Secondary Structure Composition Research Articles

Mechanistic insights into combined effects of continuous microwave heating and tremella powder addition on physiochemical properties of Nemipterus virgatus surimi gel

The physicochemical properties of Nemipterus virgatus surimi gel were investigated, with tremella powder (TP) at concentrations ranging from 0 to 0.5% (w/w) combined with continuous microwave heating (CMH) using water-bath heating (WBH) as control. Results showed that TP addition (0.1%–0.3%, w/w) could significantly enhance the water holding capacity and reduce whiteness and cooking loss, attributed to the changed lateral relaxation time of water distribution. Notably, at 0.3% TP and 80 °C, the gel strength significantly increased by 96.84%, and the hardness, chewiness, and adhesiveness improved, but the quality of surimi decreased above 0.3% TP. The gel network structure was influenced by protein secondary structure composition, especially for increasing β-sheet in Raman spectra, thus promoting the gel microstructure density and uniform protein distribution. These findings offer insights for enhancing surimi gel quality and broadening tremella application in product processing.

Evaluating the 3D structure prediction tools to identify optimal MEBPVC structure models

Multi-Epitope based Peptide Vaccines Candidates (MEBPVCs) are peptide sequences with the immunogenic epitopes interspersed with linkers, adjuvants and other components. The optimization of one of the components of MEBPVCs, epitope order, resulted in a unique epitope ordered MEBPVC showing improved vaccine potency. In addition to the above combinatorial optimization task, predicting the 3D structure of these MEBPVCs presents a significant challenge owing to the diverse modeling approaches employed by the modeling tools. Here we embarked on evaluating the performance of the threading based ab initio modeling tool: I-TASSER, Large Language Model (LLM) based tool: ESMFold and the MSA based artificial intelligence based tool: AlphaFold2. Our investigation employed RMSD, PEA, SASA, Solubility, Globularity, secondary structure analysis, Docking, MDS and MVP. I-TASSER emerged as the superior performer. The analysis revealed a strong correlation between predicted tertiary structure and secondary structure composition, especially the β-turns and random coils. It appears that accurate secondary structure identification to be a pivotal step, dictating the propagation of accuracy (or inaccuracy). This finding highlights the critical importance of secondary structure prediction/assignment algorithms for enhanced protein structure modeling accuracy especially to integrate predictions where evolutionary information is minimal and or lacking. Small sample size is attributed to the low significance of the parameters though Globularity and SASA have shown good p-values. Overall, the results support the need and utilization of the LLMs in biological research, underscoring their potential to revolutionize protein structure prediction and advance diverse biomolecular applications, accelerating Synthetic Biology research.

Purification and structural analysis of tannase from novel bacteria of Bacillus cereus strain KMS3-1 isolated in marine sediment

Tannases are a family of esterases that catalyze the hydrolysis of ester and depside bonds of hydrolyzable tannins to release small compounds such as glucose. In this work, tannase production from Bacillus cereus strain KMS3-1 was purified and characterized. The standard methods, such as ammonium sulfate precipitation followed by gel filtration chromatography, were used to purify the enzyme tannase from KMS3-1. With 40% enzyme recovery, the purification process yielded a 1.93-fold increase in specific activity. Analysis using SDS-PAGE confirmed a molecular weight of about 40 kDa. Circular dichroism analysis revealed the secondary structure composition α-helix (12.03%), indicating a dominant role of β-turns (38.4%). A pH of 6.0 and a temperature between 50 and 60 °C were ideal for enzyme activity. Tannic acid has an enzyme Km value of 3.0 x 10−3 M and a Vmax of 4.45 U/mL. Surfactants, metal ions, organic solvents, and inhibitors significantly influence the enzyme activity. Cytotoxicity assays revealed the non-toxic nature of the purified enzyme on Vero cells and rat animal models. These results explore and contribute to a better understanding of tannase enzyme properties and their potential applications in the food and beverage industries.

Reference Data Set for Circular Dichroism Spectroscopy Comprised of Validated Intrinsically Disordered Protein Models.

