Protein structure-function continuum model: Emerging nexuses between specificity, evolution, and structure.

The rationale for replacing the old binary of structure-function with the trinity of structure, disorder, and function has gained considerable ground in recent years. A continuum model based on the expanded form of the existing paradigm can now subsume importance of both conformational flexibility and intrinsic disorder in protein function. The disorder is actually critical for understanding the protein-protein interactions in many regulatory processes, formation of membrane-less organelles, and our revised notions of specificity as amply illustrated by moonlighting proteins. While its importance in formation of amyloids and function of prions is often discussed, the roles of intrinsic disorder in infectious diseases and protein function under extreme conditions are also becoming clear. This review is an attempt to discuss how our current understanding of protein function, specificity, and evolution fit better with the continuum model. This integration of structure and disorder under a single model may bring greater clarity in our continuing quest for understanding proteins and molecular mechanisms of their functionality.

Reexamining the diverse functions of arginine in biochemistry.

Arginine in a free-state and as part of peptides and proteins shows distinct tendency to form clusters. In free-form, it has been found useful in cryoprotection, as a drug excipient for both solid and liquid formulations, as an aggregation suppressor, and an eluent in protein chromatography. In many cases, the mechanisms by which arginine acts in all these applications is either debatable or at least continues to attract interest. It is quite possible that arginine clusters may be involved in many such applications. Furthermore, it is possible that such clusters are likely to behave as intrinsically disordered polypeptides. These considerations may help in understanding the roles of arginine in diverse applications and may even lead to better strategies for using arginine in different situations.

Biological importance of arginine: A comprehensive review of the roles in structure, disorder, and functionality of peptides and proteins.

Arginine shows Jekyll and Hyde behavior in several respects. It participates in protein folding via ionic and H-bonds and cation-pi interactions; the charge and hydrophobicity of its side chain make it a disorder-promoting amino acid. Its methylation in histones; RNA binding proteins; chaperones regulates several cellular processes. The arginine-centric modifications are important in oncogenesis and as biomarkers in several cardiovascular diseases. The cross-links involving arginine in collagen and cornea are involved in pathogenesis of tissues but have also been useful in tissue engineering and wound-dressing materials. Arginine is a part of active site of several enzymes such as GTPases, peroxidases, and sulfotransferases. Its metabolic importance is obvious as it is involved in production of urea, NO, ornithine and citrulline. It can form unusual functional structures such as molecular tweezers in vitro and sprockets which engage DNA chains as part of histones in vivo. It has been used in design of cell-penetrating peptides as drugs. Arginine has been used as an excipient in both solid and injectable drug formulations; its role in suppressing opalescence due to liquid-liquid phase separation is particularly very promising. It has been known as a suppressor of protein aggregation during protein refolding. It has proved its usefulness in protein bioseparation processes like ion-exchange, hydrophobic and affinity chromatographies. Arginine is an amino acid, whose importance in biological sciences and biotechnology continues to grow in diverse ways.

Do vaccines increase or decrease susceptibility to diseases other than those they protect against?

Contrary to the long-held belief that the effects of vaccines are specific for the disease they were created; compelling evidence has demonstrated that vaccines can exert positive or deleterious non-specific effects (NSEs). In this review, we compiled research reports from the last 40 years, which were found based on the PubMed search for the epidemiological and immunological studies on the non-specific effects (NSEs) of the most common human vaccines. Analysis of information showed that live vaccines induce positive NSEs, whereas non-live vaccines induce several negative NSEs, including increased female mortality associated with enhanced susceptibility to other infectious diseases, especially in developing countries. These negative NSEs are determined by the vaccination sequence, the antigen concentration in vaccines, the type of vaccine used (live vs. non-live), and also by repeated vaccination. We do not recommend stopping using non-live vaccines, as they have demonstrated to protect against their target disease, so the suggestion is that their detrimental NSEs can be minimized simply by changing the current vaccination sequence. High IgG4 antibody levels generated in response to repeated inoculation with mRNA COVID-19 vaccines could be associated with a higher mortality rate from unrelated diseases and infections by suppressing the immune system. Since most COVID-19 vaccinated countries are reporting high percentages of excess mortality not directly attributable to deaths from such disease, the NSEs of mRNA vaccines on overall mortality should be studied in depth.

Moonlighting enzymes: when cellular context defines specificity.

