Extrinsic Fluorescence Spectroscopy Research Articles

Expression, Purification and Biophysical Characterisation of Klebsiella Pneumoniae Protein Adenylyltransferase: A Systematic Integration of Empirical and Computational Modelling Approaches

Infections that are acquired due to a prolonged hospital stay and manifest 2 days following the admission of a patient to a health-care institution can be classified as hospital-acquired infections. Klebsiella pneumoniae (K. pneumoniae) has become a critical pathogen, posing serious concern globally due to the rising incidences of hypervirulent and carbapenem-resistant strains. Glutaredoxin is a redox protein that protects cells from oxidative stress as it associates with glutathione to reduce mixed disulfides. Protein adenylyltransferase (PrAT) is a pseudokinase with a proposed mechanism of transferring an AMP group from ATP to glutaredoxin. Inducing oxidative stress to the bacterium by inhibiting the activity of PrAT is a promising approach to combating its contribution to hospital-acquired infections. Thus, this study aims to overexpress, purify, and analyse the effects of ATP and Mg2+ binding to Klebsiella pneumoniae PrAT (KpPrAT). The pET expression system and nickel affinity chromatography were effective in expressing and purifying KpPrAT. Far-UV CD spectroscopy demonstrates that the protein is predominantly α-helical, even in the presence of Mg2+. Extrinsic fluorescence spectroscopy with ANS indicates the presence of a hydrophobic pocket in the presence of ATP and Mg2+, while mant-ATP studies allude to the potential nucleotide binding ability of KpPrAT. The presence of Mg2+ increases the thermostability of the protein. Isothermal titration calorimetry provides insight into the binding affinity and thermodynamic parameters associated with the binding of ATP to KpPrAT, with or without Mg2+. Conclusively, the presence of Mg2+ induces a conformation in KpPrAT that favours nucleotide binding.

Conformational changes to zein and its binding interactions with sodium caseinate during the pH-driven self-assembly using multi-spectroscopic and hydrostatic methods

Stable zein-based nanocomplexes with sodium caseinate (NaCas) could be formed using a pH-cycle treatment without organic solvents, but structural changes of water-insoluble zein was not clearly studied. The objective of this work was to investigate the conformational changes of zein and its binding with NaCas using 8-anilinonaphthalene-1-sulfonate (ANS) as a probe that could exclusively bind to zein. Extrinsic fluorescence and three-dimensional fluorescence spectroscopy showed the acidification induced the zein re-folding, as revealed by the increased polarity around tyrosine/tryptophan residues. The UV–Vis spectroscopy and morphological observations in scanning/transmission electron microscopy as well as the goniometer further confirmed these changes. The Raman spectroscopy and increased contents of sulfhydryl groups indicated that the interactions between zein and NaCas were favored by a combination of hydrophobic interaction and disulfide/hydrogen bonds. Information derived from this work could help rationalize the design of zein-NaCas nanocomplex for encapsulation, protection, and release of bioactives using pH-cycle treatment.

Effect of Divalent Metal Ion on the Structure, Stability and Function of Klebsiella pneumoniae Nicotinate-Nucleotide Adenylyltransferase: Empirical and Computational Studies.

The continuous threat of drug-resistant Klebsiella pneumoniae justifies identifying novel targets and developing effective antibacterial agents. A potential target is nicotinate nucleotide adenylyltransferase (NNAT), an indispensable enzyme in the biosynthesis of the cell-dependent metabolite, NAD+. NNAT catalyses the adenylation of nicotinamide/nicotinate mononucleotide (NMN/NaMN), using ATP to form nicotinamide/nicotinate adenine dinucleotide (NAD+/NaAD). In addition, it employs divalent cations for co-substrate binding and catalysis and has a preference for different divalent cations. Here, the biophysical structure of NNAT from K. pneumoniae (KpNNAT) and the impact of divalent cations on its activity, conformational stability and substrate-binding are described using experimental and computational approaches. The experimental study was executed using an enzyme-coupled assay, far-UV circular dichroism, extrinsic fluorescence spectroscopy, and thermal shift assays, alongside homology modelling, molecular docking, and molecular dynamic simulation. The structure of KpNNAT revealed a predominately α-helical secondary structure content and a binding site that is partially hydrophobic. Its substrates ATP and NMN share the same binding pocket with similar affinity and exhibit an energetically favourable binding. KpNNAT showed maximum activity and minimal conformational changes with Mg2+ as a cofactor compared to Zn2+, Cu2+ and Ni2+. Overall, ATP binding affects KpNNAT dynamics, and the dynamics of ATP binding depend on the presence and type of divalent cation. The data obtained from this study would serve as a basis for further evaluation towards designing structure-based inhibitors with therapeutic potential.

