Charged Protein Research Articles

Single-molecule diffusivity quantification in Xenopus egg extracts elucidates physicochemical properties of the cytoplasm

The living cell creates a unique internal molecular environment that is challenging to characterize. By combining single-molecule displacement/diffusivity mapping (SMdM) with physiologically active extracts prepared from Xenopus laevis eggs, we sought to elucidate molecular properties of the cytoplasm. Quantification of the diffusion coefficients of 15 diverse proteins in extract showed that, compared to in water, negatively charged proteins diffused ~50% slower, while diffusion of positively charged proteins was reduced by ~80 to 90%. Adding increasing concentrations of salt progressively alleviated the suppressed diffusion observed for positively charged proteins, signifying electrostatic interactions within a predominately negatively charged macromolecular environment. To investigate the contribution of RNA, an abundant, negatively charged component of cytoplasm, extracts were treated with ribonuclease, which resulted in low diffusivity domains indicative of aggregation, likely due to the liberation of positively charged RNA-binding proteins such as ribosomal proteins, since this effect could be mimicked by adding positively charged polypeptides. Interestingly, in extracts prepared under typical conditions that inhibit actin polymerization, negatively charged proteins of different sizes showed similar diffusivity suppression consistent with our separately measured 2.22-fold higher viscosity of extract over water. Restoring or enhancing actin polymerization progressively suppressed the diffusion of larger proteins, recapitulating behaviors observed in cells. Together, these results indicate that molecular interactions in the crowded cell are defined by an overwhelmingly negatively charged macromolecular environment containing cytoskeletal networks.

Balancing Group 1 Monoatomic Ion-Polar Compound Interactions in the Polarizable Drude Force Field: Application in Protein and Nucleic Acid Systems.

An accurate force field (FF) is the foundation of reliable results from molecular dynamics (MD) simulations. In our recently published work, we developed a protocol to generate atom pair-specific Lennard-Jones (known as NBFIX in CHARMM) and through-space Thole dipole screening (NBTHOLE) parameters in the context of the Drude polarizable FF based on readily accessible quantum mechanical (QM) data to fit condensed phase experimental thermodynamic benchmarks, including the osmotic pressure, diffusion coefficient, ionic conductivity, and solvation free energy, when available. In the present work, the developed protocol is applied to generate NBFIX and NBTHOLE parameters for interactions between monatomic ions (specifically Li+, Na+, K+, Rb+, Cs+, and Cl-) and common functional groups found in proteins and nucleic acids. The parameters generated for each ion-functional group pair were then applied to the corresponding functional groups within proteins or nucleic acids followed by MD simulations to analyze the distribution of ions around these biomolecules. The modified FF successfully addresses the issue of overbinding observed in a previous iteration of the Drude FF. Quantitatively, the model accurately reproduces the effective charge of proteins and demonstrates a level of charge neutralization for a double-helix B-DNA in good agreement with the counterion condensation theory. Additionally, simulations involving ion competition correlate well with experimental results, following the trend Li+ > Na+ ≈ K+ > Rb+. These results validate the refined model for group 1 ion-biomolecule interactions that will facilitate the application of the polarizable Drude FF in systems in which group 1 ions play an important role.

Dissecting the Roles of Electrostatic Interactions in Modulating the Folding Stability and Cooperativity of Engrailed Homeodomain.

Engrailed homeodomain (EngHD), a highly charged transcription factor regulating over 200 genes, is a fast-folding protein. Recent studies have shown that the abundant charged residues in EngHD not only facilitate protein-DNA interactions but also influence the conformational disorder of its native structure. However, the mechanisms by which electrostatic interactions modulate the folding of EngHD remain unclear. Here, we employ a coarse-grained structure-based model that incorporates the salt-dependent Debye-Hückel model to investigate the (un)folding behavior of EngHD under various salt concentrations. Our findings demonstrate that increasing salt concentrations enhance both folding stability and cooperativity, while the folding barrier height remains relatively constant due to the distinct electrostatic effects on individual residues. By modulating the energetic balance between local and nonlocal interactions, we shift the folding of EngHD from a downhill process to a two-state process. Notably, we observe a nonmonotonic relationship between the strength of local interactions and residue-level coupling degree during (un)folding, likely attributed to the repulsive electrostatic interactions present in the native structure of EngHD. Additionally, we identify a critical turning point in the dependence of folding cooperativity on salt concentration, classified by the energetic balance of local and nonlocal interactions. Our results provide valuable insights into how electrostatic interactions influence the folding of EngHD, contributing to the theoretical framework for engineering highly charged proteins.