It is not often realized that the absolute protein specificity is an exception rather than a rule. Two major kinds of protein multi-specificities are promiscuity and moonlighting. This review discusses the idea of enzyme specificity and then focusses on moonlighting. Some important examples of protein moonlighting, such as crystallins, ceruloplasmin, metallothioniens, macrophage migration inhibitory factor, and enzymes of carbohydrate metabolism are discussed. How protein plasticity and intrinsic disorder enable the removing the distinction between enzymes and other biologically active proteins are outlined. Finally, information on important roles of moonlighting in human diseases is updated.

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.

Drugs, host proteins and viral proteins: how their promiscuities shape antiviral design.

The reciprocal nature of drug specificity and target specificity implies that the same is true for their respective promiscuities. Protein promiscuity has two broadly different types of footprint in drug design. The first is relaxed specificity of binding sites for substrates, inhibitors, effectors or cofactors. The second involves protein-protein interactions of regulatory processes such as signal transduction and transcription, and here protein intrinsic disorder plays an important role. Both viruses and host cells exploit intrinsic disorder for their survival, as do the design and discovery programs for antivirals. Drug action, strictly speaking, always relies upon promiscuous activity, with drug promiscuity enlarging its scope. Drug repurposing searches for additional promiscuity on the part of both the drug and the target in the host. Understanding the subtle nuances of these promiscuities is critical in the design of novel and more effective antivirals.

How Corona Formation Impacts Nanomaterials as Drug Carriers.

As drugs/drug carriers, upon encountering physiological fluids, nanoparticles adsorb biological molecules almost immediately to form a biocorona, which is often simply called a corona. Once the corona is formed, it dictates the subsequent fate of the drug nanoparticle as a therapeutic agent. Protein adsorption on micron-size or even bigger particles was originally described by the Vroman effect. It has served as a useful framework to understand the corona formation. Proteins that are irreversibly adsorbed on nanoparticles form what is called a hard corona. Beyond that is the exchangeable population of proteins, which constitute the dynamic structure called a soft corona. More than the abundance, the affinity of the proteins toward the nanoparticles decides which ones end up in the corona. For example, the more common serum albumin, which is deposited initially, is displaced by fibrinogen, which has a higher affinity for gold nanoparticles. The curvature of the particle is a crucial parameter with bigger particles generally able to bind a more diverse population of proteins from the physiological milieu. The earlier perception of the corona formation being a challenge for drug targeting, etc. has been turned into an opportunity by engineering corona to manipulate properties like circulating half-lives, capacity to evade the immune system, and targeting or even overcoming the blood-brain barrier. The most commonly used techniques for particle characterization, including dynamic light scattering (DLS), differential sedimentation centrifugation, transmission electron microscopy (TEM), and SDS-PAGE, have been adopted to study corona formation in the past. Many newer tools, for example, a combination of capillary electrophoresis with mass spectrometry, are being used to study the corona composition. The comparison of interlaboratory results is a problem because of the lack of standard protocols. This has hindered the ability to obtain more precise information about the corona composition. That, in turn, affects our prospects to use nanoparticles as drugs/drug carriers. This overview is an attempt to assess our understanding of corona formation critically and to outline the complexities involved in gaining precise information. The discussion is largely focused on findings of the last year or so.

Three phase partitioning and spectroscopic characterization of bioactive constituent from halophilic Bacillus subtilis EMB M15.

In the present study, a halophilic Bacillus subtilis subsp. spizizenii (NCBI GenBank accession number KX109607) was isolated from the Sambhar Salt Lake, Rajasthan India. This organism exhibited significance antibacterial and antifungal activity against Proteus vulgaris, Bacillus subtilis, Aspergillus niger, Rhizopus oligosporus and Penicillium chrysogenum respectively. The bioactive constituent responsible for it was extracted by three phase partitioning and purified by column chromatography. The purified compound was further characterized by FTIR-ATR, NMR and Mass spectrometry. The mass spectra show a molecular ion of m/z 301.14. The compound has very high antimicrobial activity showing 35mm zone of inhibition against Bacillus subtilis.

Protein aggregates: Forms, functions and applications.