Structure characteristics and functionality of water-soluble fraction from high-intensity ultrasound treated pea protein isolate

High-intensity ultrasound (HIUS) has been widely utilized as an emerging technique to modify the functionality of proteins. However, those findings exclusively pertained to the whole protein after the treatment of HIUS. Such protein consists of not only water-soluble proteins but other insoluble constituents, which cannot reflect the role of HIUS on the protein functionality. The objective of this study is to understand the influences of ultrasonic treatment (20 kHz) at varying power (200, 300 or 500 W) and time (5, 10 or 20 min) exclusively on structural and functional properties of water-soluble fraction (WPPI) harvested from HIUS treated pea protein isolate (PPI). Relevant parameters of ultrasound including ultrasonic intensity and ultrasonic density were determined. No significant changes on the primary structure of WPPI were observed from electrophoresis and size exclusion chromatography analysis. Fourier-transform infrared spectroscopy analysis as well as intrinsic and extrinsic fluorescence spectroscopy showed that HIUS was able to change the secondary and tertiary structure of WPPI. HIUS treatment drastically improved protein content and absolute solubility of pea protein. The underlying mechanism lies in the roles of HIUS to change the secondary and tertiary structure and to increase the magnitude of surface charge of WPPI, as well as to facilitate the coexistence of both smaller particle size pea protein and soluble aggregates. HIUS exerted an adverse effect on emulsification properties but improved foaming stability. The results of this study provide a theoretical basis to unravel the essential role of HIUS on modifying the structure of plant protein and the corresponding functionality.

Interactions of β-Lactoglobulin with Bovine Submaxillary Mucin vs. Porcine Gastric Mucin: The Role of Hydrophobic and Hydrophilic Residues as Studied by Fluorescence Spectroscopy.

The aim of this study was to investigate binding interactions between β-lactoglobulin (BLG) and two different mucins, bovine submaxillary mucins (BSM) and porcine gastric mucin (PGM), using intrinsic and extrinsic fluorescence spectroscopies. Intrinsic fluorescence spectra showed an enhanced decrease of fluorescence intensity of BLG at all pH conditions when BLG was mixed with PGM rather than with BSM. We propose that, unlike BSM, the tertiary structure of PGM changes and the hydrophobic regions are exposed at pH 3 due to protonation of negatively charged residues. Results suggest that PGM also facilitated the structural unfolding of BLG and its binding with PGM by a hydrophobic interaction, especially at acidic pH, which was further supported by extrinsic fluorescence spectroscopy. Hydrophobic interaction is suggested as the dominant interaction mechanism between BLG and PGM at pH 3, whereas electrostatic interaction is the dominant one between BLG and BSM.

Influence of iron binding in the structural stability and cellular internalization of bovine lactoferrin

Lactoferrin (Lf) is an iron-binding glycoprotein and a component of many external secretions with a wide diversity of functions. Structural studies are important to understand the mechanisms employed by Lf to exert so varied functions. Here, we used guanidine hydrochloride and high hydrostatic pressure to cause perturbations in the structure of bovine Lf (bLf) in apo and holo (unsaturated and iron-saturated, respectively) forms, and analyzed conformational changes by intrinsic and extrinsic fluorescence spectroscopy. Our results showed that the iron binding promotes changes on tertiary structure of bLf and increases its structural stability. In addition, we evaluated the effects of bLf structural change on the kinetics of bLf internalization in Vero cells by confocal fluorescence microscopy, and observed that the holo form was faster than the apo form. This finding may indicate that structural changes promoted by iron binding may play a key role in the intracellular traffic of bLf. Altogether, our data improve the comprehension of bLf stability and uptake, adding knowledge to its potential use as a biopharmaceutical.