Molecular insights into the interaction between a disordered protein and a folded RNA

Intrinsically disordered protein regions (IDRs) are well established as contributors to intermolecular interactions and the formation of biomolecular condensates. In particular, RNA-binding proteins (RBPs) often harbor IDRs in addition to folded RNA-binding domains that contribute to RBP function. To understand the dynamic interactions of an IDR-RNA complex, we characterized the RNA-binding features of a small (68 residues), positively charged IDR-containing protein, Small ERDK-Rich Factor (SERF). At high concentrations, SERF and RNA undergo charge-driven associative phase separation to form a protein- and RNA-rich dense phase. A key advantage of this model system is that this threshold for demixing is sufficiently high that we could use solution-state biophysical methods to interrogate the stoichiometric complexes of SERF with RNA in the one-phase regime. Herein, we describe our comprehensive characterization of SERF alone and in complex with a small fragment of the HIV-1 Trans-Activation Response (TAR) RNA with complementary biophysical methods and molecular simulations. We find that this binding event is not accompanied by the acquisition of structure by either molecule; however, we see evidence for a modest global compaction of the SERF ensemble when bound to RNA. This behavior likely reflects attenuated charge repulsion within SERF via binding to the polyanionic RNA and provides a rationale for the higher-order assembly of SERF in the context of RNA. We envision that the SERF-RNA system will lower the barrier to accessing the details that support IDR-RNA interactions and likewise deepen our understanding of the role of IDR-RNA contacts in complex formation and liquid-liquid phase separation.

The Donnan equilibrium is still valid in high-volume HDF.

Clinical studies have shown that hemodiafiltration reduces morbidity and mortality of dialysis patients compared to hemodialysis alone. This is attributed to its superior middle molecule clearance compared to standard hemodialysis. However, doubts arose as to whether a high convective flux through the dialyzer membrane has an influence on the equilibrium concentration of small ions, especially that of sodium. Due to the presence of negatively charged impermeable proteins on the blood side, the Gibbs-Donnan effect leads to an asymmetric distribution of membrane permeable ions on both sides of the membrane. In thermodynamic equilibrium, the concentrations of those ions can easily be calculated. However, the convective fluid flow leads to deviations from thermodynamic equilibrium. In this article, the effect of a convective flow on the ion distribution across a semipermeable membrane is analyzed in a theoretical model. Starting from the extended Nernst-Planck equation, including diffusive, convective, and electrostatic effects, a set of differential equations is derived. An approximate solution for flow speeds up to 0.1 ms-1 as well as a numerical solution are given. The results show that in any practical dialysis setting the convective flow has negligible influence on the electrolyte concentrations.

Effects of mono- and divalent-ions and their strength on foaming properties of infant formula protein model system