Protein aggregation is implicated in diverse biochemical phenomena which include formation of inclusion bodies and amyloids. In recent years, inclusion bodies of many enzymes have been found to be catalytically active. Enzyme precipitates and their crystalline aggregates have found extensive applications in Biocatalysis in low water media. Protein aggregates also play a useful role in processed food. Enzymes are incorporated in detergents in the form of granulates. This review also looks at the various techniques which are used for characterizing protein aggregates.

Adding an appropriate amino acid during crosslinking results in more stable crosslinked enzyme aggregates.

Carrier free immobilization, especially crosslinked enzyme aggregates (CLEAs), has become an important design for biocatalysis in several areas. Adding amino acids during formation of CLEAs was found to give biocatalysts more stable at 55 °C and in the presence of 60% acetonitrile. The half-lives of CLEAs prepared with and without Arg addition were 21 and 15 h (subtilisin) and 4 and 1.6 h (α-chymotrypsin) at 55 °C, respectively. The corresponding half-lives during acetonitrile presence were 4.1 and 3.0 h (subtilisin) and 39 and 22 min (α-chymotrypsin), respectively. CLEAs made with Arg had higher percentages of alpha helix. CLEAs made by adding Lys, Ala, or Asp also were more stable. In the case of Thermomyces lanuginosus lipase (TLL), CLEA with Ala was even more stable than CLEA with Arg. The addition of a suitable amino acid, thus, enhances CLEA stabilities. The results are discussed in the light of earlier results on chemical modification of proteins and the observation that the Arg/Lys ratio is invariably high in the case of enzymes from thermophiles.

Lipase coated clusters of iron oxide nanoparticles for biodiesel synthesis in a solvent free medium.

Methyl or ethyl esters of long chain fatty acids are called biodiesel. Biodiesel is synthesized by the alcoholysis of oils/fats. In this work, lipase from Thermomyces lanuginosus was precipitated over the clusters of Fe3O4 nanoparticles. This biocatalyst preparation was used for obtaining biodiesel from soybean oil. After optimization of both immobilization conditions and process parameters, complete conversion to biodiesel was obtained in 3h and on lowering the enzyme amount, as little as 1.7U of lipase gave 96% conversion in 7h. The solvent free media with oil:ethanol (w/w) of 1:4 and 40°C with 2% (w/w) water along with 20% (w/w) silica (for facilitating acyl migration) were employed for reaching this high % of conversion. The biocatalyst design enables one to use a rather small amount of lipase. This should help in switching over to a biobased production of biodiesel.

Dual bioimprinting of Thermomyces lanuginosus lipase for synthesis of biodiesel.

Use of biodiesel as an alternative to non-renewable sources of energy has become an attractive option in recent years. The enzymatic synthesis of biodiesel by transesterification of fats/oils with an alcohol is a much more sustainable route than the chemical method. However, cost effectiveness of the enzymatic route is a major barrier in its commercialization. In this work, a high activity biocatalyst design of Thermomyces lanuginosus lipase is made by dually bioimprinting it with substrate and a surfactant (which is believed to open up the lid covering the active site of the lipase) during precipitation of the lipase in organic solvent. When the lipase was bioimprinted with only the surfactants, 28 U of the enzyme/g of oil could yield 99% biodiesel from soybean oil in about 4 h. However, when dually bioimprinted even very low enzyme load 1.4 U/g of oil, yielded 99% biodiesel within 48 h.

Molecular bioimprinting of lipases with surfactants and its functional consequences in low water media.

Lipases from Thermomyces lanuginosa (TLL), Candida rugosa (CRL) and Burkholderia cepacia (BCL) were obtained in the 'open lid' form by adding surfactant molecules like n-octyl-ß-d-glucopyranoside (OG), hexadecyl trimethyl ammonium bromide (CTAB), Bis(2-ethylhexyl) sulfosuccinate sodium salt (AOT) and triton X-100 for this purpose. The enzymes were 'dried' by precipitating with 4× (v/v) excess of organic solvents. The imprint surfactant molecules were removed by extensive washing with organic solvents. TLL imprinted with 0.05% CTAB showed 11-fold increase in the transesterification activity and was a better preparation to kinetically resolve (±)-1-phenylethanol. Fluorescence emission spectra confirmed that Trp89 of the lid was indeed affected during bioimprinting. With CRL, bioimprinting with OG gave 7-fold increase in the transesterification rates and resulted in reversal of enantioselectivity of CRL and gave R-phenylethyl acetate instead of the S-product as with the unimprinted precipitate. Bioimprinted BCL was also a 7-fold better catalyst for transesterification as well as enantioselectivity.

