Compact Globular Structure Research Articles

Influence of macromolecules and electrolytes on heteroaggregation kinetics of polystyrene nanoplastics and goethite nanoparticles in aquatic environments

Fate and transport of nanoplastics in aquatic environments are affected by their heteroaggregation with minerals in the presence of macromolecules. This study investigated the heteroaggregation of polystyrene nanoplastics (PSNPs) with goethite nanoparticles (GNPs) under the influence of macromolecules [humic acid (HA), bovine serum albumin (BSA), and DNA] and electrolytes. Under 1 mg C/L macromolecule, raising electrolyte concentration promoted heteroaggregation via charge screening, except that calcium bridging with HA also enhanced heteroaggregation at CaCl2 concentration above 5 mM. At all NaCl concentrations and CaCl2 concentration below 5 mM, 1 mg C/L macromolecules strongly retarded heteroaggregation, ranking BSA > DNA > HA. Raising macromolecule concentration strengthened such stabilization effect of all macromolecules in NaCl solution and that of DNA and BSA in CaCl2 solution by enhancing steric hindrance. However, 0.1 mg C/L BSA slightly promoted heteroaggregation in CaCl2 solution due to stronger electrostatic attraction than steric hindrance. In CaCl2 solution, raising HA concentration strengthened its destabilization effect via calcium bridging. Macromolecules having more compact globular structure and higher molecular weight may exert greater steric hindrance to inhibit heteroaggregation more effectively. This study provides new insights on the effects of macromolecules and electrolytes on heteroaggregation between nanoplastics and iron minerals in aquatic environments.

Conformations of a highly expressed Z19 α-zein studied with AlphaFold2 and MD simulations.

α-zeins are amphiphilic maize seed storage proteins with material properties suitable for a multitude of applications e.g., in renewable plastics, foods, therapeutics and additive manufacturing (3D-printing). To exploit their full potential, molecular-level insights are essential. The difficulties in experimental atomic-resolution characterization of α-zeins have resulted in a diversity of published molecular models. However, deep-learning α-zein models are largely unexplored. Therefore, this work studies an AlphaFold2 (AF2) model of a highly expressed α-zein using molecular dynamics (MD) simulations. The sequence of the α-zein cZ19C2 gave a loosely packed AF2 model with 7 α-helical segments connected by turns/loops. Compact tertiary structure was limited to a C-terminal bundle of three α-helices, each showing notable agreement with a published consensus sequence. Aiming to chart possible α-zein conformations in practically relevant solvents, rather than the native solid-state, the AF2 model was subjected to MD simulations in water/ethanol mixtures with varying ethanol concentrations. Despite giving structurally diverse endpoints, the simulations showed several patterns: In water and low ethanol concentrations, the model rapidly formed compact globular structures, largely preserving the C-terminal bundle. At ≥ 50 mol% ethanol, extended conformations prevailed, consistent with previous SAXS studies. Tertiary structure was partially stabilized in water and low ethanol concentrations, but was disrupted in ≥ 50 mol% ethanol. Aggregated results indicated minor increases in helicity with ethanol concentration. β-sheet content was consistently low (∼1%) across all conditions. Beyond structural dynamics, the rapid formation of branched α-zein aggregates in aqueous environments was highlighted. Furthermore, aqueous simulations revealed favorable interactions between the protein and the crosslinking agent glycidyl methacrylate (GMA). The proximity of GMA epoxide carbons and side chain hydroxyl oxygens simultaneously suggested accessible reactive sites in compact α-zein conformations and pre-reaction geometries for methacrylation. The findings may assist in expanding the applications of these technologically significant proteins, e.g., by guiding chemical modifications.