Metal ions may affect the foaming properties of infant formula products that including milk protein during reconstitution. In this study, an infant formula protein model system (IFPMS) composed of both whey protein and casein was constructed, and the effect of mono (Na+, K+) and divalent (Mg2+, Ca2+) ions and their strength (0–100 mM) on foaming properties was investigated. The mono ions had no significantly impact on foaming capacity (FC), MgCl2 slightly increase FC and reaching a maximum of 116.09 ± 7.46% at 25 mM, whereas CaCl2 significantly decreased FC from 105.78 ± 7.42% to 83.14 ± 6.75% as 0–25 mM (p < 0.05). Foam stability (FS) was significantly improved as all salts above 10 mM (p < 0.05). The dramatically decreased net charge and larger protein aggregates (>25 mM) of IFPMS with divalent ions indicating their stronger charge-shielding effect, which caused slow adsorption of protein and increased surface tension. Multiple spectral results confirmed that salts enhanced protein interactions via inter-molecular and form a viscoelastic layer to stabilize foam. Absorbed protein and SDS-PAGE analysis revealed that MgCl2 promoted protein adsorption, while CaCl2 reduced casein in the foam phase. Correlation analysis further implied that the FC showed highly significant positive with absorbed protein, solubility, and β-turn (p ≤ 0.01), and FS was significantly correlated with secondary structure and viscosity (p ≤ 0.05). This study may provide useful information for an in-depth understanding of the foaming properties of infant formula.

Driving Forces in the Formation of Biocondensates of Highly Charged Proteins: A Thermodynamic Analysis of the Binary Complex Formation.

A thermodynamic analysis of the binary complex formation of the highly positively charged linker histone H1 and the highly negatively charged chaperone prothymosin α (ProTα) is detailed. ProTα and H1 have large opposite net charges (-44 and +53, respectively) and form complexes at physiological salt concentrations with high affinities. The data obtained for the binary complex formation are analyzed by a thermodynamic model that is based on counterion condensation modulated by hydration effects. The analysis demonstrates that the release of the counterions mainly bound to ProTα is the main driving force, and effects related to water release play no role within the limits of error. A strongly negative Δcp (=-0.87 kJ/(K mol)) is found, which is due to the loss of conformational degrees of freedom.

Rapid and non-invasive renal injury diagnosis unlocked by a glimpse into urinary protein particle size and charge

Urinary protein, an important marker for early detection of kidney injury, would change in type and content dynamically with the degree of kidney injury due to the particle size and charge selectivity of the glomerular filtration system, making it significantly valuable for accurate classification and early diagnosis. In this study, we developed a fluorescence sensor (Ami-AuNP/DNAs) based on charge interaction to rapidly identify the progression of kidney injury. When the positively charged Ami-AuNP combines with negatively charged DNAs, fluorescence quenching occurs, and urine proteins that appear compete with the DNAs, leading to fluorescence recovery. Based on these signal changes, PCA and PSO-BP neural network analysis were used to successfully identified kidney injury progression in 197 animal kidney injury and 62 clinical chronic kidney disease urine samples through a simple urine sample drop. Additionally, the sensor could also evaluate the effect of Huangkui capsule on kidney injury in adriamycin nephropathy model mice. Accordingly, this method transforms complex biological signals in vivo into macroscopic visual optical signals, amplifying differences of urinary protein, making up for the deficiency of the traditional method in hysteresis and low accuracy, and promoting urinary protein as the potential noninvasive biomarker for evaluating kidney injury.

Phase behavior and interaction of strong polyelectrolyte dextran sulfate and whey protein isolation: Effects of pH, protein/polysaccharide ratio, and salt addition

The strong polyelectrolyte dextran sulfate (DS) is an anionic polysaccharide with a high negative charge, characterized by high stability and pH independence. DS and whey protein isolate (WPI) were selected to study the specific effects of highly negatively charged polysaccharides on the phase behavior and interaction of WPI/DS complexes (1 % w/v) under varying external conditions (pH, WPI:DS ratio, and salt addition). The phase diagrams, zeta potential, and laser confocal scanning microscopy measurements indicated that the WPI/DS complexes did not dissociate even at pH 1 due to the pH independence of DS. The exclusion volume effect of DS promoted WPI self-aggregation at high salt concentrations, which inhibited acidification-induced dissociation. Isothermal titration calorimetry indicated that the WPI/DS interaction is a spontaneous exothermic reaction driven by both enthalpy and entropy changes due to electrostatic interactions. This study provides valuable information on the interactions between highly negatively charged polysaccharides and proteins.

Nanoetched Stainless Steel Architecture Enhances Cell Uptake of Biomacromolecules and Alters Protein Corona Abundancy.

