. Marks were poorly visible in all non‐lateral line scales from both CAL‐ and ARS‐treated groups. Fluorescent marks were readily detected in lateral line scales at 100–200 mg L^?1 CAL or 150–300 mg L^?1 ARS, and in fin rays at 150–200 mg L^?1 CAL or 150–300 mg L^?1 ARS. In particular, optimal marks were observed at the highest concentrations investigated in sagittae (300 mg L^?1 ARS), lateral line scales (150–200 mg L^?1 CAL or 250–300 mg L^?1 ARS), and fin rays (200 mg L^?1 CAL or 250–300 mg L^?1 ARS). However, fluorescent marks visible to the naked eye were not produced by any of the CAL or ARS treatments in sagittae, scales, or fin rays during this experiment. In addition, there was no significant difference on survival and growth of marked fish compared to controls throughout the experiment (P > 0.05).     

Macromolecular crowding effects on the kinetics of opposing reactions catalyzed by alcohol dehydrogenase

In order to better understand how the complex, densely packed, heterogeneous milieu of a cell influences enzyme kinetics, we exposed opposing reactions catalyzed by yeast alcohol dehydrogenase (YADH) to both synthetic and protein crowders ranging from 10 to 550 kDa. The results reveal that the effects from macromolecular crowding depend on the direction of the reaction. The presence of the synthetic polymers, Ficoll and dextran, decrease V_max and K_m for ethanol oxidation. In contrast, these crowders have little effect or even increase these kinetic parameters for acetaldehyde reduction. This increase in V_max is likely due to excluded volume effects, which are partially counteracted by viscosity hindering release of the NAD⁺ product. Macromolecular crowding is further complicated by the presence of a depletion layer in solutions of dextran larger than YADH, which diminishes the hindrance from viscosity. The disparate effects from 25 g/L dextran or glucose compared to 25 g/L Ficoll or sucrose reveals that soft interactions must also be considered. Data from binary mixtures of glucose, dextran, and Ficoll support this “tuning” of opposing factors. While macromolecular crowding was originally proposed to influence proteins mainly through excluded volume effects, this work compliments the growing body of evidence revealing that other factors, such as preferential hydration, chemical interactions, and the presence of a depletion layer also contribute to the overall effect of crowding.     

Macromolecular Crowding Modulates Folding Mechanism of α/β Protein Apoflavodoxin

Protein dynamics in cells may be different from those in dilute solutions in vitro, because the environment in cells is highly concentrated with other macromolecules. This volume exclusion because of macromolecular crowding is predicted to affect both equilibrium and kinetic processes involving protein conformational changes. To quantify macromolecular crowding effects on protein folding mechanisms, we investigated the folding energy landscape of an α/β protein, apoflavodoxin, in the presence of inert macromolecular crowding agents, using in silico and in vitro approaches. By means of coarse-grained molecular simulations and topology-based potential interactions, we probed the effects of increased volume fractions of crowding agents (ϕ_c) as well as of crowding agent geometry (sphere or spherocylinder) at high ϕ_c. Parallel kinetic folding experiments with purified Desulfovibro desulfuricans apoflavodoxin in vitro were performed in the presence of Ficoll (sphere) and Dextran (spherocylinder) synthetic crowding agents. In conclusion, we identified the in silico crowding conditions that best enhance protein stability, and discovered that upon manipulation of the crowding conditions, folding routes experiencing topological frustrations can be either enhanced or relieved. Our test-tube experiments confirmed that apoflavodoxin''s time-resolved folding path is modulated by crowding agent geometry. Macromolecular crowding effects may be a tool for the manipulation of protein-folding and function in living cells.     