Structural, emulsifying and curcumin-embedded properties of arachin nanoparticles induced by ultrasound and heat

Different types of arachin (one of the major globulins in peanut proteins) nanoparticles were prepared by ultrasound (US) and heat treatments, respectively. The results showed that short-term US treatment induced large nanoparticles (named US200 and US500, ∼267 nm) that maintained a compact globular structure; long-term US treatment induced small nanoparticles (named US500L, ∼197 nm) with a moderately unfolded structure; heat treatment induced medium-sized nanoparticles with an unfolded structure. The large nanoparticles had significantly lower surface hydrophobicity, β-sheet content, and λmax but higher enthalpy of denaturation (ΔH) than other nanoparticles. US500L and US500 exhibited the highest emulsifying activity and stability indices, respectively. As for curcumin-embedded properties, both US and heat treatments enhanced the curcumin encapsulation efficiency (from 88.53 to 94.08–97.88%) and load amount (from 1.77 to 1.88–1.96 μg/mg) of arachin. However, only curcumin in large nanoparticles was effectively protected from damage by heat and light. In simulated digestion tests, curcumin in US500L and heat-induced nanoparticles showed higher stability and bioavailability, which was mainly attributed to the slower digestion of these nanocomplexes. We have established a simple method for the preparation of arachin nanoparticles by US and the nanoparticles have potential applications in the food industry due to their favorable bifunctional properties.

Improvement of emulsifying and loading properties of whey protein isolate via ultrasound-assisted alkali pretreatment and carboxymethyl cellulose interaction

Whey protein isolate (WPI) is a by-product of the dairy industry. It exists as compact globular structure that always limits its wide applications. This research aimed to improve the emulsifying and loading properties of WPI through ultrasound-assisted alkali pretreatment and carboxymethyl cellulose (CMC) interaction. The results demonstrated enhanced solubility and reduced size of WPI following the pretreatment of ultrasound-assisted alkali. Circular dichroism spectra showed that α-helix content decreased from 14.8% to 12.8%, and the number of random coils increased from 30.6% to 37.2% (P < 0.05). Fluorescence spectra showed more hydrophobic amino acids were exposed. When WPI underwent ultrasound-assisted 0.01 mol L−1 NaOH and was complexed with CMC (4:1, w/w), the resulting emulsifying properties were improved compared to the control. The emulsions stabilized by modified WPI/CMC composites had greater encapsulation efficiency on curcumin, resulting in improved protection against UV light and thermal degradation. This study confirmed that ultrasound-assisted alkali pretreatment and CMC complexation could improve the emulsifying and loading ability of WPI.

Characterization of the interaction between allicin and soy protein isolate and functional properties of the adducts.

Soybean meal, a by-product of the soybean oil production industry, has a high protein content but the compact globular structure of the protein from soybean meal limits its wide application in food processing. Allicin has been found to have numerous functional properties. In this study, allicin was interacted with soy protein isolate (SPI). The functional properties of the adducts were investigated. Binding with allicin significantly quenched the fluorescence intensity of SPI. Static quenching was the main quenching mechanism. The stability of adducts decreased with increasing temperature. The greatest extent of binding between allicin and sulfhydryl groups (SH) of SPI was obtained at an allicin/SH molar ratio of 1:2. The amino groups of SPI did not bind with allicin covalently. Soy protein isolate was modified by allicin through covalent and non-covalent interactions. Compared with SPI, the emulsifying activity index and foaming capacity of adducts with a ratio of 3:1 were improved by 39.91% and 64.29%, respectively. Soy protein isolate-allicin adducts also exhibited obvious antibacterial effects. The minimum inhibitory concentrations (MICs) of SPI-allicin adducts on Escherichia coli and Staphylococcus aureus were 200 and 160 μg mL-1 , respectively. The interaction of allicin with SPI is beneficial for the functional properties of SPI. These adducts can be used in different food formulations as emulsifiers, foamers, and transport carriers. © 2023 Society of Chemical Industry.

Structural Characterization of Degraded Lycium barbarum L. Leaves' Polysaccharide Using Ascorbic Acid and Hydrogen Peroxide.