Nanotexture on biocompatible surfaces promotes cell adhesion and proliferation. High aspect ratio nanoachitecture serves as an ideal interface between implant materials and host cells that is well-suited for localized therapeutic delivery. Despite this potential, nanotextured surfaces have not been widely applied for biomacromolecule delivery. Here, we employed a low-cost, industrially relevant nanoetching process to modify the surface of biocompatible stainless steel 316 (SS316L), creating nanotextured SS316L (NT-SS316L) as a material for intracellular biomacromolecule delivery. As biomacromolecule cargoes are adsorbed to the steel and ultimately would be used in protein-rich environments, we performed serum protein corona analysis on unmodified SS316L and NT-SS316L using tandem mass spectrometry. We observed an increase in proteins associated with cell adhesion on the surface of NT-SS316L compared to that of SS316L, supporting literature reports of enhanced adhesion on nanotextured materials. For delivery to adherent cells, a "hard corona" of model biomacromolecule cargoes including superfolder green fluorescent protein (sfGFP) charge variants, cytochrome c, and siRNA was adsorbed on NT-SS316L to assess delivery. Nanotextured surfaces enhanced cellular biomacromolecule uptake and delivered cytosolic-functional proteins and nucleic acids through energy-dependent endocytosis. Collectively, these findings indicate that NT-SS316L holds potential as a surface modification for implants to achieve localized drug delivery for a variety of biomedical applications.

Adenosine Triphosphate Inhibits Cold-Responsive Aggregation.

Adenosine triphosphate (ATP), ubiquitous in all living organisms, is conventionally recognized as a fundamental energy currency essential for a myriad of cellular processes. While its traditional role in energy metabolism requires only micromolar concentrations, the cellular content of ATP has been found to be significantly higher at the millimolar level. Recent studies have attempted to correlate this higher concentration of ATP with its nonenergetic role in maintaining protein homeostasis, leaving the investigation of ATP's nontrivial activities in biology an open question. Here, by coupling computer simulations and experiments, we uncover new insights into ATP's role as a cryoprotectant against cold-salt stress, highlighting the necessity for higher cellular ATP concentrations. We present direct evidence at charged silica interfaces, demonstrating ATP's ability to restore native intersurface interactions disrupted by combined cold-salt stress, thereby inhibiting cold-responsive aggregation in high-salt conditions. ATP desorbs salt cations from negatively charged surfaces through predominant interactions between ATP and the salt cations. Although the mode of ATP's action remains unchanged with temperature, the extent of interaction scales with temperature, requiring less ATP activity at lower temperatures, justifying the reason for reduction in cellular ATP content due to the cold effect, reported in previous experimental studies. The trend observed in inorganic nanostructures is recurrent and robustly transferable to charged protein interfaces. A thorough comparison of ATP's cryoprotective activity with traditionally known biological cryoprotectants (glycine and betaine) reveals ATP's greater efficiency. In retrospect, our findings highlight ATP's additional biological role in cryopreservation, expanding its potential biomedical applications by offering effective protection of cells from cryoinjuries and avoiding the significant challenges associated with the toxicity of organic cryoprotectants.

Characterization of a Charged Biomimetic Lipid Membrane for Unique Antifouling Effects against Clinically Relevant Matrices in Biosensing.

Clinically relevant matrices such as human blood and serum can cause substantial interference in biosensing measurements, severely compromising the effectiveness of the sensors. We report the characterization of a positively charged lipid membrane that has demonstrated unique features to suppress the nonspecific signal for antifouling effects by using SPR, fluorescence recovery after photobleaching (FRAP), and MALDI-TOF-MS. The ethylphosphocholine (EPC) lipid membrane proved to be exceptionally effective at reducing irreversible interactions from human serum on a Protein A surface. The membrane formation conditions and their effects on membrane fluidity and mobility were characterized for understanding the antifouling functions when various capture molecules were immobilized. Specifically, EPC lipid membranes on a Protein A substrate appear to exhibit a strong interaction, likely through the electrostatic effect with the negatively charged proteins that resulted in a stable hydration layer. The strong interaction also limited lipid mobility, contributing to a robust, protective interface that remained undamaged in undiluted serum. Tailoring a surface with antifouling lipid membranes allows for a range of biosensing applications in highly complex biological media.