Circular dichroism (CD) spectroscopy is an analytical technique that measures the wavelength-dependent differential absorbance of circularly polarized light and is applicable to most biologically important macromolecules, such as proteins, nucleic acids, and carbohydrates. It serves to characterize the secondary structure composition of proteins, including intrinsically disordered proteins, by analyzing their recorded spectra. Several computational tools have been developed to interpret protein CD spectra. These methods have been calibrated and tested mostly on globular proteins with well-defined structures, mainly due to the lack of reliable reference structures for disordered proteins. It is therefore still largely unclear how accurately these computational methods can determine the secondary structure composition of disordered proteins. Here, we provide such a required reference data set consisting of model structural ensembles and matching CD spectra for eight intrinsically disordered proteins. Using this set of data, we have assessed the accuracy of several published CD prediction and secondary structure estimation tools, including our own CD analysis package, SESCA. Our results show that for most of the tested methods, their accuracy for disordered proteins is generally lower than for globular proteins. In contrast, SESCA, which was developed using globular reference proteins, but was designed to be applicable to disordered proteins as well, performs similarly well for both classes of proteins. The new reference data set for disordered proteins should allow for further improvement of all published methods.

Effect of camellia oil gel on rheology, water distribution and microstructure of flour dough for crispy biscuits

This study investigated the effect of camellia oil gel on rheology, water distribution and microstructure of flour dough for crispy biscuits. Camellia oil gels, numbered 6#, 8#, and 10#, were formed by mixed plant-based wax and camellia oil (6 g/100 g, 8 g/100 g, 10 g/100 g). The results indicated that with the increase of mixed plant-based wax, the fat distribution in the dough changed from an unevenly dispersed droplet to a uniformly distributed film, and the gluten network structure changed from dense to sparse (P < 0.05). The adsorbed bound water coalesced in the multimolecular water layer of oil gel dough was reduced, but the free water was significantly higher than that of the lard added dough. The gluten matrix surface of the dough was partially exposed, the continuity between gluten protein and starch granules was broken, the gluten network structure was weakened, and the protein secondary structure composition and relative content were better than other oil samples (P < 0.05). As a result, 10# gel could be considered to replace or partially replace lard in baked goods to reduce the intake of saturated fatty acids, providing some theoretical support for the development of new plastic fats.

Refining quinoa storage stability through microwave-induced structural alterations and activity suppression of key enzymes

This study investigated the effects of microwave on preserving the quality of quinoa during storage. Quinoa treated with 9W/60s exhibited a significant decrease in fatty acid values compared to hot air treatment. Microwave effectively delayed lipid oxidation during quinoa storage by suppressing the increase in peroxide values. MDA gradually accumulated from peroxides during storage, reaching its peak at 0.423 μmol/L in the second week. Microwave disrupted the original hydrogen bonds in lipase, causing the unwinding of the α-helix and resulting in the loss of its regular structure. Microwave reduced the stability of the β-sheet structure in lipoxygenase, breaking the natural secondary structure composition. The observed fluorescence and UV spectra features were similar, indicating that microwave alter the peptide chain of the enzyme's skeletal structure, increasing the exposure of hydrophobic chromophores. These results indicated the potential of microwave to enhance the stability of quinoa during storage.

Surface-mediated spontaneous emulsification of the acylated peptide semaglutide

Acylated peptides composed of glucagon-like peptide-1 receptor agonists modified with a fatty acid side chain are an important class of therapeutics for type 2 diabetes and obesity but are susceptible to an unusual physical instability in the presence of hydrophobic surfaces, i.e., spontaneous emulsification, also known as ouzo formation in practice. In this work, light scattering, small-angle X-ray scattering, and circular dichroism measurements are used to characterize the physical properties of the semaglutide colloidal phase, including size distribution, shape, secondary structure, internal structure, and internal composition, as a function of solution physico-chemical conditions. The existence and size of the colloids formed are successfully predicted by a classical Rayleigh model, which identifies the parameters controlling their size and formation. Colloid formation is found to be catalyzed by hydrophobic surfaces, and formation rates are modeled as an autocatalytic reaction, enabling the formation of a master curve for various surfaces that elucidates the mechanism. Surfaces differ due to differences in surface wettability, which can be correlated with Hansen solubility parameters. This work provides insights into this unusual colloidal phenomenon and guides the peptide synthesis process and drug product formulation in the pharmaceutical industry.