Evaluation of Luciferase Thermal Stability by Arginine Saturation in the Flexible Loops

Background: The firefly luciferase enzyme is widely used in protein engineering and diverse areas of biotechnology, but the main problem with this enzyme is low-temperature stability. Previous reports indicated that surface areas of thermostable proteins are rich in arginine, which increased their thermal stability. In this study, this aspect of thermophilic proteins evaluated by mutations of surface residues to Arg. Here, we report the construction, purification, and studying of these mutated luciferases. Methods: For mutagenesis, the QuikChange site-directed mutagenesis was used and the I108R, T156R, and N177R mutant luciferases were created. In the following, the expression and purification of wild-type and mutant luciferases were conducted and their kinetic and structural properties were analyzed. To analyze the role of these Arg in these loops, the 3D models of these mutants’ enzymes were constructed in the I-TASSER server and the exact situation of these mutants was studied by the SPDBV and PyMOL software. Results: Overall, the optimum temperature of these mutated enzymes was not changed. However, after 30 min incubation of these mutated enzymes at 30°C, the I108R, T156R, N177R, and wild-type kept the 80%, 50%, 20%, and 20% of their original activity, respectively. It should be noted that substitution of these residues by Arg preserved the specific activity of firefly luciferase. Conclusion: Based on these results, it can be concluded that T156R and N177R mutants by compacting local protein structure, increased the thermostability of luciferase. However, insertion of positively charged residues in these positions create the new hydrogen bonds that associated with a series of structural changes and confirmed by intrinsic and extrinsic fluorescence spectroscopy and homology modeling studies.

Soluble complexes between chenopodins and alginate/chitosan: Intermolecular interactions and structural-physicochemical properties

The research aim was to study the nature of intermolecular interactions involved in the formation of soluble complexes between chenopodins (QP) and anionic/cationic polysaccharides, sodium alginate (Alg) and chitosan (CH), above the pI of QP at different protein-polysaccharide ratios; and to evaluate their structural-physicochemical properties. The interactions and properties analysed by electrical conductivity, zeta potential, hydrodynamic particle size, intrinsic and extrinsic fluorescence spectroscopy, circular dichroism spectroscopy, differential scanning calorimetry and infrared spectroscopy. The protein-polysaccharide ratio and the type of polysaccharide influenced the complexes properties. In QP-CH complexes, the main interactions were electrostatic, while in QP-Alg complexes, hydrophobic interactions, hydrogen bonds and weak electrostatic interactions were detected. Changes in secondary and tertiary structure and increased thermal stability of chenopodins in the presence of both polysaccharides were observed. QP-Alg and QP-CH complexes seemed to have achieved the strongest interactions at 1:4, 2:3 and 3:2 ratios and at 1:4 and 2:3 ratios, respectively.

Characterization of Protein‐Ligand Interactions in MsrA from E. coli

The amino acid methionine is highly susceptible to oxidation via reactive oxygen species, resulting in loss of function in proteins containing oxidized methionines. This loss of protein function has been implicated in numerous neurodegenerative and age‐related diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (Lou Gehrig's Disease), emphysema, and arthritis. The enzyme methionine sulfoxide reductase (MsrA) catalyzes the stereospecific reduction of oxidized methionine in free amino acids, peptides, and proteins, restoring function in the latter. The catalytic mechanism of MsrA has been studied extensively, but less is known about how MsrA is able to recognize its large variety of substrates, from single amino acids or small molecules to oxidized methionines in the context of a protein. Earlier work from our lab using intrinsic and extrinsic fluorescence spectroscopy suggests that the presence of sulfoxide group on a methionine in a target protein enhances existing weak hydrophobic interactions with MsrA. The current study builds on these findings using fluorescence spectroscopy to characterize how MsrA of E. coli recognizes its substrates and repairs specific oxidized methionine residues in an intrinsically‐disordered form of staphylococcal nuclease and a nonapeptide derived from it.Support or Funding InformationThis work was supported by a Research Corporation award (grant number CC5482) to VFS. The Office of Naval Research supported Trident Scholar research by JS. MJW was supported by a Garrigues Program scholarship award.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Self-assembled nanoparticles from heat treated ovalbumin as nanocarriers for polyunsaturated fatty acids