Inhibition of RNA splicing triggers CHMP7 nuclear entry, impacting TDP-43 function and leading to the onset of ALS cellular phenotypes.

Amyotrophic lateral sclerosis (ALS) is linked to the reduction of certain nucleoporins in neurons. Increased nuclear localization of charged multivesicular body protein 7 (CHMP7), a protein involved in nuclear pore surveillance, has been identified as a key factor damaging nuclear pores and disrupting transport. Using CRISPR-based microRaft, followed by gRNA identification (CRaft-ID), we discovered 55 RNA-binding proteins (RBPs) that influence CHMP7 localization, including SmD1, a survival of motor neuron (SMN) complex component. Immunoprecipitation-mass spectrometry (IP-MS) and enhanced crosslinking and immunoprecipitation (CLIP) analyses revealed CHMP7's interactions with SmD1, small nuclear RNAs, and splicing factor mRNAs in motor neurons (MNs). ALS induced pluripotent stem cell (iPSC)-MNs show reduced SmD1 expression, and inhibiting SmD1/SMN complex increased CHMP7 nuclear localization. Crucially, overexpressing SmD1 in ALS iPSC-MNs restored CHMP7's cytoplasmic localization and corrected STMN2 splicing. Our findings suggest that early ALS pathogenesis is driven by SMN complex dysregulation.

A biodegradable lipid nanoparticle delivers a Cas9 ribonucleoprotein for efficient and safe in situ genome editing in melanoma

The development of melanoma is closely related to Braf gene, which is a suitable target for CRISPR/Cas9 based gene therapy. CRISPR/Cas9-sgRNA ribonucleoprotein complexes (RNPs) stand out as the safest format compared to plasmid and mRNA delivery. Similarly, lipid nanoparticles (LNPs) emerge as a safer alternative to viral vectors for delivering the CRISPR/Cas9-sgRNA gene editing system. Herein, we have designed multifunctional cationic LNPs specifically tailored for the efficient delivery of Cas9 RNPs targeting the mouse Braf gene through transdermal delivery, aiming to treat mouse melanoma. LNPs are given a positive charge by the addition of a newly synthesized polymer, deoxycholic acid modified polyethyleneimine (PEI-DOCA). Positive charge enables LNPs to be delivered in vivo by binding to negatively charged cell membranes and proteins, thereby facilitating efficient skin penetration and enhancing the delivery of RNPs into melanoma cells for gene editing purposes. Our research demonstrates that these LNPs enhance drug penetration through the skin, successfully delivering the Cas9 RNPs system and specifically targeting the Braf gene. Cas9 RNPs loaded LNPs exert a notable impact on gene editing in melanoma cells, significantly suppressing their proliferation. Furthermore, in mice experiments, the LNPs exhibited skin penetration and tumor targeting capabilities. This innovative LNPs delivery system offers a promising gene therapy approach for melanoma treatment and provides fresh insights into the development of safe and effective delivery systems for Cas9 RNPs in vivo. Statement of significanceCRISPR/Cas9 technology brings new hope for cancer treatment. Cas9 ribonucleoprotein offers direct genome editing, yet delivery challenges persist. For melanoma, transdermal delivery minimizes toxicity but faces skin barrier issues. We designed multifunctional lipid nanoparticles (LNPs) for Cas9 RNP delivery targeting the Braf gene. With metal microneedle pretreatment, our LNPs effectively edited melanoma cells, reducing Braf expression and inhibiting tumor growth. Our study demonstrates LNPs' potential for melanoma therapy and paves the way for efficient in vivo Cas9 RNP delivery systems in cancer therapy.