Plant-derived polysaccharide’s conformation and chain structure play a key role in their various biological activities. Lycium barbarum L. leaves’ polysaccharide is well renowned for its health functions. However, its functional bioactivities are greatly hindered by its compact globular structure and high molecular weight. To overcome such issue and to improve the functional bioactivities of the polysaccharides, degradation is usually used to modify the polysaccharides conformation. In this study, the ethanol extract containing crude Lycium barbarum L. leaves’ polysaccharide was first extracted, further characterized, and subsequently chemically modified with vitamin C (Ascorbic acid) and hydrogen peroxide (H2O2) to produce degraded Lycium barbarum L. leaves’ polysaccharide. To explore the degradation effect, both polysaccharides were further characterized using inductively coupled plasma mass spectrometry (ICP-MS), gas chromatography–mass spectrometry (GC–MS), Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), high performance gel permeation chromatography (HPGPC), and scanning electron microscope (SEM). Results shown that both polysaccharides were rich in sugar and degradation had no significant major functional group transformation effect on the degraded product composition. However, the molecular weight (Mw) had decreased significantly from 223.5 kDa to 64.3 kDa after degradation, indicating significant changes in the polysaccharides molecular structure caused by degradation.

High voltage electrical treatments can eco-efficiently promote the production of high added value peptides during chymotryptic hydrolysis of β-lactoglobulin

Chymotryptic hydrolysis of β-lactoglobulin (β-lg) leads to the release of a wide range of biofunctional peptides. However, the proteolysis of native β-lg is limited due to its compact globular structure, thus ending with low yields of peptides. Whereas our previous study showed that eco-efficient High Voltage Electric Treatments (HVET), namely Pulsed Electric Fields (PEF) and Electric Arc (ARC), of β-lg substantially improved its enzymatic hydrolysis, no information regarding the nature of released peptides is available up to date. Hence, the time-dependent release of peptides and the dynamics of this release were discussed in the current study. Chromatographic analysis of the final hydrolysates showed that chymotrypsinolysis of HVET samples released 19% more peptides with higher concentration than the native and preheated ones. Furthermore, 1-min HVET were found to be more efficient than 10-min ones as they led to the release of bioactive peptides (even without chymotrypsin) with higher eco-efficiency scores.

Mutual Information in Molecular and Macromolecular Systems.

The relaxation properties of viscous liquids close to their glass transition (GT) have been widely characterised by the statistical tool of time correlation functions. However, the strong influence of ubiquitous non-linearities calls for new, alternative tools of analysis. In this respect, information theory-based observables and, more specifically, mutual information (MI) are gaining increasing interest. Here, we report on novel, deeper insight provided by MI-based analysis of molecular dynamics simulations of molecular and macromolecular glass-formers on two distinct aspects of transport and relaxation close to GT, namely dynamical heterogeneity (DH) and secondary Johari–Goldstein (JG) relaxation processes. In a model molecular liquid with significant DH, MI reveals two populations of particles organised in clusters having either filamentous or compact globular structures that exhibit different mobility and relaxation properties. In a model polymer melt, MI provides clearer evidence of JG secondary relaxation and sharper insight into its DH. It is found that both DH and MI between the orientation and the displacement of the bonds reach (local) maxima at the time scales of the primary and JG secondary relaxation. This suggests that, in (macro)molecular systems, the mechanistic explanation of both DH and relaxation must involve rotation/translation coupling.

Substantial Improvement of Tryptic and Chymotryptic Hydrolysis of β-Lactoglobulin Pretreated with High Voltage Electrical Treatments

β-lactoglobulin (β-lg) is an important source of biologically and functionally active peptides that are of particular interest in agri-food, nutraceutical, and pharmaceutical industries. Enzymatic hydrolysis of proteins remains the most common mode of peptide production. However, efficient hydrolysis of proteins and particularly β-lg is difficult to perform due to their compact globular structure. Thus, the current study deals with green high-voltage electric treatments (HVET): pulsed electric fields and electric arc to pretreat β-lg and improve its proteolysis by trypsin and chymotrypsin. The results demonstrate substantial improvement of hydrolysis performance after the pretreatment of β-lg with HVET compared to the non-pretreated and preheated β-lg. Moreover, HVET led to the peptide release from β-lg even before the addition of proteases. Furthermore, pioneer studies of proteolysis kinetics and structural changes of β-lg molecules subjected to HVET allowed a better understanding of the mechanisms underlying hydrolysis improvement.