The objective of the present work was to obtain and characterize two heat induced (85 °C, 5 min) ovalbumin nanoparticles (OVAn1 formed at pH 11.35 and OVAn2 formed at pH 7.50). In addition, the impact of these OVA nanosized aggregates on the formation and characterization of inclusion nanocomplexes formed with two polyunsaturated fatty acids (PUFAs): linoleic acid (LA) and its isomer, conjugated linoleic acid (CLA) was studied. The pH in which heat treatment was performed had a great impact on nanoparticles hydrodynamic diameter (dH). Nanoparticles dH values were 24.63 ± 0.04 nm for OVAn1 and 92.0 ± 0.2 nm for OVAn2, respectively, and no significant differences in ζ potential values (p > 0.05) were detected. Nanocomplexes colloidal characteristics were studied by particle size distribution (PSD) and ζ potential determinations. In all cases, size measurements showed that PUFAs binding promoted an increased nanoparticles size. In addition, atomic force microscopy (AFM) examination for nanoparticles and nanocomplexes showed a rounded shape and an increase in dimensions of the nanocomplexes which was attributed to LA and CLA binding. On the other hand, nanocomplex formation was monitored by intrinsic and extrinsic fluorescence spectroscopy. Results highlighted that both nanoparticles bound LA and CLA in greater amount than OVA did. Besides, both nanoparticles were able to bind more amount of LA than CLA. Results suggested that knowledge of differences in OVA nanoparticles properties and PUFAs molecular structure is a relevant requisite for the design of inclusion nanocomplexes with especial colloidal properties.

Amorphous aggregation of tau in the presence of titanium dioxide nanoparticles: biophysical, computational, and cellular studies.

BackgroundNanoparticles (NPs) when injected into the body can reach target tissues like nervous system and interact with tau proteins and neurons. This can trigger conformational changes of tau and may affect NP toxicity.MethodsIn this study, we used several biophysical techniques (extrinsic and intrinsic fluorescence spectroscopy, circular dichroism (CD) spectroscopy, ultraviolet (UV)-visible spectroscopy), transmission electron microscopy (TEM) investigations, molecular docking and molecular dynamics studies, and cellular assays [3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) and flow cytometry) to reveal how structural changes of tau protein can change the cytotoxicity of titanium dioxide (TiO2) NPs against neuron-like cells (SH-SY5Y) cells.ResultsIt was shown that TiO2 NPs result in hydrophilic interactions, secondary and tertiary structural changes, and the formation of amorphous tau aggregates. Conformational changes of tau increased the induced cytotoxicity by TiO2 NPs. These data revealed that the denatured adsorbed protein on the NP surface may enhance NP cytotoxicity.ConclusionTherefore, this study provides useful insights on the NP–protein interactions and discusses how the protein corona can increase cytotoxicity to determine the efficacy of targeted delivery of nanosystems.

Biophysical, bioinformatical, cellular, and molecular investigations on the effects of graphene oxide nanosheets on the hemoglobin structure and lymphocyte cell cytotoxicity.

BackgroundImplementations of nanoparticles have been receiving great interest in medicine and technology due to their unique characteristics. However, their toxic impacts on the biological system are not well explored.AimThis study aims to investigate the influence of fabricated nano graphene oxide (NGO) sheets on the secondary and quaternary structural alterations of human hemoglobin (Hb) and cytotoxicity against lymphocyte cells.Materials and methodsDifferent spectroscopic methods, such as extrinsic and synchronous fluorescence spectroscopy and far circular dichroism (CD) spectroscopy, molecular docking investigation, cellular assays (trypan blue exclusion, cellular uptake, ROS, cell cycle, and apoptosis), and molecular assay (fold changes in anti/proapoptotic genes [B-cell lymphoma-2 {BCL2}/BAX] expression levels) were used in this study.ResultsTransmission electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and zeta potential investigations revealed the nano-sized nature of NGOs with good colloidal stability. Extrinsic fluorescence spectroscopy by using 8-anilinonaphthalene-1 -sulfonic acid and synchronous fluorescence spectroscopy showed that NGOs can unfold the quaternary structure of Hb in the vicinity of Tyr residues. The CD investigation demonstrated that the α-helicity of Hb experienced substantial alteration upon interaction with increasing concentrations of NGOs. The molecular docking study showed that NGOs interacted with polar residues of Hb. Cellular and molecular assays revealed that NGOs lead to ROS formation, cell cycle arrest, and apoptosis through the BAX and BCL2 pathway.ConclusionThese data reveal that NGOs can induce some protein structural changes and stimulate cytotoxicity against normal cell targets. Therefore, their applications in healthy systems should be limited.