Macromolecular crowding modulates α-synuclein amyloid fiber growth

The crowdedness of living cells, hundreds of milligrams per milliliter of macromolecules, may affect protein folding, function, and misfolding. Still, such processes are most often studied in dilute solutions in vitro. To assess consequences of the in vivo milieu, we here investigated the effects of macromolecular crowding on the amyloid fiber formation reaction of α-synuclein, the amyloidogenic protein in Parkinson’s disease. For this, we performed spectroscopic experiments probing individual steps of the reaction as a function of the macromolecular crowding agent Ficoll70, which is an inert sucrose-based polymer that provides excluded-volume effects. The experiments were performed at neutral pH at quiescent conditions to avoid artifacts due to shaking and glass beads (typical conditions for α-synuclein), using amyloid fiber seeds to initiate reactions. We find that both primary nucleation and fiber elongation steps during α-synuclein amyloid formation are accelerated by the presence of 140 and 280 mg/mL Ficoll70. Moreover, in the presence of Ficoll70 at neutral pH, secondary nucleation appears favored, resulting in faster overall α-synuclein amyloid formation. In contrast, sucrose, a small-molecule osmolyte and building block of Ficoll70, slowed down α-synuclein amyloid formation. The ability of cell environments to modulate reaction kinetics to a large extent, such as severalfold faster individual steps in α-synuclein amyloid formation, is an important consideration for biochemical reactions in living systems.     

Simultaneous real‐time assay of copper and cadmium ions by infrared photo diode electrode implanted in the muscle of live fish

The electrical circuit of an infrared photodiode electrode (IPE) was used in the simultaneous assay of copper and cadmium ions. The electrode's cyclic voltammetry (CV), chronoamperometry and square‐wave (SW) stripping voltammetric optimum conditions were examined. Results for 0–160 mg L^?1 and 50–400 μg L^?1 SW Cu(II) Cd(II), the relative standard deviation of 0.158 Cu(II), 0.077 Cd(II) (n = 15) using 20.0 mg L^?1 have been obtained at optimum conditions. The low detection limit (S/N) was attained to be at 14.71 μg L^?1(2.31 × 10^?7 mol L^?1) Cu(II) and 18.42 μg L^?1(1.63 × 10^?7 mol L^?1) Cd(II). The handmade electrode was implanted deep in the muscle of live fish and interfaced with an electrochemical workstation. Real‐time analytical application was performed on the online assay of living tissue as the specimen was moving. The methods are deemed useful in interfaced assay for physiological control, nanodiode fabrication, and in the production of laboratory on a biochip. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:256–262, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20287     

Corynebacterium glutamicum,a natural overproducer of succinic acid?

Corynebacterium glutamicum is well known as an important industrial amino acid producer. For a few years, its ability to produce organic acids, under micro‐aerobic or anaerobic conditions was demonstrated. This study is focused on the identification of the culture parameters influencing the organic acids production and, in particular, the succinate production, by this bacterium. Corynebacterium glutamicum 2262, used throughout this study, was a wild‐type strain, which was not genetically designed for the production of succinate. The oxygenation level and the residual glucose concentration appeared as two critical parameters for the organic acids production. The maximal succinate concentration (4.9 g L^?1) corresponded to the lower k_La value of 5 h^?1. Above 5 h^?1, a transient accumulation of the succinate was observed. Interestingly, the stop in the succinate production was concomitant with a lower threshold glucose concentration of 9 g L^?1. Taking into account this threshold, a fed‐batch culture was performed to optimize the succinate production with C. glutamicum 2262. The results showed that this wild‐type strain was able to produce 93.6 g L^?1 of succinate, which is one of the highest concentration reported in the literature.     