Ribosylation induced structural changes in Bovine Serum Albumin: understanding high dietary sugar induced protein aggregation and amyloid formation

Non-enzymatic glycation of proteins is believed to be the root cause of high dietary sugar associated pathophysiological maladies. We investigated the structural changes in protein during progression of glycation using ribosylated Bovine Serum Albumin (BSA). Non enzymatic attachment of about 45 ribose molecules to BSA resulted in gradual reduction of hydrophobicity and aggregation as indicated by red-shifted tryptophan fluorescence, reduced ANS binding and lower anisotropy of FITC-conjugated protein. Parallely, there was a significant decrease of alpha helicity as revealed by Circular Dichroism (CD) and Fourier transformed-Infra Red (FT-IR) spectra. The glycated proteins assumed compact globular structures with enhanced Thioflavin-T binding resembling amyloids. The gross structural transition affected by ribosylation led to enhanced thermostability as indicated by melting temperature and Transmission Electron Microscopy. At a later stage of glycation, the glycated proteins developed non-specific aggregates with increase in size and loss of amyloidogenic behaviour. A parallel non-glycated control incubated under similar conditions indicated that amyloid formation and associated changes were specific for ribosylation and not driven by thermal denaturation due to incubation at 37 °C. Functionality of the glycated protein was significantly altered as probed by Isothermal Titration Calorimetry using polyphenols as substrates. The studies demonstrated that glycation driven globular amyloids form and persist as transient intermediates during formation of misfolded glycated adducts. To the best of our knowledge, the present study is the first systematic attempt to understand glycation associated changes in a protein and provides important insights towards designing therapeutics for arresting dietary sugar induced amyloid formation.

Simulating the ultrafiltration of whey proteins isolate using a mixture model

Ultrafiltration (UF) is widely used in the fractionation and concentration of milk proteins. During this process, proteins concentrate at the membrane surface, increasing the resistance of the membrane system, and decreasing permeate flux leaving the process unit. In this study we use Computational Fluid Dynamics (CFD) to examine the UF of whey proteins. Specifically we employ a multiphase mixture model to predict the solid concentration and velocity distributions near the membrane, under both dead-end and crossflow modes. As whey proteins have a fairly compact globular structure we use hard sphere theory to model the suspension permeability and osmotic pressure: Notably these properties depend only on parameters that can be independently measured, such as protein size and concentration. Model predictions are found to be in good agreement with experimental flux data for both flow modes, significantly in the absence of any additional resistance models representing (for example) pore blockage, surface adsorption or gel/cake formation. We find that during dead-end filtration, the extent of flux decline is logically dependent on the concentration film thickness and maximum solids concentration attained at the membrane surface. Under crossflow conditions suspension advection from the bulk towards the membrane increases the permeate flux, particularly along any upstream membrane area edges. Simulating the system using simplified 2D geometries produces qualitative, rather than quantitative agreement with experiment, highlighting the need to capture the system geometry accurately when modelling concentration polarisation (CP) effects. This work shows that industrially relevant whey protein filtration can be predicted via the multiphase modelling of hard sphere suspensions, using as-measured particle sizes, and points to more accurate methods for optimising and designing whey UF systems.

