Native State Research Articles

The sizing and suitability of nonspherical ellipsoid humeral heads for total shoulder arthroplasty.

Total shoulder arthroplasty (TSA) implants have evolved to include more anatomically shaped components that better replicate the native state. The geometry of the humeral head is nonspherical, with the frontal diameter of the base of the head being up to 6% larger than the sagittal diameter. Despite this, most TSA humeral head implants are spherical, meaning that the diameter must be oversized to achieve complete coverage, resulting in articular overhang, or portions of the resection plane will remain uncovered. It is suggested that implant-bone load transfer between the backside of the humeral head and the cortex on the resection plane may yield better load-transfer characteristics if resection coverage were properly matched without overhang, thereby mitigating proximal stress shielding. Eight paired cadaveric humeri were prepared for TSA by an orthopedic surgeon who selected and prepared the anatomic humeral resection plane using a cutting guide and a reciprocating sagittal saw. The humeral head was resected, and the resulting cortical boundary of the resection plane was digitized using a stylus and an optical tracking system. To simulate optimal sizing of both circular and elliptical humeral heads, both circles and ellipses were fit to the traces. Two extreme scenarios were also investigated: upsizing until 100% total coverage and downsizing until 0% overhang. By switching from a spherical (circular) to an ellipsoid (elliptical) humeral head, a small, 2.3%±0.3% increase in total coverage occurred (P<.001), which led to a large, 19.5%±1.3% increase in cortical coverage (P<.001). Using a circular head resulted in 2.0%±0.1% greater overhang (P<.001), defined as a percentage of the total enclosed area that exceeded the bounds of the humeral resection. As a result of increasing the head size until 100% resection coverage occurred, the ellipse produced 5.4%±3.5% less overhang than the circle (P<.001). When the head size was decreased until 0% overhang occurred, total coverage was 7.5%±2.8% greater for the ellipse (P<.001) and cortical coverage was 7.9%±8.2% greater for the ellipse (P=.01). Cortical coverage was greater for circular heads when the head size was shrunk below -2.25% of the optimal fitted size. Reconstruction with ellipsoid humeral heads can provide greater total resection and cortical coverage than spherical humeral heads while avoiding excessive overhang; however, cortical coverage can be inferior when undersized. These initial findings suggest that resection-matched humeral heads may yield benefits worth pursuing in the next generation of TSA implant design.

Functional biomechanical comparison of Latarjet vs. distal tibial osteochondral allograft for anterior glenoid defect reconstruction.

Glenoid reconstruction is indicated for recurrent glenohumeral instability with significant glenoid bone deficiency. Coracoid autograft (Latarjet) and distal tibial osteochondral allograft (DTA) reconstructions have been used to successfully restore glenohumeral stability. Relative advantages and disadvantages associated with each reconstruction technique have been described. However, direct comparisons of functional glenohumeral biomechanics associated with Latarjet vs. DTA reconstruction are lacking. This study was designed to compare these 2 glenoid reconstruction techniques with respect to joint kinematics and cartilage pressure mapping using a robotic testing system. In accordance with institutional review board policies, human cadaveric shoulders (n=8) were cyclically tested in the neutral position and 90° of external rotation with 60° and 90° of abduction under a 45-N joint-compression load to measure clinically relevanttranslations, loads, and torques. Joint contact pressure maps were obtained under a 120-N joint-compression load using pressure mapping sensors. After confirming that a 25% anterior glenoid defect resulted in glenohumeral dislocation, testing was performed to compare 3 conditions: native intact glenoid, 25% anterior glenoid defect with Latarjet reconstruction, and 25% anterior glenoid defect with DTA reconstruction. Analyses of variance and t tests were used to analyze data with statistical significance set at P< .05. Significant differences in anterior translation, inferior drawer, anterior drawer, compression loads, horizontal abduction, negative elevation (adduction), and external rotation torques during cyclical testing in 90° of external rotation with 60° and/or 90° of abduction were noted when comparing the 2 different glenoid bone reconstruction techniques to native, intact shoulders. The only significant difference between Latarjet and DTA reconstructions for measured translations, loads, and torques was a significantly higher absolute maximum compressive load for Latarjet compared to DTA at 60° of abduction. Latarjet coracoid osseous autograft and distal tibial osteochondral allograft reconstructions of large (25%) glenoid bone defects prevent failure (dislocation) and are associated with significant glenohumeral kinematic differences that largely confer less translation, load, and torque on the joint in abduction when compared to the native state. These findings suggest that these 2 surgical techniques exhibit similar glenohumeral kinematics such that each provides adequate functional stability following anterior glenoid bone reconstruction. Joint compression load and articular contact pressure distribution may favor distal tibial osteochondral allograft reconstruction for treatment of large (25%) anterior glenoid bone defects associated with shoulder instability.