Macromolecular crowding modulates α-synuclein amyloid fiber growth

The crowdedness of living cells, hundreds of milligrams per milliliter of macromolecules, may affect protein folding, function, and misfolding. Still, such processes are most often studied in dilute solutions in vitro. To assess consequences of the in vivo milieu, we here investigated the effects of macromolecular crowding on the amyloid fiber formation reaction of α-synuclein, the amyloidogenic protein in Parkinson’s disease. For this, we performed spectroscopic experiments probing individual steps of the reaction as a function of the macromolecular crowding agent Ficoll70, which is an inert sucrose-based polymer that provides excluded-volume effects. The experiments were performed at neutral pH at quiescent conditions to avoid artifacts due to shaking and glass beads (typical conditions for α-synuclein), using amyloid fiber seeds to initiate reactions. We find that both primary nucleation and fiber elongation steps during α-synuclein amyloid formation are accelerated by the presence of 140 and 280 mg/mL Ficoll70. Moreover, in the presence of Ficoll70 at neutral pH, secondary nucleation appears favored, resulting in faster overall α-synuclein amyloid formation. In contrast, sucrose, a small-molecule osmolyte and building block of Ficoll70, slowed down α-synuclein amyloid formation. The ability of cell environments to modulate reaction kinetics to a large extent, such as severalfold faster individual steps in α-synuclein amyloid formation, is an important consideration for biochemical reactions in living systems.     

Metabolism in 1,3‐propanediol fed‐batch fermentation by a D‐lactate deficient mutant of Klebsiella pneumoniae

Klebsiella pneumoniae HR526, a new isolated 1,3‐propanediol (1,3‐PD) producer, exhibited great productivity. However, the accumulation of lactate in the late‐exponential phase remained an obstacle of 1,3‐PD industrial scale production. Hereby, mutants lacking D ‐lactate pathway were constructed by knocking out the ldhA gene encoding fermentative D ‐lactate dehydrogenase (LDH) of HR526. The mutant K. pneumoniae LDH526 with the lowest LDH activity was studied in aerobic fed‐batch fermentation. In experiments using pure glycerol as feedstock, the 1,3‐PD concentrations, conversion, and productivity increased from 95.39 g L^?1, 0.48 and 1.98 g L^?1 h^?1 to 102. 06 g L^?1, 0.52 mol mol^?1 and 2.13 g L^?1 h^?1, respectively. The diol (1,3‐PD and 2,3‐butanediol) conversion increased from 0.55 mol mol^?1 to a maximum of 0.65 mol mol^?1. Lactate would not accumulate until 1,3‐PD exceeded 84 g L^?1, and the final lactate concentration decreased dramatically from more than 40 g L^?1 to <3 g L^?1. Enzymic measurements showed LDH activity decreased by 89–98% during fed‐batch fermentation, and other related enzyme activities were not affected. NADH/NAD⁺ enhanced more than 50% in the late‐exponential phase as the D ‐lactate pathway was cut off, which might be the main reason for the change of final metabolites concentrations. The ability to utilize crude glycerol from biodiesel process and great genetic stability demonstrated that K. pnemoniae LDH526 was valuable for 1,3‐PD industrial production. Biotechnol. Bioeng. 2009; 104: 965–972. © 2009 Wiley Periodicals, Inc.     

Combined Effects of Agitation,Macromolecular Crowding,and Interfaces on Amyloidogenesis

Amyloid formation and accumulation is a hallmark of protein misfolding diseases and is associated with diverse pathologies including type II diabetes and Alzheimer''s disease (AD). In vitro, amyloidogenesis is widely studied in conditions that do not simulate the crowded and viscous in vivo environment. A high volume fraction of most biological fluids is occupied by various macromolecules, a phenomenon known as macromolecular crowding. For some amyloid systems (e.g. α-synuclein) and under shaking condition, the excluded volume effect of macromolecular crowding favors aggregation, whereas increased viscosity reduces the kinetics of these reactions. Amyloidogenesis can also be catalyzed by hydrophobic-hydrophilic interfaces, represented by the air-water interface in vitro and diverse heterogeneous interfaces in vivo (e.g. membranes). In this study, we investigated the effects of two different crowding polymers (dextran and Ficoll) and two different experimental conditions (with and without shaking) on the fibrilization of amyloid-β peptide, a major player in AD pathogenesis. Specifically, we demonstrate that, during macromolecular crowding, viscosity dominates over the excluded volume effect only when the system is spatially non homogeneous (i.e. an air-water interface is present). We also show that the surfactant activity of the crowding agents can critically influence the outcome of macromolecular crowding and that the structure of the amyloid species formed may depend on the polymer used. This suggests that, in vivo, the outcome of amyloidogenesis may be affected by both macromolecular crowding and spatial heterogeneity (e.g. membrane turn-over). More generally, our work suggests that any factors causing changes in crowding may be susceptibility factors in AD.     