Structure and thermal properties of arachin from six varieties: effect of 35.5 kDa subunit

ABSTRACT The subunit composition, and thermal and molecular conformation properties of arachin from six peanut varieties are characterized and compared. The thermal and molecular conformation properties including the thermal stability, steady flow properties, surface hydrophobicity, and sulfhydryl content were determined. Two groups of arachin were identified: one containing the 35.5 kDa subunit and the other one without this specific subunit. The results showed that arachin with the 35.5 kDa subunit was more heat-sensitive (Tonset <100°C), had a significant lower initial denaturation temperature, less disulfide bonds, and more hydrophobic groups. The arachin without the 35.5 kDa subunit was less heat-sensitive to temperature (Tonset >100°C), and a more compact globular structure. The presence or absence of 35.5 kDa subunit in arachin significantly influenced the thermal and molecular conformation properties of arachin. Furthermore, the 35.5 kDa subunit was sequenced by Q-TOF and identified as an isoform of Ara h3. This study can provide useful information for processing peanut protein products with good thermal stability and hypoallergenic properties.

Exploring the structural basis of conformational heterogeneity and autoinhibition of human cGMP-specific protein kinase Iα through computational modelling and molecular dynamics simulations

Protein kinase Iα (PKGIα) is a pivotal cyclic guanosine monophosphate (cGMP) signalling protein. Major steps related to the structural plasticity of PKGIα have been inferred but the structural aspects of the auto-inhibition and multidomain tertiary organization of human PKGIα in active and inactive form are not clear. Here we combine computational comparative modelling, protein–protein docking and molecular dynamics (MD) simulations to investigate structural details of the repressed state of the catalytic domain of PKGIα. Exploration of the potential inhibitory conformation of the auto-inhibitory domain (AI) within the catalytic cleft reveals that the pseudo-substrate motif binds with residues of the glycine rich loop and substrate-binding lobe. Dynamic changes as a result of coupling of the catalytic and AI domains are also investigated. The three-dimensional homodimeric models of PKGIα in the active and inactive state indicate that PKGIα in its inactive-state attains a compact globular structure where cyclic nucleotide binding (CNB-A/B) domains are buried, whereas the catalytic domains are inaccessible with their substrate-binding pockets facing the N-terminal of CNB-A. Contrary to this, the active-state model of PKGIα shows an extended conformation where CNB-A/B domains are slightly rearranged and the catalytic domains of homodimer flanking the C-terminal with their substrate binding lobes free to entrap downstream proteins. These findings are consistent with previously reported static images of the multidomain organization of PKGIα. Structural insights pertaining to the conformational heterogeneity and auto-inhibition of PKGIα provided in this study may help to understand the dynamics-driven effective regulation of PKGIα.

Degradation of polysaccharides from Lycium barbarum L. leaves improves bioaccessibility and gastrointestinal transport of endogenous minerals

Acidic heteropolysaccharide (LP) from Lycium barbarum L. leaves has compact globular structure which wrapped abundant endogenous minerals inside by ionic interactions with uronic acid. This study investigated the efficacy of chemical degradation of LP on the bioaccessibility and transport of endogenous minerals in simulated gastrointestinal fluids. Results showed that the degradation using vitamin C and hydrogen peroxide mildly decreased LP molecular weight from 162.0 kDa to 94.3 kDa, and the structure of degraded LP (LPD) was converted to loose coil. After the simulated intestinal digestion, the accessibility of Ca, Fe, Zn, and Mg in LPD increased by119%, 52%, 103% and 112.5% compared with the intact LP, respectively, and in particular, the uptake rate increased by 15.8%, 8.1%, 23.4% and 21.6% for Ca, Fe, Zn, and Mg, respectively. These results demonstrated that the chemical degradation is a helpful strategy to improve the uptake of endogenous minerals wrapped in polysaccharide.

Sterol and Triterpene Profiles of the Callus Culture of Solanum mammosum

This study aimed to compare the sterol and triterpene profiles of two types of Solanum mammosum callus cultures, i.e., compact globular structure (CGS) and normal fine (F) calluses. The CGS callus resulted from the differentiation of the F callus culture after many years of subculturing. The growth rate, microscopic characteristics, and morphologies of the two callus types were determined and compared. Sterols and triterpenes were identified through thin-layer chromatography, gas chromatography–flame ionization detection, and gas chromatography–mass spectrometry analyses. The growth rate of the CGS callus was lower than that of the F callus. Microscopic identification revealed that thick, lignin-containing cell walls formed in the CGS callus but not in the F callus. The chromatographic analysis suggested that the CGS and F callus cultures had different sterol and triterpenoid profiles. The sterols and triterpenes produced by the CGC culture were more diverse than those produced by the F callus culture.