Separating the thermodynamic impact of non-specific interfacial interactions from confinement effects on protein stability.

The intracellular milieu is incredibly complex, containing myriad degrees of spatial restriction and highly variable interfacial chemistry. It is well established that this environmental complexity impacts diffusive properties, stability, and functional rates of proteins. The relative thermodynamic contributions of confinement versus non-specific interfacial interactions (quinary interactions), however, remain poorly understood. We are using reverse micelles (RMs) as a tool to differentiate between these effects. RMs are spontaneously organizing nanopools of water stabilized by surfactants in bulk non-polar solvent. By using optimized surfactant mixtures, the native states of proteins can be maintained in the RM water core. We have executed folding studies on a wide variety of proteins using varied surfactant mixtures with uniform degrees of confinement. We investigate both equilibrium folding thermodynamics and folding kinetics using various spectroscopic approaches. Our findings begin to unveil the complex relationship between a protein's thermodynamic stability and its local solvating environment, helping to dissect the differential impacts of confinement and quinary interactions.

Imaging of interactions of biomolecules with nanomaterials with interferometric scattering microscopy.

Interferometric scattering microscopy (iSCAT) is a label-free imaging technique that achieves single-molecule spatial and milli-second temporal resolution. In this technique, we use the combination of reflected and scattered light of the nanoparticle itself as our signal obviating the need of a label or tags as commonly used in fluorescence microscopy. With iSCAT, photobleaching is not an issue as the signal donor (the particle of interest) provides an infinite budget of scattered photons and hence indefinite imaging times. We apply this method to image and track biomolecules, such as DNA and proteins, without any fluorescent tags in their native state for upwards of seconds. We characterize and tracked the motion of DNA from the initial attachment, diffusion along the surface and finally detachment with single-molecule resolution. This allows us to image the nanoscopic interactions of native DNA with different 2D nanomaterials such as graphene and hBN. Together with other surface characterization techniques such as AFM, we identify sites of increased trapping potential and elucidate the nanoscopic mechanism behind the interaction between biomolecules and the 2D material surface. We envision that our studies allow us to inform on future biosensor design with engineered surface interactions.

HDX-MS guided ensemble reweighting approach reveals cryptic drug binding sites in the cytoplasmic heme binding protein, PhuS.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a valuable approach to probe protein structure and dynamics. However, interpretation of HDX-MS data is often qualitative and typically limited to peptide-level resolution. Ongoing efforts aim to integrate experimental HDX-MS data with molecular dynamics (MD) approaches, to provide a high-resolution interpretation of the HDX-MS data. One such approach is an HDX-MS based maximum-entropy reweighting approach (HDXer) to reweight computationally generated ensembles to HDX-MS data. Here, HDXer is used in conjunction with enhanced MD (eMD) to characterize the structural dynamics of PhuS. PhuS is a cytoplasmic heme binding protein from P. aeruginosa which shuttles exogenous heme to Heme Oxygenase (HemO) for degradation and is essential for virulence. Through the implementation of a workflow that integrates eMD, HDXer, and dimensional reduction, we had previously modeled the native state ensemble of apo-PhuS revealing a locally diverse apo-PhuS ensemble characterized by large rearrangements of helices surrounding the heme binding. Here, the representative structures from the modeled native ensemble were used as starting points for computer aided drug design (CADD). Implementing the Site Identification by Ligand Competitive Saturation (SILCS) CADD method approximately 1.3 million compounds were screened for each representative structure as well as the published apo PhuS crystal structure, yielding a library of 50 chemically representative candidate compounds. The candidate compounds were then screened using a fluorescence quenching assay. From these assays, several hits were confirmed to bind apo PhuS at nanomolar binding affinities. Most significantly, none of the compounds were derived from CADD using the published apo PhuS crystal structure, demonstrating the utility of HDX-MS guiding ensemble reweighting in revealing cryptic drug binding site in CADD approaches.

Probing the Energy Landscape of Spectrin R15 and R16 and the Effects of Non-native Interactions.

