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This study investigates the interface between ice and organic solvents using atomic force microscopy (AFM). Atomically flat ice surfaces were prepared and observed by AFM in 1-octanol, 1-hexanol, and 1-butanol. The results show differences in surface roughness influenced by the interaction of ice and alcohols. Young's modulus of ice was analyzed by force curve measurements, providing valuable insights into the properties of ice in liquid environments. The results showed the characteristics of the ice surface in different solvents, suggesting potential applications in understanding surface and interface phenomena associated with ice under realistic conditions. [ABSTRACT FROM AUTHOR]

Antigen-presenting cells (APCs) play a crucial role in the immune system by breaking down antigens into peptide fragments that subsequently bind to major histocompatibility complex (MHC) molecules. Previous studies indicate that stable proteins can impede CD4+ T cell stimulation by hindering antigen processing and presentation. Conversely, certain proteins require stabilization in order to activate the immune response. Several factors, including the characteristics of the protein and the utilization of different adjuvants in animal experiments, may contribute to this disparity. In this study, we investigated the impact of adjuvants on antigen administration in mice, specifically focusing on the stability of the CH2 domain. Consequently, the CH2 domain induced a stronger IgG response in comparison to the stabilized one when using Alum and PBS (without adjuvant). On the other hand, animal experiment using Freund's adjuvant showed the opposite results. These findings indicate the significance of considering the intrinsic conformational stability of a protein when eliciting its immunogenicity, particularly within the context of vaccine development. [ABSTRACT FROM AUTHOR]

Protein aggregations decrease production yields and impair the efficacy of therapeutics. The CH2 domain is a crucial part of the constant region of human IgG. But, it is also the least stable domain in IgG, which can result in antibody instability and aggregation problems. We created a novel mutant of the CH2 domain (T250C/L314C, mut10) by introducing a disulfide bond and expressed it using Pichia pastoris. The mut10 variant exhibited enhanced thermal stability, resistance to enzymatic degradation, and reduced aggregation in comparison to the original CH2 domain. However, it was less stable than mut20 (L242C/K334C), which is the variant prepared in a previous study (Gong et al., J. Biol. Chem., 2009). A more advanced mutant, mut25, was created by combining mut10 and mut20. Mut25 artificially contains two disulfide bonds. The new mutant, mut25, showed enhanced thermal stability, increased resistance to enzymatic digestion, and reduced aggregation in comparison to mut20. According to our knowledge, mut25 achieves an unprecedented level of stability among the humanized whole CH2 domains that have been reported so far. Mut25 has the potential to serve as a new platform for antibody therapeutics due to its ability to reduce immunogenicity by decreasing aggregation. [ABSTRACT FROM AUTHOR]

The interaction of egg-white lysozyme with N-ovalbumin, the native form of egg-white ovalbumin with the denaturation temperature, [T.sub.m], of 78°C, was investigated by the inhibition of lysozyme muramidase activity, differential scanning calorimetry, and circular dichroism assay as indicators. Signals for the interaction were the most prominent when the mixture of lysozyme and N-ovalbumin was co-heated at 72°C, slightly lower than the [T.sub.m] of N-ovalbumin. The interaction was also marked when unheated lysozyme was mixed with N-ovalbumin preheated at 72°C. Moreover, the mixture rapidly formed fibrous precipitates, which were positive for thioflavin T fluorescent emission, a marker for the amyloid fibril formation. Also electron microscopic observation exhibited features of fibrils. The interaction potency of ovalbumin was ascribed to the tryptic fragment ILELPFASGT MSMLVLLPDE VSGLEQLESIINFEK (residues 229-263), derived from the 2B strands 2 and 3 of ovalbumin. From lysozyme, on the other hand, the chymotryptic peptide RNRCKGTDVQAW (residues 112-123), including cluster 6, and the chymotryptic/tryptic peptide GILQINSRW (residues 54-62), including cluster 3, were responsible for the interaction with N-ovalbumin. Interestingly, this nonamer peptide was found to have the ability to self-aggregate. To the authors knowledge, this may be the first report to document the possible involvement of dual proteins in the formation of amyloid-like fibrils. Key words: lysozyme, ovalbumin, egg white, amyloid fibril formation, core peptide, protein-protein interaction. L'interaction entre le lysozyme du blanc d'oeuf et la N-ovalbumine, la forme native de l'ovalbumine du blanc d'oeuf dont la temperature de denaturation, [T.sub.m], est de 78[degre]C, a ete etudiee par l'inhibition de l'activite muraminidase du lysozyme, par calorimetrie differentielle a balayage et par dichroisme circulaire. Les signaux indicateurs de l'interaction etaient plus forts lorsque le melange de lysozyme et de N-ovalbumine etait chauffe a 72[degre]C, soit une temperature legerement inferieure au [T.sub.m] de la N-ovalbumine. L'interaction etait aussi marquee lorsque le lysozyme non chauffe etait melange a la Novalbumine prechauffee a 72[degre]C. De plus, le melange formait rapidement des precipites fibreux fluorescents en presence de thioflavine T, un marqueur de la formation des fibrilles amyloides. Des elements caracteristiques des fibrilles etaient aussi observes en microscopie electronique. La force de l' interaction de l'ovalbumine a ete attribuee au fragment tryptique ILELPFASGT MSMLVLLPDE VSGLEQLESIINFEK (residus 229-263), derive des portions 2 et 3 du fragment 2B de l'o valbumine. D'un autre cote, sur le lysozyme, le peptide chymotriptique RNRCKGTDVQAW (residus 112-123), comprenant le cluster 6, et le peptide chymotryptique/tryptique GILQINSRW (residus 54-62) comprenant le cluster 3, etaient responsables de l'interaction avec la N-ovalbumine. Fait interessant, ce peptide nonamere avait la propriete de s'auto-agreger. A la connaissance des auteurs, ceci pourrait constituer le premier rapport a documenter l'implication possible de deux proteines dans la formation de fibrilles de type amyloide. Mots-cles : lysozyme, ovalbumine, blanc d'oeuf, formation de fibrilles amyloides, peptide central, interaction proteineproteine. [Traduit par la Redaction], Introduction Misfolding and aggregation of proteins often cause serious pathological disorders via the deposition of amyloid fibrils (Chiti and Dobson 2006). These possess a common cross-β structural motif, having β-strands [...]