Natural deep eutectic solvents as a storage medium for human interferon-α2: a green and improved strategy for room-temperature biologics

The inherent vulnerability of protein biologics to high temperature is the origin of their limited use in clinical settings where proper storage and handling systems are not available. Herein, we demonstrate the first application of natural deep eutectic solvents (NADES) as a temperature-stable storage medium for a real therapeutic protein, human interferon-alpha 2 (IFN-α2). A formulation of IFN-α2 based on a NADES composed of choline chloride and fructose at a 1:1 molar ratio (CCl:Fru) had no influence on the activity of IFN-α2. The NADES provided an appropriate environment for effective protection of IFN-α2 from thermal denaturation at mid (37°C) and high (70°C) temperatures during long-term (90 days) and short-term (2h) storage. Protein stability in the presence of CCl:Fru was demonstrated by orthogonal or complementary methods, including in vitro activity assay, circular dichroism spectroscopy and extrinsic fluorescence spectroscopy. In view of their biocompatibility, customizable properties, and the simplicity of synthesis and use, NADESs are suggested as a green and improved short-term and long-term storage medium that could facilitate the development of room-temperature biologics.

Minichaperone‐mediated folding of MalZ proceeds by binding and release of native and functional intermediates Neha Jain and and Tapan K. Chaudhuri Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, India.

It is well understood that molecular chaperones play an essential role in the prevention of aggregation and also in the correct folding of newly synthesized polypeptides in vivo as well as in vitro. Among the variety of molecular chaperones identified in E.coli, GroEL/GroES is the mostly investigated chaperone system reported in the literature. The essential chaperonin GroEL, acting with its cochaperonin GroES, has the ability to bind and assist the folding of a wide variety of client proteins. This process requires a complex allosteric reaction cycle, driven by ATP binding and hydrolysis, which involves concerted movements of the domains of this large molecular machine. It has long been known that isolated apical domains of GroEL (known as “minichaperone”) also show some chaperone ability, but relatively little attention has been paid to the mechanism by which these much simpler chaperones act. Therefore, in order to probe the mechanism of minichaperone action in detail, efficiency in the folding of relatively larger aggregation prone multidomain proteins and the fate of a polypeptide during a minichaperone-mediated folding reaction remain to be identified. Here, using surface plasmon resonance measurements provide an estimate for the equilibrium dissociation constant KD for the MalZ-minichaperone complex of 0.21 ± 0.04 μM, significantly higher than for most GroEL clients, showing that minichaperone interacts loosely with MalZ during the refolding process. Noticeably, we also found that, upon addition of GroES as an interference trap, MalZ is released from minichaperone in a predominantly non-native conformation that can be trapped by mutant forms of GroEL that are capable of binding but not releasing substrate. Released polypeptide undergoes kinetic partitioning: a fraction completes folding while the remainder is rebound by other minichaperone molecules. Folding appears to occur in an all-or-none manner, since, our evidence by using intrinsic and extrinsic fluorescence spectroscopy, trypsin sensitivity, enzymatic activity and gel filtration chromatography suggest that the refolded MalZ has the same structure as the native MalZ, but that its structure when bound to minichaperone is different. In conclusion, our results indicate that the minichaperone works through a process of iterative annealing, with repeated cycles of binding and release of the client protein. By this mechanism, minichaperone may lower the effective concentration of free, unfolded intermediates and reduce off-pathway processes such as aggregation or misfolding. Since folding intermediates of MalZ could not fold by itself in solution and hence requires assistance to fold, the transient release and binding of folding intermediates with minichaperone proceeds until it reaches the correct folded form. These finding will be value for researcher as minichaperone is less complex, and does not require high energy co-factors like ATP, for its function as compared to conventional GroEL-GroES system, it can act as a very good in vitro as well as in vivo chaperone model for monitoring assisted protein folding phenomenon. Support or Funding Information Neha jain acknowledges joint Council of Scientific and Industrial Research-University of Grant Commission, Govt. of India for doctoral fellowship award no. 20-6/2008/ 2009. The authors acknowledge Indian Institute of Technology Delhi and the University of Birmingham for infrastructural support. Neha jain gratefully acknowledges financial support from Boeheringer Ingelheim Fonds to facilitate my experiments (SPR and thermofluor) at the University of Birmingham, United Kingdom This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lag phase alteration in the modified bovine serum albumin under the inducing and inhibitory effect of vitamin C