Understanding the details of a protein folding mechanism can be a challenging and complex task. One system with an interesting folding behavior is the α-spectrin domain, where the R15 folds three-orders of magnitude faster than its homologues R16 and R17, despite having similar structures. The molecular origins that explain these folding rate differences remain unclear, but our previous work revealed that a combined effect produced by non-native interactions could be a reasonable cause for these differences. In this study, we explore further the folding process by identifying the molecular paths, metastable states, and the collective motions that lead these unfolded proteins to their native state conformation. Our results uncovered the differences between the folding pathways for the wild-type R15 and R16 and an R16 mutant. The metastable ensembles that speed down the folding were identified using an energy landscape visualization method (ELViM). These ensembles correspond to similar experimentally reported configurations. Our observations indicate that the non-native interactions are also associated with secondary structure misdocking. This computational methodology can be used as a fast, straightforward protocol for shedding light on systems with unclear folding or conformational traps.

Pre-Molten, Wet, and Dry Molten Globules en Route to the Functional State of Proteins.

Transitions between the unfolded and native states of the ordered globular proteins are accompanied by the accumulation of several intermediates, such as pre-molten globules, wet molten globules, and dry molten globules. Structurally equivalent conformations can serve as native functional states of intrinsically disordered proteins. This overview captures the characteristics and importance of these molten globules in both structured and intrinsically disordered proteins. It also discusses examples of engineered molten globules. The formation of these intermediates under conditions of macromolecular crowding and their interactions with nanomaterials are also reviewed.

Differential effects of DTT on HEWL amyloid fibrillation and fibril morphology at different pH

Proteins can transform from their native state to a state having fibrillar aggregates characterized by cross β sheet structure. The fibrillar aggregates are known as amyloid and have been linked to several disorders. Disulfide bonds in proteins are one of the important factors that determine the propensity of aggregation. Hen Egg White Lysozyme (HEWL) was used by us as a model protein to decipher the role disulfide bonds play in the amyloid fibril formation and fibril morphology by using Dithiothreitol (DTT) as reducing agent at pH 2.7 and pH 7.4. We found that DTT can have different effects on HEWL amyloid depending on pH and the buffer used for preparing the amyloid fibrils. Our studies highlight the critical role of non-native disulfide bonds in amyloidogenesis and how disruption of these bonds can greatly affect the fibrillation process. Overall, these studies throw light on the fibrillation mechanism and can be explored further in designing effective inhibitors against amyloidosis.

History of an observatory on the Agasthiyar hill top

Remarkable feats of the past often leave behind historical pointers leaving us mesmerised. One such episode is that of the establishment of a magnetic and meteorological observatory on the peak of the Agasthiyar mountain 6200 feet above the sea level on the western Ghats in the year 1855 by John Allan Broun under the princely state of Travancore. It had facilities far ahead of its times representing the commitment to scientific universalism by its patrons. Broun established the observatory on the Agasthiyar mountain peak and undertook a meticulous study overcoming daunting geographical constrains. Broun was awarded the Keith Medal from the Royal Society of Edinburgh for the period 1859–1861. He received the Royal Medal from the Royal Society of London in 1878. He passed away in 1879. Remnants of the magnetic observatory which earned Broun scientific acclaim remain undiscovered, clouded within the Agasthiyar mountains.

Ageing-induced shrinkage of intervessel pit membranes in xylem of Clematis vitalba modifies its mechanical properties as revealed by atomic force microscopy.

Bordered pit membranes of angiosperm xylem are anisotropic, mesoporous media between neighbouring conduits, with a key role in long distance water transport. Yet, their mechanical properties are poorly understood. Here, we aim to quantify the stiffness of intervessel pit membranes over various growing seasons. By applying an AFM-based indentation technique "Quantitative Imaging" we measured the effective elastic modulus (E effective) of intervessel pit membranes of Clematis vitalba in dependence of size, age, and hydration state. The indentation-deformation behaviour was analysed with a non-linear membrane model, and paired with magnetic resonance imaging to visualise sap-filled and embolised vessels, while geometrical data of bordered pits were obtained using electron microscopy. E effective was transformed to the geometrically independent apparent elastic modulus E apparent and to aspiration pressure P b. The material stiffness (E apparent) of fresh pit membranes was with 57 MPa considerably lower than previously suggested. The estimated pressure for pit membrane aspiration was 2.20+28 MPa. Pit membranes from older growth rings were shrunken, had a higher material stiffness and a lower aspiration pressure than current year ones, suggesting an irreversible, mechanical ageing process. This study provides an experimental-stiffness analysis of hydrated intervessel pit membranes in their native state. The estimated aspiration pressure suggests that membranes are not deflected under normal field conditions. Although absolute values should be interpreted carefully, our data suggest that pit membrane shrinkage implies increasing material stiffness, and highlight the dynamic changes of pit membrane mechanics and their complex, functional behaviour for fluid transport.