HrpY protein of Ralstonia solanacearum exhibits spontaneous formation of pilus like assembly: analysis of its stability

Type 3 secretory system (T3SS), a complex protein machinery has a unique virulence mechanism that involves injecting effector proteins directly into host cells. The T3SS effector proteins are transported through an extracellular long hollow needle made up of multiple copies of a small protein. In T3SS of the plant pathogen Ralstonia solanacearum, the 8.6 kDa HrpY protein assembles into a large needle like apparatus (pilus) for transporting effector proteins. To study structural details of HrpY, we recombinantly expressed and purified HrpY in E. coli. The dynamic light scattering (DLS) analysis showed that rHrpY has spontaneously formed oligomers of large order (>100 nm). Transmission electron microscopy of rHrpY samples revealed that the large structures are tube like assembly having dimensions 86.3–166.6 nm and 5.8–6.8 nm in length and width respectively. Different molecular sizes of the purified rHrpY hindered the crystallization of the protein. The stability of oligomer assembly was studied with denaturants and surfactants. Denaturants like urea and guanidine HCl could not break them apart; however, detergents like SDS, sarkosyl, Octyl-β-Glucoside, CHAPS, Tween 20, Tween 80 and Triton X-100 showed disassembly of the oligomer. rHrpY assembly was found to withstand up to 50 °C and the circular dichroism analysis revealed that there is no significant change in the secondary structural composition with increase in temperature. However, change in the secondary structure was observed with the addition of SDS. Communicated by Ramaswamy H. Sarma

Recombinant Full-Length TDP-43 Oligomers Retain Their Ability to Bind RNAs, Are Not Toxic, and Do Not Seed TDP-43 Aggregation in Vitro.

TAR DNA-binding protein with 43 kD (TDP-43) is a partially disordered protein that misfolds and accumulates in the brains of patients affected by several neurodegenerative diseases. TDP-43 oligomers have been reported to form due to aberrant misfolding or self-assembly of TDP-43 monomers. However, very little is known about the molecular and structural basis of TDP-43 oligomerization and the toxic properties of TDP-43 oligomers due to several reasons, including the lack of conditions available for isolating native TDP-43 oligomers or producing pure TDP-43 oligomers in sufficient quantities for biophysical, cellular, and in vivo studies. To address these challenges, we developed new protocols to generate different stable forms of unmodified and small-molecule-induced TDP-43 oligomers. Our results showed that co-incubation of TDP-43 with small molecules, such as epigallocatechin gallate (EGCG), dopamine, and 4-hydroxynonenal (4-HNE), increased the production yield of TDP-43 stable oligomers, which could be purified by size-exclusion chromatography. Interestingly, despite significant differences in the morphology and size distribution of the TDP-43 oligomer preparations revealed by transmission electron microscopy (TEM) and dynamic light scattering (DLS), they all retained the ability to bind to nucleotide DNA. Besides, circular dichroism (CD) analysis of these oligomers did not show much difference in the secondary structure composition. Surprisingly, none of these oligomer preparations could seed the aggregation of TDP-43 core peptide 279-360. Finally, we showed that all four types of TDP-43 oligomers exert very mild cytotoxicity to primary neurons. Collectively, our results suggest that functional TDP-43 oligomers can be selectively stabilized by small-molecule compounds. This strategy may offer a new approach to halt TDP-43 aggregation in various proteinopathies.