Steps Toward High‐Performance PLA: Economical Production of d‐Lactate Enabled by a Newly Isolated Sporolactobacillus terrae Strain

Optically pure d ‐lactate production has received much attention for its critical role in high‐performance polylactic acid production. However, the current technology can hardly meet the comprehensive demand of industrialization on final titer, productivity, optical purity, and raw material costs. Here, an efficient d ‐lactate producer strain, Sporolactobacillus terrae (S. terrae) HKM‐1, is isolated for d ‐lactate production. The strain HKM‐1 shows extremely high d ‐lactate fermentative capability by using peanut meal, soybean meal, or corn steep liquor powder as a sole nitrogen source; the final titers (205.7 g L^?1, 218.9 g L^?1, and 193.9 g L^?1, respectively) and productivities (5.56 g L^?1 h^?1, 5.34 g L^?1 h^?1, and 3.73 g L^?1 h^?1, respectively) of d ‐lactate reached the highest level ever reported. A comparative genomic analysis between S. terrae HKM‐1 and previously reported d ‐lactate high‐producing Sporolactobacillus inulinus (S. inulinus) CASD is conducted. The results show that many unrelated genetic features may contribute to the superior performance in d ‐lactate production of S. terrae HKM‐1. This d ‐lactate producer HKM‐1, along with its fermentation process, is promising for sustainable d ‐lactate production by using agro‐industrial wastes.     

Impact of light quality on a native Louisiana Chlorella vulgaris/Leptolyngbya sp. co‐culture

Light effect on cultures of microalgae has been studied mainly on single species cultures. Cyanobacteria have photosynthetic pigments that can capture photons of wavelengths not available to chlorophylls. A native Louisiana microalgae (Chlorella vulgaris ) and cyanobacteria (Leptolyngbya sp.) co‐culture was used to study the effects of light quality (blue–467 nm, green–522 nm, red–640 nm and white–narrow peak at 450 nm and a broad range with a peak at 550 nm) at two irradiance levels (80 and 400 μmol m^?2 s^?1) on the growth, species composition, biomass productivity, lipid content and chlorophyll‐a production. The co‐culture shifted from a microalgae dominant culture to a cyanobacteria culture at 80 μmol m^?2 s^?1. The highest growth for the cyanobacteria was observed at 80 μmol μmol m^?2 s^?1 and for the microalgae at 400 μmol m^?2 s^?1. Red light at 400 μmol m^?2 s^?1 had the highest growth rate (0.41 d^?1), biomass (913 mg L^?1) and biomass productivity (95 mg L^?1 d^?1). Lipid content was similar between all light colors. Green light had the highest chlorophyll‐a content (1649 μg/L). These results can be used to control the species composition of mixed cultures while maintaining their productivity.     

Variability of responses to 1-methylcyclopropene by banana: influence of time of year at harvest and fruit position in the bunch