A protein engineering approach towards understanding FKBP51 conformational dynamics and mechanisms of ligand binding.

Most proteins are flexible molecules that coexist in an ensemble of several conformations. Point mutations in the amino acid sequence of a protein can trigger structural changes that drive the protein population to a conformation distinct from the native state. Here, we report a protein engineering approach to better understand protein dynamics and ligand binding of the FK506-binding protein 51 (FKBP51), a prospective target for stress-related diseases, metabolic disorders, some types of cancers and chronic pain. By randomizing selected regions of its ligand-binding domain and sorting yeast display libraries expressing these variants, mutants with high affinity to conformation-specific FKBP51 selective ligands were identified. These improved mutants are valuable tools for the discovery of novel selective ligands that preferentially and specifically bind the FKBP51 active site in its open conformation state. Moreover, they will help us understand the conformational dynamics and ligand binding mechanics of the FKBP51 binding pocket.

Functional Analyses of Rare Germline Missense BRCA1 Variants Located within and outside Protein Domains with Known Functions.

The BRCA1 protein is implicated in numerous important cellular processes to prevent genomic instability and tumorigenesis, and pathogenic germline variants predispose carriers to hereditary breast and ovarian cancer (HBOC). Most functional studies of missense variants in BRCA1 focus on variants located within the Really Interesting New Gene (RING), coiled-coil and BRCA1 C-terminal (BRCT) domains, and several missense variants in these regions have been shown to be pathogenic. However, the majority of these studies focus on domain specific assays, and have been performed using isolated protein domains and not the full-length BRCA1 protein. Furthermore, it has been suggested that BRCA1 missense variants located outside domains with known function are of no functional importance, and could be classified as (likely) benign. However, very little is known about the role of the regions outside the well-established domains of BRCA1, and only a few functional studies of missense variants located within these regions have been published. In this study, we have, therefore, functionally evaluated the effect of 14 rare BRCA1 missense variants considered to be of uncertain clinical significance, of which 13 are located outside the well-established domains and one within the RING domain. In order to investigate the hypothesis stating that most BRCA1 variants located outside the known protein domains are benign and of no functional importance, multiple protein assays including protein expression and stability, subcellular localisation and protein interactions have been performed, utilising the full-length protein to better mimic the native state of the protein. Two variants located outside the known domains (p.Met297Val and p.Asp1152Asn) and one variant within the RING domain (p.Leu52Phe) were found to make the BRCA1 protein more prone to proteasome-mediated degradation. In addition, two variants (p.Leu1439Phe and p.Gly890Arg) also located outside known domains were found to have reduced protein stability compared to the wild type protein. These findings indicate that variants located outside the RING, BRCT and coiled-coiled domains could also affect the BRCA1 protein function. For the nine remaining variants, no significant effects on BRCA1 protein functions were observed. Based on this, a reclassification of seven variants from VUS to likely benign could be suggested.

Nicked tRNAs are stable reservoirs of tRNA halves in cells and biofluids

Nonvesicular extracellular RNAs (nv-exRNAs) constitute the majority of the extracellular RNAome, but little is known about their stability, function, and potential use as disease biomarkers. Herein, we measured the stability of several naked RNAs when incubated in human serum, urine, and cerebrospinal fluid (CSF). We identified extracellularly produced tRNA-derived small RNAs (tDRs) with half-lives of several hours in CSF. Contrary to widespread assumptions, these intrinsically stable small RNAs are full-length tRNAs containing broken phosphodiester bonds (i.e., nicked tRNAs). Standard molecular biology protocols, including phenol-based RNA extraction and heat, induce the artifactual denaturation of nicked tRNAs and the consequent invitro production of tDRs. Broken bonds are roadblocks for reverse transcriptases, preventing amplification and/or sequencing of nicked tRNAs in their native state. To solve this, we performed enzymatic repair of nicked tRNAs purified under native conditions, harnessing the intrinsic activity of phage and bacterial tRNA repair systems. Enzymatic repair regenerated an RNase R-resistant tRNA-sized band in northern blot and enabled RT-PCR amplification of full-length tRNAs. We also separated nicked tRNAs from tDRs by chromatographic methods under native conditions, identifying nicked tRNAs inside stressed cells and in vesicle-depleted human biofluids. Dissociation of nicked tRNAs produces single-stranded tDRs that can be spontaneously taken up by human epithelial cells, positioning stable nv-exRNAs as potentially relevant players in intercellular communication pathways.