Uninterrupted control of coating thickness during the wear process of vehicle units

In the course of this research in the field of automotive technology, systematic patterns and features of wear of structural materials with wear-resistant coatings that were applied using various technologies, depending on the change in friction modes, were revealed. Our research led to the identification of physical parameters of tribological properties and their changes in the friction process.&#x0D; An interesting property discovered in the course of our work is the fact that changing the contact area several times does not significantly affect the change in the friction coefficient. Instead, changes in the coefficient of friction are mainly related to the chemical composition of the secondary structures that are formed during friction.&#x0D; An important aspect of our findings is that the change in the coefficient of friction is usually due to the chemical composition of the secondary structures, which in turn depend on the chemical composition of the base material, the characteristics of the coating, the characteristics of the environment and the temperature conditions in the friction zone. An analysis of the chemical composition of secondary structures formed during friction was carried out, which allows for a deeper understanding of wear mechanisms and creates opportunities for optimizing materials and coatings in the field of automotive technology.

IDPpred: a new sequence-based predictor for identification of intrinsically disordered protein with enhanced accuracy

Discovery of intrinsically disordered proteins (IDPs) and protein hybrids that contain both intrinsically disordered protein regions (IDPRs) along with ordered regions has changed the sequence–structure–function paradigm of protein. These proteins with lack of persistently fixed structure are often found in all organisms and play vital roles in various biological processes. Some of them are considered as potential drug targets due to their overrepresentation in pathophysiological processes. The major bottlenecks for characterizing such proteins are their occasional overexpression, difficulty in getting purified homogeneous form and the challenge of investigating them experimentally. Sequence-based prediction of intrinsic disorder remains a useful strategy especially for many large-scale proteomic investigations. However, worst accuracy still occurs for short disordered regions with less than ten residues, for the residues close to order–disorder boundaries, for regions that undergo coupled folding and binding in presence of partner, and for prediction of fully disordered proteins. Annotation of fully disordered proteins mostly relies on the far-UV circular dichroism experiment which gives overall secondary structure composition without residue-level resolution. Current methods including that using secondary structure information failed to predict half of target IDPs correctly in the recent Critical Assessment of protein Intrinsic Disorder prediction (CAID) experiment. This study utilized profiles of random sequential appearance of physicochemical properties of amino acids and random sequential appearance of order and disorder promoting amino acids in protein together with the existing CIDER feature for the prediction of IDP from sequence input. Our method was found to significantly outperform the existing predictors across different datasets.Communicated by Ramaswamy H. Sarma

Identification of a cryptic functional apolipophorin-III domain within the Prominin-1 gene of Litopenaeus vannamei

The Apolipophorin-III (apoLp-III) is reported as an essential protein element in lipids transport and incorporation in lepidopterans. Structurally, apoLp-III has an α-helix bundle structure composed of five α-helices. Interestingly, classic studies proposed a structural switch triggered by its interaction with lipids, where the α-helix bundle opens. Currently, the study of the apoLp-III has been limited to insects, with no homologs identified in other arthropods. By implementing a structure-based search with the Phyre2 algorithm surveying the shrimp Litopenaeus vannamei's transcriptome, we identified a putative apoLp-III in this farmed penaeid (LvApoLp-III). Unlike canonical apoLp-III, the LvApoLp-III was identified as an internal domain within the transmembrane protein Prominin-1. Structural modeling using the template-based Phyre2 and template-free AlphaFold algorithms rendered two distinct structural topologies: the α-helix bundle and a coiled-coil structure. Notably, the secondary structure composition on both models was alike, with differences in the orientation and distribution of the α-helices and hydrophobic moieties. Both models provide insights into the classical structural switch induced by lipids in apoLp-III. To corroborate structure/function inferences, we cloned the synthetic LvApoLp-III domain, overexpressed, and purified the recombinant protein. Circular dichroism measurements with the recombinant LvApoLp-III agreed with the structural models. In vitro liposome interaction demonstrated that the apoLp-III domain within the PROM1 of L.vannamei associated similarly to exchangeable apolipoproteins. Altogether, this work reports the presence of an apolipophorin-III domain in crustaceans for the first time and opens questions regarding its function and importance in lipid metabolism or the immune system.