Advancing Protein Analysis: A Low-Pressure Drift Tube Orbitrap Mass Spectrometer for Ultraviolet Photodissociation-Based Structural Characterization.

Owing to its ability to generate extensive fragmentation of proteins, ultraviolet photodissociation (UVPD) mass spectrometry (MS) has emerged as a versatile ion activation technique for the structural characterization of native proteins and protein complexes. Interpreting these fragmentation patterns provides insight into the secondary and tertiary structures of protein ions. However, the inherent complexity and diversity of proteins often pose challenges in resolving their numerous conformations. To address this limitation, we combined UVPD-MS with drift tube ion mobility, offering potential to acquire conformationally selective MS/MS information. A low-pressure drift tube (LPDT) Orbitrap mass spectrometer equipped with 193 nm UVPD capabilities enables the analysis of protein conformers through the analysis of arrival time distributions (ATDs) of individual fragment ions. ATDs of fragment ions are compared for different backbone cleavage sites of the protein or different precursor charge states to give information about regions of potential folding or elongation. This integrated platform offers promise for advancing our understanding of protein structures in the gas phase.

Monitoring Binding of Protamine with DNA Using Protein Charge Transfer Spectra.

In this work, novel intrinsic electronic absorption (250-400 nm) with a molar extinction coefficient of 752 M-1cm-1 at 250 nm, arising from photoinduced electron transfer involving charged amino acid side chains and the polypeptide backbone, along with luminescence (300-500 nm) with quantum yield of 0.011 from subsequent charge recombination, was observed in salmon sperm Protamine (PRM). The absorption of PRM was attributed to the previously identified Peptide Backbone-to-Side chain Charge Transfer (PBS-CT) from the polypeptide backbone to the abundant cationic headgroups of Arginine in PRM, while the luminescence was believed to originate from charge recombination within the charge-separated excited states of PRM. Remarkably, since Arg is the only charged residue in the PRM sequence, the PRM Protein Charge Transfer Spectra (ProCharTS) is both totally and uniquely Arg specific. Interestingly, the peak of PRM luminescence emission spectrum was independent of the excitation wavelength, unlike other proteins such as human serum albumin, displaying unconventional luminescence. Aggregation-induced effects on PRM absorbance and luminescence were ruled out, as both PRM absorbance and luminescence increase maintained linearity with increasing concentration in the 25-150 μM range. Nucleoprotamine complex formation, resulting from the binding of PRM with calf-thymus genomic DNA (gDNA), was monitored through increased scattering by the nucleoprotamine complex, a decrease in gDNA/PRM absorbance, a decrease in gDNA/PRM ellipticity, and shifts of nucleoprotamine complex band upon agarose gel electrophoresis. Upon binding with gDNA (700 μM base pair concentration), PRM ProCharTS absorbance at 260 nm decreased by 72%. This decrease was attributed to the formation and subsequent precipitation of nucleoprotamine complex upon PRM-gDNA binding. The application of ProCharTS absorbance to indirectly monitor DNA-protein binding in a label-free approach was thus demonstrated.

Statistical Thermodynamic Description of Self-Assembly of Large Inclusions in Biological Membranes.

Recent studies revealed anomalous underscreening in concentrated electrolytes, and we suggest that the underscreened electrostatic forces between membrane proteins play a significant role in the process of self-assembly. In this work, we assumed that the underscreened electrostatic forces compete with the thermodynamic Casimir forces induced by concentration fluctuations in the lipid bilayer, and developed a simplified model for a binary mixture of oppositely charged membrane proteins with different preference to liquid-ordered and liquid-disordered domains in the membrane. In the model, like macromolecules interact with short-range Casimir attraction and long-range electrostatic repulsion, and the cross-interaction is of the opposite sign. We determine energetically favored patterns in a system in equilibrium with a bulk reservoir of the macromolecules. Different patterns consisting of clusters and stripes of the two components and of vacancies are energetically favorable for different values of the chemical potentials. Effects of thermal flutuations at low temperature are studied using Monte Carlo simulations in grand canonical and canonical ensembles. For fixed numbers of the macromolecules, a single two-component cluster with a regular pattern coexists with dispersed small one-component clusters, and the number of small clusters depends on the ratio of the numbers of the molecules of the two components. Our results show that the pattern formation is controlled by the shape of the interactions, the density of the proteins, and the proportion of the components.