Enrichment cultivation of VOC-degrading bacteria using diffusion bioreactor and development of bacterial-immobilized biochar for VOC bioremediation

Volatile organic compounds (VOCs) have been globally reported at various sites. Currently, limited literature is available on VOC bioremediation using bacterial-immobilized biochar (BC–B). In this study, multiple VOC-degrading bacteria were enriched and isolated using a newly designed diffusion bioreactor. The most effective VOC-degrading bacteria were then immobilized on rice husk-derived pristine biochar (BC) to develop BC-B. Finally, the performances of BC and BC-B for VOCs (benzene, toluene, xylene, and trichloroethane) bioremediation were evaluated by establishing batch microcosm experiments (Control, C; bioconsortium, BS; pristine biochar, BC; and bacterial-immobilized biochar, BC-B). The results revealed that the newly designed diffusion bioreactor effectively simulated native VOC-contaminated conditions, easing the isolation of 38 diverse ranges of VOC-degrading bacterial strains. Members of the genus Pseudomonas were isolated in the highest (26.33%). The most effective bacterial strain was Pseudomonas sp. DKR-23, followed by Rhodococcus sp. Korf-18, which degraded multiple VOCs in the range of 52–75%. The batch microcosm experiment data showed that BC-B remediated the highest >90% of various VOCs, which was comparatively higher than that of BC, BS, and C. In addition, compared with C, the BS, BC, and BC-B microcosms abundantly reduced the half-life of various VOCs, implying a beneficial impact on the degradation behavior of VOCs. Altogether, this study suggests that a diffusion bioreactor system can be used as a cultivation device for the isolation of a wide range of VOC-degrading bacterial strains, and a compatible combination of biochar and bacteria may be an attractive and promising approach for the sustainable bioremediation of multiple VOCs.

NF-YB family transcription factors in Arabidopsis: Structure, phylogeny, and expression analysis in biotic and abiotic stresses.

Nuclear factor-Y (NF-Y) transcription factors (TFs) are conserved heterotrimeric complexes present and widespread across eukaryotes. Three main subunits make up the structural and functional aspect of the NF-Y TFs: NF-YA, NF-YB and NF-YC, which bind to the conserved CCAAT- box of the promoter region of specific genes, while also interacting with each other, thereby forming myriad combinations. The NF-YBs are expressed differentially in various tissues and plant development stages, likely impacting many of the cellular processes constitutively and under stress conditions. In this study, ten members of NF-YB family from Arabidopsis thaliana were identified and expression profiles were mined from microarray data under different biotic and abiotic conditions, revealing key insights into the involvement of this class of proteins in the cellular and biological processes in Arabidopsis. Analysis of cis-acting regulatory elements (CAREs) indicated the presence of abiotic and biotic stress-related transcription factor binding sites (TFBs), shedding light on the multifaceted roles of these TFs. Microarray data analysis inferred distinct patterns of expression in various tissues under differing treatments such as drought, cold and heat stress as well as bacterial, fungal, and viral stress, indicating their likelihood of having an expansive range of regulatory functions under native and stressed conditions; while quantitative real-time PCR (qRT-PCR) based expression analysis revealed that these TFs get real-time-modulated in a stress dependent manner. This study, overall, provides an understanding of the AtNF-YB family of TFs in their regulation and participation in various morphogenetic and defense- related pathways and can provide insights for development of transgenic plants for trait dependent studies.

Characterization of the aroma profile of quinoa (Chenopodium quinoa Willd.) and assessment of the impact of malting on the odor-active volatile composition.

Quinoa (Chenopodium quinoa Willd.) is a gluten-free pseudocereal, rich in starch and high-quality proteins. It can be used as a cereal. Recently, a variety of nontraditional food products were developed; however, the sharp bitterness and the earthy aroma of unprocessed quinoa interfered with the acceptance of these products. Malting of cereals is known to improve their processing properties and enhance their sensory quality. To evaluate the acceptance and potential of quinoa malt as a raw material for beverage production, malt quality indicators (e.g., soluble protein) and the aroma profiles of different quinoa malts were compared. Initial sensory assessment of quinoa in its native and malted state identified differences in their aroma profiles and revealed that pleasant nutty and caramel aromas were formed by malting. Subsequently, three complementary isolation techniques and gas chromatography-olfactometry/mass spectrometry (GC-O/MS) were used for volatile analysis. Instrumental analysis detected 34 and 62 odor-active regions in native quinoa and quinoa malt, respectively. In the second part, storage and the impact of three malting parameters on volatile formation were examined. By varying the malting parameters, seven additional odor-active malting byproducts were revealed. Three naturally occurring methoxypyrazines were identified as important contributors to the characteristic quinoa aroma. In all fresh quinoa malts a similar number of volatile compounds was perceived but their intensity and composition varied. Higher germination temperature promoted the formation of lipid oxidation products. Fatty smelling compounds and carboxylic acids, formed during storage, were classified as aging indicators of quinoa malt. © 2022 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Magnetic corner states in a two-dimensional gyromagnetic photonic crystal