Amyloid models for quantitative x‐ray brain amyloid imaging

AbstractBackgroundA range of human neurological disorders are related to amyloid fibrillar deposits including Alzheimer’s disease (AD). AD is linked to the presence of amyloid‐β (Aβ) fibrils and those are comprised of β‐sheet motifs. The precise mechanism through which Aβ leads to AD are not completely understood. The scarcity of human derived Aβ fibrils and high cost of recombinant Aβ fibrils further restrict the research in this area. However, intrinsically disordered proteins (IDPs) have been reported as amyloid model proteins. Even though, these models qualitatively mimic β‐sheet structure of Aβ fibrils but often fail to quantitatively match the β‐sheet content. Therefore, this study aimed to develop model proteins to closely mimic Aβ fibrils structure and produce them in a large quantity to evaluate emerging X‐ray methods to detect and quantify amyloid plaques.MethodsWe used a commercially available β‐lactoglobulin A and B genetic mixture (LGAB) protein as our amyloid model to mimic the structural properties of Aβ fibrils. Here, the alteration in the protein conformation was induced by altering the net charge on the polypeptide chain by changing the pH from 2 to 7. Additionally, we studied the fibrillization of these proteins in presence of more cooperative transitions at 90°C. The secondary structural compositions of LGAB were studied using attenuated total reflectance Fourier transform infrared (ATR‐FTIR) spectroscopy. The nanostructures of LGAB fibrils were characterized using small angle X‐ray scattering (SAXS). We further varied the mass fraction of LGAB fibrils (amyloid model) and quantitatively measured their amyloid content using a SAXS based method.ResultsATR‐FTIR (Fig.1) and SAXS (Fig.2) studies confirm that thermally aggregated LGAB fibrils exhibited parallel β‐sheet secondary structure similar to Aβ fibrils. Systematic variation in LGAB fibril concentration in the tissue mimicking environment showed a linear correlation between protein mass fraction and area under the curve of a peak (∼ 6 nm−1) corresponding to inter β‐sheet spacing (Fig.3).ConclusionOur study suggests that the amyloid model based on LGAB fibrils can likely be used to simulate Aβ fibrils in phantom studies for the evaluation of emerging X‐ray methods to detect and quantify amyloid plaques.

Characteristics and molecular properties of crude hemeproteins extracted from Asian seabass gills using an ultrasound-assisted process.

The development of a safe and effective iron supplement is important for the treatment of iron-deficient anemia. Therefore, the crude hemeprotein extract (CHPE) from Asian seabass gills wasextracted without (CON) and with ultrasound (US)-assisted process, followed by freeze-drying. The resulting freeze-dried crude hemeprotein extract (FDCHPE) powders were determined for trace mineral content, color, secondary structure, protein pattern, size distribution, volatile compounds, and amino acid composition. The extraction yields of CON-FDCHPE and US-FDCHPE were 6.76% and 13.65%, respectively. Highest heme iron (0.485 mg/mL) and non-heme iron (0.023 mg/mL) contents were found when US at 70% amplitude for 10 min (US 70/10) was applied. Both CON-FDCHPE and US-FDCHPE had no heavy metals, but higher iron content (432.8 mg/kg) was found in US-FDCHPE (P < 0.05). Typical red color was observed in CON-FDCHPE and US-FDCHPE with a*-values of 9.72 and 10.60, respectively. Ultrasonication affected protein structure, in which β-sheet upsurged, whereas random coil, α-helix, and β-turn were reduced. Protein pattern confirmed that both samples had myoglobin as the major protein. US-FDCHPE also showed a higher abundance of volatile compounds, especially propanal, hexanal, heptanal, and so forth, compared to CON-FDCHPE. Amino acid composition of US-FDCHPE was comparable to Food and Agriculture Organization of the United Nations (FAO) values. Overall, FDCHPE extracted using ultrasonication could be safe and effective for fortification in food products as an iron supplement to alleviate iron-deficient anemia. Additionally, gills as leftovers could be better exploited rather than being disposed. © 2023 Society of Chemical Industry.

Adsorption behavior of serum proteins on anodized titanium is driven by surface nanomorphology.