In situ formation of biomolecular condensates as intracellular drug reservoirs for augmenting chemotherapy.

Biomolecular condensates, which arise from liquid-liquid phase separation within cells, may provide a means of enriching and prolonging the retention of small-molecule drugs within cells. Here we report a method for the controlled in situ formation of biomolecular condensates as reservoirs for the enrichment and retention of chemotherapeutics in cancer cells, and show that the approach can be leveraged to enhance antitumour efficacies in mice with drug-resistant tumours. The method involves histones as positively charged proteins and doxorubicin-intercalated DNA strands bioorthogonally linked via a click-to-release reaction between trans-cyclooctene and tetrazine groups. The reaction temporarily impaired the phase separation of histones in vitro, favoured the initiation of liquid-liquid phase separation within cells and led to the formation of biomolecular condensates that were sufficiently large to be retained within tumour cells. The controlled formation of biomolecular condensates as drug reservoirs within cells may offer new options for boosting the efficacies of cancer therapies.

Remarkable Insensitivity of Protein Diffusion to Protein Charge.

Friction to translational diffusion of ionic particles in polar liquids should scale linearly with the squared ion charge, according to standard theories. Substantial slowing of translational diffusion is expected for proteins in water. In contrast, our simulations of charge mutants of green fluorescent proteins in water show remarkable insensitivity of the translational diffusion constant to protein's charge in the range of charges between -29 and +35. The friction coefficient is given as a product of the force variance and the memory function relaxation time. We find remarkably accurate equality between the variance of the electrostatic force and the negative cross-correlation of the electrostatic and van der Waals forces. The charge invariance of the diffusion constant is a combined effect of the force variance and relaxation time invariances with the protein charge. The temperature dependence of the protein diffusion constant is highly non-Arrhenius, with a fragile-to-strong crossover at the glass transition.

An innovative method for fractionating the milk fat globule membrane and the major buttermilk proteins

Buttermilk (BM) is a by-product of butter manufacturing that shares a similar composition to skim milk but contains a higher concentration of phospholipids due to milk fat globule membrane (MFGM) fragments released during cream churning. Despite its high added-value components, MFGM is underutilized mainly due to the challenge in separating its components from the other constituents of buttermilk. Our work has focused on the use of calcium phosphate particles, called hydroxyapatite (HA), to extract and separate the different components from buttermilk. This study investigated the impact of various physicochemical parameters (temperature, ionic strength, and pH) on the adsorption of a mixture of micellar casein (CM), whey proteins, and MFGM from BM onto HA surfaces. A box-Behnken design was utilized to predict optimal physicochemical conditions for selectively separating components in buttermilk. Ionic strength (IS) played a crucial role, with low IS having a minimal positive influence and high IS negatively influencing adsorption. pH influenced adsorption by altering protein and HA surface charges. The optimal conditions for adsorption were an IS of 100 mM for a pH of 7, for 90 % of CM, 37 % of β-lactoglobulin, 11 % of α-lactalbumin and 7 % of MFGM. Desorption experiments using citrate and alkaline pH showed promising results for CM recovery, with citrate efficiently releasing the adsorbed CM. Additionally, MFGM were separated from whey proteins through selective precipitation at a pH of 4.0, allowing sedimentation of MFGM (100 %) while keeping whey proteins soluble. The presence of β-lactoglobulin in the protein fraction of MFGM (31 %) and CM (37 %) was attributed to pasteurization, which denatured a portion of β-lactoglobulin and led to its attachment to MFGM surfaces and interaction with CM. This protocol produces a nearly pure fraction of MFGM with a simple method which is suitable for industrial production, representing significant valorization for the dairy sector.