Recently discovered higher-order states extensively enrich the study of topological phases, allowing one to manipulate wave propagating or localizing in more than one dimension lower than the bulk. To date, vast higher-order topological phases have been demonstrated in photonics, acoustics, mechanics, and electronics; however, these are usually in magnetic-free scenarios. Here, we report our realization of magnetic corner states in a two-dimensional honeycomb lattice gyromagnetic photonic crystal. By simultaneously breaking both time-reversal and parity symmetries with one-sublattice magnetic bias, we elaborately gap the native gapless chiral edge states of the quantum-Hall phase. In the complete band gaps, we experimentally observe a pair of unconventional corner states originating from the hybridization of two Wannier centers to shape symmetrical and antisymmetrical modes. Furthermore, we can switch on (off) the corner field localizations solely by adding (removing) the magnetic fields of the corner sites without changing other configurations.

Nanopore single-molecule biosensor in protein denaturation analysis

Unclear issues in protein studies include but not limited to the stability and denaturation mechanism in the presence of denaturants. Herein, we report a dynamic monitoring approach based on nanopore single-molecule biosensor, which can detect the protein's folding and unfolding transitions by recording a nanopore ionic current. When gradually increasing the concentration of denaturant guanidine hydrochloride (GdmCl), sensitive responses were observed with lysozyme unfolding. The emergence of the featured biphasic-pulse demonstrated the existence of a stable intermediate. It was the first time to experimentally confirm the dynamic equilibrium between the intermediate and the native states at single molecule level, therefore consolidating the standpoint of lysozyme denaturation process following the three-state model. Additionally, we got more insights into the conformation about the intermediate as globular-like structure, larger gyration radius, and enhanced positive charge density. We considered that the manner of denaturant toward lysozyme adopts the “direct” model based on stronger electrostatic and van der Waals forces. Nanopore biosensor exhibited excellent sensitivity with a low detection concentration of 280 pM and reproducibility in analysing the folding intermediate of lysozyme.

Correlated Evolution of Low-Frequency Vibrations and Function in Enzymes.

Previous studies of the flexibility of ancestral proteins suggest that proteins evolve their function by altering their native state ensemble. Here, we propose a more direct method to analyze such changes during protein evolution by comparing thermally activated vibrations at frequencies below 6 THz, which report on the dynamics of collective protein modes. We analyzed the backbone vibrational density of states of ancestral and extant β-lactamases and thioredoxins and observed marked changes in the vibrational spectrum in response to evolution. Coupled with previously observed changes in protein flexibility, the observed shifts of vibrational mode densities suggest that protein dynamics and dynamical allostery are critical factors for the evolution of enzymes with specialized catalytic and biophysical properties.

Comparative study on molecular and higher-order structures of legume seed protein isolates: Lentil, mungbean and yellow pea

Lentils and mungbean proteins are under-researched compared to pea and soybean. Lentils (green, red and black-lentils), mungbean and yellow pea protein isolates were obtained by alkaline extraction (pH 9)-isoelectric precipitation (pH 4.5) and investigated for molecular and higher-order structures using complementary and novel approaches. These extracted isolates showed comparable protein content but significantly greater nitrogen solubility index (NSI > 85 %) than commercial pea and soy protein isolates (NSI < 60 %). Based on molecular weight estimations from sodium dodecyl sulphate–polyacrylamide gel electrophoresis analysis, the soluble proteins of lentils and yellow pea were identified as legumin-like and vicilin-like, while mungbean was dominated by vicilin-like proteins. The soluble extracts were confirmed to be in native structural condition by size exclusion chromatography and nano-differential scanning calorimetry, unlike commercial extracts. Further differences in secondary structure were evident on circular dichroism spectra of the soluble extracts and deconvolution of the Amide I region (1700–1600 cm−1) from Fourier Transform Infrared of the total protein.