Increasing importance of protein flexibility in designing biocatalytic processes.

Enzymes require some flexibility for catalysis. Biotechnologists prefer stable enzymes but often this stabilization comes at the cost of reduced efficiency. Enzymes from thermophiles have low flexibility but poor catalytic rates. Enzymes from psychrophiles are less stable but show good catalytic rates at low temperature. In organic solvents enzymes perform poorly as the prior drying makes the enzyme molecules very rigid. Adding water or increasing reaction temperature improves flexibility and catalytic rates. In case of hydrolases, flexibility and enantioselectivity have interdependence. Understanding the complex role of protein flexibility in biocatalysis can help in designing biotechnological processes.

Preparation, characterization and biocatalytic activity of a nanoconjugate of alpha amylase and silver nanoparticles.

The primary challenge in developing nanoparticle based enzymatic devices is to be able to chemically immobilize an enzyme, which will retain its activity or improve its function while being attached to the nanoparticle. This would be of even greater significance if the whole process could be performed under benign conditions without having to resort to functionalization of key molecules at various steps. In the present study the conjugates of amylase and silver nanoparticles were synthesized using neem leaf extract as the reducing and stabilizing agent. The silver nanoparticles were characterized using Surface Plasmon Resonance Spectra, Dynamic Light Spectroscopy (DLS), Fourier Transform Infrared Spectroscopy (FTIR), Circular Dichroism (CD) and Surface Tunneling Microscopy (STM). The silver nanoparticles retained 85% amylase activity. The nanobiocatalyst was further characterized in terms of kinetic parameters and thermal stability. It was thermally more stable as compared to the free alpha amylase enzyme.

Immobilization of Candida rugosa lipase on superparamagnetic Fe3O4 nanoparticles for biocatalysis in low-water media.

A simple immobilization method for Candida rugosa lipase on superparamagnetic Fe3O4 nanoparticles is described. The Fe3O4 nanoparticles were coated with PEI and Candida rugosa lipase was adsorbed on these particles via electrostatic interactions. The immobilization resulted in marginal simultaneous purification. However, the immobilized preparation showed 110× higher transesterification activity in low-water media. It was also efficient in kinetic resolution of (±)-1-phenylethanol with eep of 99 % and E = 412 within 24 h.

Immobilization of enzymes by bioaffinity layering.

Bioaffinity immobilization exploits the affinity of the enzyme to a macro-(affinity ligand). Such a macro-(affinity ligand) could be a lectin, a water-soluble polymer, or a bioconjugate of a water-soluble polymer and the appropriate affinity ligand. Successive layering of the enzyme and the macro-(affinity ligand) on a matrix allows deposition of a large amount of enzyme activity on a small surface. Illustrative protocols show affinity layering of a pectinase and horseradish peroxidase on Concanavalin A-agarose and Concanavalin A-Sephadex matrices, respectively.

High activity preparations of lipases and proteases for catalysis in low water containing organic solvents and ionic liquids.

Simple precipitation of enzymes has shown impressive catalytic efficiencies in organic solvents. In asmuch as these can be recovered after the reaction, these can be viewed as immobilized preparations just like more extensively used cross-linked enzyme aggregates (CLEAs). This chapter describes three protocols which use these enzyme precipitated and rinsed with propanol/some other appropriate organic solvent. The first two protocols show their applications in ionic liquids for a transesterification reaction and a kinetic resolution. The third protocol presumably incorporates an "imprinting" effect so that the precipitates are now able to efficiently catalyze transesterification of tributyrin with tertiary alcohols.

Three phase partitioning leads to subtle structural changes in proteins.

Three phase partitioning consists of precipitation of proteins due to simultaneous presence of ammonium sulphate and t-butanol. The technique has been successfully used for purification and refolding of proteins. There are however indications that the structures of proteins subjected to three phase partitioning are different from native structure of proteins. Taking several proteins, the present work examines the structural changes in proteins by comparing their thermal stabilities, secondary structure contents, surface hydrophobicities, hydrodynamic radii and solubilities in the presence of ammonium sulphate. The results show that while the nature or extent of structural changes may vary, in all the cases the changes are rather subtle and not drastic in nature. Hence, the technique can be safely used for protein purification and refolding.