Diacylglycerol kinases (DGKs) are multi‐domain lipid kinases that modulate the levels of lipid messengers, diacylglycerol, and phosphatidic acid. Recently, increasing attention has been paid to its α isozyme (DGKα) as a potential target for cancer immunotherapy. However, little progress has been made on the structural biology of DGKs, and a detailed understanding of the Ca2+‐triggered activation of DGKα, for which the N‐terminal domains likely play a critical role, remains unclear. We have recently shown that Ca2+ binding to DGKα‐EF induces conformational changes from a protease‐susceptible "open" conformation in the apo state to a well‐folded one in its holo state. Here, we further studied the structural properties of DGKα N‐terminal (RVH and EF) domains using a series of biophysical techniques. We first revealed that the N‐terminal RVH domain is a novel Ca2+‐binding domain, but the Ca2+‐induced conformational changes mainly occur in the EF domain. This was corroborated by NMR experiments showing that the EF domain adopts a molten‐globule like structure in the apo state. Further analyses using SEC‐SAXS and NMR indicate that the partially unfolded EF domain interacts with RVH domain, likely via hydrophobic interactions in the absence of Ca2+, and this interaction is modified in the presence of Ca2+. Taken together, these results present novel insights into the structural rearrangement of DGKα N‐terminal domains upon binding to Ca2+, which is essential for the activation of the enzyme. [ABSTRACT FROM AUTHOR]

The introduction of intermolecular disulfide bonds by amino acid mutations is an effective method for stabilizing dimeric proteins. X-ray crystal structure of Fab of a therapeutic antibody, adalimumab, revealed the first loop of the CH1 domain to be partially unsolved at position 135–141. To find new sites for the introduction of intermolecular disulfide bonds in adalimumab Fab, Fab mutants targeting the unsolved region were predicted using molecular simulation software. Four Fab mutants, H:K137C-L:I117C, H:K137C-L:F209C, H:S138C-L:F116C and H:S140C-L:S114C, were expressed in the methylotrophic yeast Pichia pastoris. SDS-PAGE analysis of these mutants indicated that H:K137C-L:F209C, H:S138C-L:F116C and H:S140C-L:S114C mutants mostly formed intermolecular disulfide bonds, whereas some H:K137C-L:I117C mutants formed intermolecular disulfide bonds and some did not. Differential scanning calorimetry measurements showed increased thermal stability in all Fab mutants with engineered disulfide bonds. The bio-layer interferometry measurements, for binding of the antigen tumor necrotic factor α, indicated that Fab mutants had less antigen-binding activity than wild-type Fab. In particular, the K D value of H:K137C-L:F209C was ~17 times higher than that of wild-type Fab. Thus, we successfully introduced intermolecular disulfide bonds between the first loop region of the CH1 and CL domains and observed that it increases the thermostability of Fab and affects the antigen-binding activity. [ABSTRACT FROM AUTHOR]