Influence of macromolecules on aggregation kinetics of diesel soot nanoparticles in aquatic environments

Soot nanoparticles (SNPs) produced from incomplete combustion have strong impacts on aquatic environments as they eventually reach surface water, where their environmental fate and transport are largely controlled by aggregation. This study investigated the aggregation kinetics of SNPs in the presence of macromolecules including fulvic acid (FA), humic acid (HA), alginate polysaccharide, and bovine serum albumin (BSA, protein) under various environmentally relevant solution conditions. Our results showed that increasing salt concentrations induced SNP aggregation by suppressing electrostatic repulsion and that CaCl2 exhibited stronger effect than NaCl in charge neutralization, which is in agreement with the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. The aggregation rates of SNPs were variously reduced by macromolecules, and such stabilization effect was the greatest by BSA, followed by HA, alginate, and FA. Steric repulsion resulting from macromolecules adsorbed on SNP surfaces was mainly responsible for enhancing SNP stability. Such steric repulsion appeared to be affected by macromolecular structure, as BSA having a more compact globular structure on SNP surfaces imparted long-range steric repulsive forces and retarded the SNP aggregation rate by 10–100 times. In addition, alginate was shown to enhance SNP aggregation by ∼10 times at high CaCl2 concentrations due to alginate gel formation via calcium bridging. The results may bear strong significance for the fate and transport of SNPs in both natural and controlled environmental systems.

Chromatographic separation of similar post-translationally modified metallothioneins reveals the changing conformations of apo-MT upon cysteine alkylation by high resolution LC-ESI-MS

Metallothioneins (MTs) are a class of small cysteine-rich proteins essential for Zn and Cu homeostasis, heavy metal detoxification, and cellular redox chemistry. Herein, we describe the separation and characterization of MTs differentially modified with N-ethylmaleimide (NEM) by liquid chromatography-mass spectrometry (LC-MS). The full-length recombinant MT isoform 1a as well as is isolated domain fragments were first alkylated, then separated on column with subsequent detection by ultra-high resolution ESI-MS. Different behavior was observed for the three peptides with the full-length protein and the isolated α-domain exhibiting similar separation characteristics. For the isolated β-domain, the smallest peptide with 9 cysteines in the sequence, each alkylated species was well separated, indicating large changes in protein conformation. For the full-length (20 cysteines in the sequence) and α-domain (11 cysteiens in the sequence) peptides, the apo- and lightly alkylated species co-eluted, indicating similar structural properties. However, the more extensively alkylated species were well separated from each other, indicating the sequential unfolding of the apo-MT peptides and providing evidence for the mechanistic explanation for the cooperative alkylation reaction observed for NEM and other bulky and hydrophobic alkylation reagents. We show for the first time clear separation of highly similar MTs, differing by only +125 Da, and can infer structural properties from the LC-MS data, analogous to more complicated and less ubiquitous ion-mobility experiments. The data suggest a compact globular structure for each of the apo-MTs, but where the β-domain is more easily unfolded. This differential folding stability may have biological implications in terms of domain-specific participation of MT in cellular redox chemistry and resulting metal release.

Structural Basis for Interaction of the Tandem Zinc Finger Domains of Human Muscleblind with Cognate RNA from Human Cardiac Troponin T.