Protein adsorption behavior can play a critical role in defining the outcome of a material by affecting the subsequent in vivo response to it. To date, the effect of surface properties on protein adsorption behavior has been mainly focused on surface chemistry, but research on the effect of nanoscale surface topography remains limited. In this study, the adsorption behavior of human serum albumin, immunoglobulin G, and fibrinogen in terms of the adsorbed amount and conformational changes were investigated on bare and anodized titanium (Ti) samples (40 and 60 V applied voltages). While the surface chemistry, RMS surface roughness, and arithmetic surface roughness of the anodized samples were similar, they had distinctly different nanomorphologies identified by atomic force microscopy and scanning electron microscopy, and the surface statistical parameters, surface skewness Ssk and kurtosis Sku. The Feret pore size distribution was more uniform on the 60 V sample, and surface nanostructures were more symmetrical with higher peaks and deeper pores. On the other hand, the 40 V sample surface presented a nonuniform pore size distribution and asymmetrical surface nanostructures with lower peaks and shallower pores. The amount of surface-adsorbed protein increased on the sample surfaces in the order of Ti < 40 V < 60 V with the predominant factor affecting the amount of surface-adsorbed protein being the increased surface area attained by pore formation. The secondary structure of all adsorbed proteins deviated from that of their native counterparts. While comparing the secondary structure components of proteins on anodized surfaces, it was observed that all three proteins retained more of their secondary structure composition on the surface with more uniform and symmetrical nanofeatures than the surface having asymmetrical nanostructures. Our results suggest that the nanomorphology of the peaks and outer walls of the nanotubes can significantly influence the conformation of adsorbed serum proteins, even for surfaces having similar roughness values.

Effects of walnut seed coat polyphenols on walnut protein hydrolysates: Structural alterations, hydrolysis efficiency, and acetylcholinesterase inhibitory capacity

The walnut meal is rich in nutrients such as protein from the kernel and polyphenolic compounds from the seed coat. However, the influences of seed coat polyphenols on walnut protein (WP) hydrolysis remained unclear. In this study, our findings indicated that polyphenols induced alterations in the secondary structure and amino acid composition of WP. These changes resulted in both a hindrance of hydrolysis and an enhancement of acetylcholinesterase (AChE) inhibition. Furthermore, four peptides of 119 identified peptides (LR, SF, FQ, and FR) were synthesized based on higher predicted bioactivity and Vinascores in silico. Among them, FQ showed interaction with amino acid residues in AChE through the formation of four π-π stacking bonds and two hydrogen bonds, resulting in the highest AChE inhibitory capacity. The combination index showed that chlorogenic acid derived from the seed coat and FQ at the molar ratio of 1:4 exhibited synergistic effects of AChE inhibition.

Slow-digestive yeast protein concentrate: An investigation of its in vitro digestibility and digestion behavior

Yeast protein concentrate, a by-product of the fermentation industry waste, is a potential alternative protein source with high nutritional quality, environmental sustainability, and functional properties. However, its digestibility and digestion behavior are poorly understood. In this study, we compared the in vitro digestion behavior of yeast protein concentrate and whey protein concentrate using simulated gastrointestinal conditions. We found that yeast protein concentrate had lower digestibility than whey protein concentrate (31.25% vs. 86.23% at 120 min of pepsin digestion and 75.12% vs. 95.2% at 120 min of pancreatin digestion). Yeast protein concentrate differed from whey protein concentrate in microstructure, secondary structure, and amino acid composition, which may affect its digestion behavior. Compared to whey protein concentrate, a higher level of β-sheets and a lower zeta potential explain the slow-digesting property of yeast protein concentrate. Yeast protein concentrate also underwent depolymerization and Plastein reaction during digestion. These results provided valuable information for developing and applying yeast protein concentrate as an alternative to conventional animal protein.

The Prenucleation Equilibrium of the Parathyroid Hormone Determines the Critical Aggregation Concentration and Amyloid Fibril Nucleation.