To examine the effect of early‐climacteric (postripening) 1‐methylcyclopropene (1‐MCP) exposure on the shelf‐life and quality of green Cavendish bananas (Musa acuminata cv. Williams) from the middle section of the bunch, bananas were harvested bimonthly and treated with 100 μL L^?1 ethylene for 2 consecutive days prior to exposure to 0, 100, 300, 1000, 3000 or 10 000 nL L^?1 1‐MCP for 24 h prior to storage at 22°C. 1‐MCP treatment at a concentration of 300 nL L^?1 or above increased banana shelf‐life significantly compared with the control, regardless of the month in which fruit were harvested except March where a higher concentration was needed (3000 nL L^?1). Fruit harvested in May were the most responsive with a greater than twofold increase in shelf‐life. To examine the effect of fruit position in the bunch on 1‐MCP efficacy, green fruit from the top or bottom of bunches were treated with 100 μL L^?1 ethylene for 2 consecutive days prior to early‐climacteric 1‐MCP (300 nL L^?1) exposure for 24 h at 22°C. In spring and autumn but not in summer, application of 1‐MCP to early‐climacteric fruit was more effective in fruit from the top than in those treated from the bottom of the bunch, increasing shelf‐life. Firmness of 1‐MCP‐treated fruit was up to 19% greater than that of the control across the year, except in fruit from the bottom of the bunch. Given that 1‐MCP is less effective in extending the shelf‐life of summer‐harvested fruit (particularly those from the bottom of the bunch), we conclude that preharvest conditions and fruit position in the bunch affect their responsiveness to ethylene and their behaviour during the ripening process.     

Minimal Effects of Macromolecular Crowding on an Intrinsically Disordered Protein: A Small-Angle Neutron Scattering Study

Small-angle neutron scattering was used to study the effects of macromolecular crowding by two globular proteins, i.e., bovine pancreatic trypsin inhibitor and equine metmyoglobin, on the conformational ensemble of an intrinsically disordered protein, the N protein of bacteriophage λ. The λ N protein was uniformly labeled with ²H, and the concentrations of D₂O in the samples were adjusted to match the neutron scattering contrast of the unlabeled crowding proteins, thereby masking their contribution to the scattering profiles. Scattering from the deuterated λ N was recorded for samples containing up to 0.12 g/mL bovine pancreatic trypsin inhibitor or 0.2 g/mL metmyoglobin. The radius of gyration of the uncrowded protein was estimated to be 30 Å and was found to be remarkably insensitive to the presence of crowders, varying by <2 Å for the highest crowder concentrations. The scattering profiles were also used to estimate the fractal dimension of λ N, which was found to be ∼1.8 in the absence or presence of crowders, indicative of a well-solvated and expanded random coil under all of the conditions examined. These results are contrary to the predictions of theoretical treatments and previous experimental studies demonstrating compaction of unfolded proteins by crowding with polymers such as dextran and Ficoll. A computational simulation suggests that some previous treatments may have overestimated the effective volumes of disordered proteins and the variation of these volumes within an ensemble. The apparent insensitivity of λ N to crowding may also be due in part to weak attractive interactions with the crowding proteins, which may compensate for the effects of steric exclusion.     

Effect of crowding by Ficolls on OmpA and OmpT refolding and membrane insertion

Folding of outer membrane proteins (OMPs) has been studied extensively in vitro. However, most of these studies have been conducted in dilute buffer solution, which is different from the crowded environment in the cell periplasm, where the folding and membrane insertion of OMPs actually occur. Using OmpA and OmpT as model proteins and Ficoll 70 as the crowding agent, here we investigated the effect of the macromolecular crowding condition on OMP membrane insertion. We found that the presence of Ficoll 70 significantly slowed down the rate of membrane insertion of OmpA while had little effect on those of OmpT. To investigate if the soluble domain of OmpA slowed down membrane insertion in the presence of the crowding agent, we created a truncated OmpA construct that contains only the transmembrane domain (OmpA171). In the absence of crowding agent, OmpA171 refolded at a similar rate as OmpA, although with decreased efficiency. However, under the crowding condition, OmpA171 refolded significantly faster than OmpA. Our results suggest that the periplasmic domain slows down the rate, while improves the efficiency, of OmpA folding and membrane insertion under the crowding condition. Such an effect was not obvious when refolding was studied in buffer solution in the absence of crowding.