The human muscleblind-like proteins (MBNL) regulate tissue-specific splicing by targeting cardiac troponin T and other pre-mRNAs; aberrant targeting of CUG and CCUG repeat expansions frequently accompanies the neuromuscular disease myotonic dystrophy. We show, using biolayer interferometry (Octet) and NMR spectroscopy, that the zinc finger domains of MBNL isoform 1 (MBNL1) are necessary and sufficient for binding CGCU sequences within the pre-mRNA of human cardiac troponin T. Protein constructs containing zinc fingers 1 and 2 (zf12) and zinc fingers 3 and 4 (zf34) of MBNL1 each fold into a compact globular tandem zinc finger structure that participates in RNA binding. NMR spectra show that the stoichiometry of the interaction between zf12 or zf34 and the CGCU sequence is 1:1, and that the RNA is single-stranded in the complex. The individual zinc fingers within zf12 or zf34 are nonequivalent: the primary RNA binding surface is formed in each pair by the second zinc finger (zf2 or zf4), which interacts with the CGCU RNA sequence. The NMR structure of the complex between zf12 and a 15-base RNA of sequence 95GUCUCGCUUUUCCCC109, containing a single CGCU element, shows the single-stranded RNA wrapped around zf2 and extending to bind to the C-terminal helix. Bases C101, U102, and U103 make well-defined and highly ordered contacts with the protein, whereas neighboring bases are less well-ordered in the complex. Binding of the MBNL zinc fingers to cardiac troponin T pre-mRNA is specific and relatively simple, unlike the complex multiple dimer–trimer stoichiometries postulated in some previous studies.

Biological Semiconductors: Structural Control of Heme Redox Potentials in PpcA, a 3-Heme Cytochrome

In an effort to tune and optimize relative bandgap potentials found in biological nanowires that form from multiheme domains of members of the cytochrome c7 family, we developed and extensively characterized 12 point mutations of PpcA, a 3-heme member of the cytochrome c7 family native to Geobacter sulfurreducens. These mutations were engineered to influence the redox potential (Em) of the middle heme (heme III) in PpcA by using four different strategies: performing charge reversal mutations, decreasing solvent access to the heme plane with bulky residues, altering the native bis-histidine axial ligation of the heme, and by attempting to form hydrogen bonds with the propionates of the heme. The latter strategy is expected not only to increase Em but also to introduce a redox Bohr effect. Out of 12 mutants, 11 were successfully expressed in E.coli and show thermal stability in temperature-dependent CD experiments comparable to the wild-type protein (Tm > 90°C). HPLC-ESI-MS was used to confirm both the purity and the mass of the expressed mutants. Small-angle X-ray scattering confirmed that the mutant proteins formed the expected compact globular structures. In addition to ongoing attempts to obtain protein crystals for X-ray crystallography, we performed extensive all-atom molecular dynamics simulations for all mutants. Formation of propionate hydrogen bonds is supported by simulations for A19R and A23R, while increased solvent access is supported for A19I, A19R, A23R, H20M, and K60E. UV/Vis and NMR redox titrations were performed in order to measure the effect of the mutations on the electrochemical properties of all 3 hemes and to understand the underlying principles and viable approaches in tuning relative heme redox potentials. Successful development of this project may lead to biological semiconductors with much smaller footprints and selectively tunable bandgap properties.

Calcium ions function as a booster of chromosome condensation.

Chromosome condensation is essential for the faithful transmission of genetic information to daughter cells during cell division. The depletion of chromosome scaffold proteins does not prevent chromosome condensation despite structural defects. This suggests that other factors contribute to condensation. Here we investigated the contribution of divalent cations, particularly Ca2+, to chromosome condensation in vitro and in vivo. Ca2+ depletion caused defects in proper mitotic progression, particularly in chromosome condensation after the breakdown of the nuclear envelope. Fluorescence lifetime imaging microscopy-Förster resonance energy transfer and electron microscopy demonstrated that chromosome condensation is influenced by Ca2+. Chromosomes had compact globular structures when exposed to Ca2+ and expanded fibrous structures without Ca2+. Therefore, we have clearly demonstrated a role for Ca2+ in the compaction of chromatin fibres.