Lag phase as the initial stage of protein aggregation has an important role in fibril formation and contributes to pathogenesis of many human diseases. Harsh environments provided by some additives can change the rate and the amounts of protein abnormalities. Here we studied the lag phase as the first stage of fibrillation process of bovine serum albumin (BSA) and determined the effects of potassium sorbate (PS) a widespread industrial preservative and vitamin C as an important antioxidant on these changes. Thioflavin T binding assay, circular dichroism, intrinsic and extrinsic fluorescence spectroscopy, and atomic force microscopy techniques were used for these assessments. Our results indicated that the lag phase of BSA fibrillation was affected under different conditions. The length of lag phase in BSA protein aggregation process was short due to the generation of the advanced glycation end products (AGEs) by PS. In contrast, vitamin C by its antioxidant properties and chaperone ability dramatically prolonged BSA lag phase, preserved BSA structural changes that lead to aggregation, and dramatically decreased formation of AGEs and the amount of fibrillation.

Correlating the Effects of Antimicrobial Preservatives on Conformational Stability, Aggregation Propensity, and Backbone Flexibility of an IgG1 mAb

Multidose formulations of biotherapeutics, which offer better dosage management and reduced production costs, require the addition of antimicrobial preservatives (APs). APs have been shown, however, to decrease protein stability in solution and cause protein aggregation. In this report, the effect of 4 APs, m-cresol, phenol, phenoxyethanol, and benzyl alcohol on conformational stability, aggregation propensity, and backbone flexibility of an IgG1 mAb, mAb-4, is investigated. Compared with no preservative control, each of the APs decreased the conformational stability of mAb-4 as measured by differential scanning calorimetry and extrinsic fluorescence spectroscopy. The addition of APs resulted in increased monomer loss and aggregate accumulation at 50°C over 28 days, as monitored by size-exclusion chromatography. The extent of conformational destabilization and protein aggregation of mAb-4 induced by APs followed their calculated octanol–water partition coefficients. Increases in backbone flexibility, as measured by hydrogen exchange, of a region located in the CH2 domain of the mAb (heavy chain 237-254) in the presence of APs also correlated with hydrophobicity. Based on these results, the destabilizing effect of APs on mAb-4 correlates with the increased hydrophobicity of the APs and their ability to enhance the local backbone flexibility of an aggregation hot spot within the CH2 domain of the mAb.

Formation and colloidal stability of ovalbumin-retinol nanocomplexes

This work is aimed to obtain and characterize nanocomplexes formed by non-covalent attractive interactions between ovalbumin (OVA) and retinol (RET). In order to gain some ideas about the effect of OVA aggregation on RET complexation, OVA nanoparticles (OVAn) obtained by controlled heat treatment (85 °C, 5 min) were also evaluated. Nanocomplex formation was monitored by intrinsic and extrinsic fluorescence spectroscopy. Results suggest that OVA was more effective for binding RET than OVAn, possibly because aggregate formation could employ some hydrophobic patches needed for RET binding. Furthermore, nanocomplex colloidal stability was studied by means of absorbance (at 400 nm), particle size distribution (PSD) and ζ potential determinations. At pH values of common foods (4.0 and 7.0), nanocomplex colloidal stability mainly depended on aqueous phase behaviour of the proteins (OVA and OVAn). Finally, long-term photochemical stability against light and oxygen of RET nanocomplexes depended on solution pH and protein aggregation.