Nucleation and growth of amyloid fibrils were found to only occur in supersaturated solutions above a critical concentration (ccrit ). The biophysical meaning of ccrit remained mostly obscure, since typical low values of ccrit in the sub-μM range hamper investigations of potential oligomeric states and their structure. Here, we investigate the parathyroid hormone PTH84 as an example of a functional amyloid fibril forming peptide with a comparably high ccrit of 67±21 μM. We describe a complex concentration dependent prenucleation ensemble of oligomers of different sizes and secondary structure compositions and highlight the occurrence of a trimer and tetramer at ccrit as possible precursors for primary fibril nucleation. Furthermore, the soluble state found in equilibrium with fibrils adopts to the prenucleation state present at ccrit . Our study sheds light onto early events of amyloid formation directly related to the critical concentration and underlines oligomer formation as a key feature of fibril nucleation. Our results contribute to a deeper understanding of the determinants of supersaturated peptide solutions. In the current study we present a biophysical approach to investigate ccrit of amyloid fibril formation of PTH84 in terms of secondary structure, cluster size and residue resolved intermolecular interactions during oligomer formation. Throughout the investigated range of concentrations (1 μM to 500 μM) we found different states of oligomerization with varying ability to contribute to primary fibril nucleation and with a concentration dependent equilibrium. In this context, we identified the previously described ccrit of PTH84 to mark a minimum concentration for the formation of homo-trimers/tetramers. These investigations allowed us to characterize molecular interactions of various oligomeric states that are further converted into elongation competent fibril nuclei during the lag phase of a functional amyloid forming peptide.

HIV Protease Hinge Region Insertions at Codon 38 Affect Enzyme Kinetics, Conformational Stability and Dynamics.

HIV-1 protease is essential for the production of mature, infectious virions and is a major target in antiretroviral therapy. We successfully purified a HIV-1 subtype C variant, L38↑N↑L- 4, containing an insertion of asparagine and leucine at position 38 without the four background mutations - K20R, E35D, R57K, V82I using a modified purification protocol. Isothermal titration calorimetry indicated that 50% of the variant protease sample was in the active conformation compared to 62% of the wild type protease. The secondary structure composition of the variant protease was unaffected by the double insertion. The specific activity and kcat values of the variant protease were approximately 50% lower than the wild type protease values. The variant protease also exhibited a 1.6-fold increase in kcat/KM when compared to the wild type protease. Differential scanning calorimetry showed a 5°C increase in Tm of the variant protease, indicating the variant was more stable than the wild type. Molecular dynamics simulations indicated the variant was more stable and compact than the wild type protease. A 3-4% increase in the flexibility of the hinge regions of the variant protease was observed. In addition, increased flexibility of the flaps, cantilever and fulcrum regions of the variant protease B chain was observed. The variant protease sampled only the closed flap conformation indicating a potential mechanism for drug resistance. The present study highlights the direct impact of a double amino acid insertion in hinge region on enzyme kinetics, conformational stability and dynamics of an HIV-1 subtype C variant protease.

Characteristics of N-Glycosylation and Its Impact on the Molecular Behavior of Lupinus angustifolius γ-Conglutin.

γ-Conglutin, a lupin seed protein, is an intriguing protein both in terms of the complexity of its molecular structure and a broad spectrum of unique health-promoting properties manifested in animal and human trials. Moreover, this protein is an evolutionary cornerstone whose physiological significance for the plant has not been determined yet. Herein, a comprehensive characterization of γ-conglutin glycosylation is presented and includes (i) the identification of the N-glycan-bearing site, (ii) the qualitative and quantitative composition of glycan-building saccharides, as well as (iii) the effect of oligosaccharide removal on structural and thermal stability. The obtained results indicate the presence of glycans belonging to different classes attached to the Asn98 residue. In addition, the detachment of the oligosaccharide significantly affects secondary structure composition, which disturbs the oligomerization process. The structural changes were also reflected in biophysical parameters, i.e., at a pH value of 4.5, an increase in γ-conglutin thermal stability was observed for the deglycosylated monomeric form. Collectively, the presented results provide evidence of the high complexity of the post-translational maturation and suggest the possibility of a functional effect that glycosylation might have on γ-conglutin structure integrity.