Studies on the interaction between nanodiamond and human hemoglobin by surface tension measurement and spectroscopy methods

In this study, a novel method to probe molecular interactions and binding of human hemoglobin (Hb) with nanodiamond (ND) was introduced based on the surface tension measurement. This method complements conventional techniques, which are basically done by zeta potential and dynamic light scattering (DLS) measurements, near and far circular dichroism (CD) spectroscopy, intrinsic and extrinsic fluorescence spectroscopy. Addition of ND to Hb solution increased the surface tension value of Hb–ND complex relative to those of Hb and ND molecules. The zeta potential values reveled that Hb and ND provide identical charge distribution at pH 7.5. DLS measurements demonstrated that Hb, ND, and ND–Hb complex have hydrodynamic radiuses of 98.37 ± 4.57, 122.07 ± 7.88 nm and 62.27 ± 3.70 at pH of 7.5 respectively. Far and near UV-CD results indicated the loss of α-helix structure and conformational changes of Hb, respectively. Intrinsic fluorescence data demonstrated that the fluorescence quenching of Hb by ND was the result of the static quenching. The hydrophobic interaction plays a pivotal role in the interaction of ND with Hb. Fluorescence intensity changes over time revealed conformational change of Hb continues after the mixing of the components (Hb–ND) till 15 min, which is indicative of the denaturation of the Hb relative to the protein control. Extrinsic fluorescence data showed a considerable enhancement of the ANS fluorescence intensity of Hb–ND system relative to the Hb till 60 nM of ND, likely persuaded by greater exposure of nonpolar residues of Hb hydrophobic pocket. The remarkable decrease in Tm value of Hb in Hb–ND complex exhibits interaction of Hb with ND conducts to conformational changes of Hb. This study offers consequential discrimination into the interaction of ND with proteins, which may be of significance for further appeal of these nanoparticles in biotechnology prosecution.

Structure and membrane interactions of chionodracine, a piscidin-like antimicrobial peptide from the icefish Chionodraco hamatus

Chionodracine (Cnd) is a 22-residue peptide of the piscidin family expressed in the gills of the Chionodraco hamatus as protection from bacterial infections. Here, we report the effects of synthetic Cnd on both Psychrobacter sp. TAD1 and Escherichia coli bacteria, as well as membrane models. We found that Cnd perforates the inner and outer membranes of Psychrobacter sp. TAD1, making discrete pores that cause the cellular content to leak out. Membrane disruption studies using intrinsic and extrinsic fluorescence spectroscopy revealed that Cnd behaves similarly to other piscidins, with comparable membrane partition coefficients. Membrane accessibility assays and structural studies using NMR in detergent micelles show that Cnd adopts a canonical topology of antimicrobial helical peptides, with the hydrophobic face toward the lipid environment and the hydrophilic face toward the bulk solvent. The analysis of Cnd free energy of binding to vesicles with different lipid contents indicates a preference for charged phospholipids and a more marked binding to native E. coli extracts. Taken with previous studies on piscidin-like peptides, we conclude that Cnd first adsorbs to the membrane, and then forms pores together with membrane fragmentation. Since Cnd has only marginal hemolytic activity, it constitutes a good template for developing new antimicrobial agents.

Structural Characterization of IgG1 mAb Aggregates and Particles Generated Under Various Stress Conditions

IgG1 mAb solutions were prepared with and without sodium chloride and subjected to different environmental stresses. Formation of aggregates and particles of varying size was monitored by a combination of size-exclusion chromatography, Nanoparticle Tracking Analysis, Micro-flow Imaging (MFI), turbidity, and visual assessments. Stirring and heating induced the highest concentration of particles. In general, the presence of NaCl enhanced this effect. The morphology of the particles formed from mAb samples exposed to different stresses was analyzed from transmission electron microscopy and MFI images. Shaking samples without NaCl generated the most fibrillar particles, whereas stirring created largely spherical particles. The composition of the particles was evaluated for covalent cross-linking by SDS-PAGE, overall secondary structure by FTIR microscopy, and surface apolarity by extrinsic fluorescence spectroscopy. Freeze-thaw and shaking led to particles containing protein with native-like secondary structure. Heating and stirring produced IgG1-containing aggregates and particles with some non-native disulfide cross-links, varying levels of intermolecular beta sheet content, and increased surface hydrophobicity. These results highlight the importance of evaluating protein particle morphology and composition, in addition to particle number and size distributions, to better understand the effect of solution conditions and environmental stresses on the formation of protein particles in mAb solutions.