Hydrophobicity plays an important role in numerous physico-chemical processes, from the process of dissolution in water to protein folding, but its origin at the fundamental level is still unclear. The classical view of hydrophobic hydration is that in the presence of a hydrophobic solute, water forms transient microscopic ‘icebergs’ arising from strengthened water hydrogen bonding, yet there is no experimental evidence for enhanced hydrogen bonding and/or ‘icebergs’ in such solutions. Here we have used the redshifts and line-shapes of the isotopically decoupled infrared O-D stretching mode of small, purely hydrophobic solutes (methane, ethane, krypton, xenon) in water to study hydrophobicity at the most fundamental level. We present the first unequivocal and modelfree experimental proof for the presence of strengthened water hydrogen bonds near four hydrophobic solutes, matching those in ice and clathrates. The water molecules involved in the enhanced hydrogen bonds display extensive structural ordering resembling that in clathrates. The number of ice-like hydrogen bonds is 10 to 15 per methane molecule. Abinitio molecular dynamics simulations have confirmed that water molecules in the vicinity of methane form stronger, more numerous and more tetrahedrally oriented hydrogen bonds than those in bulk water, and that their mobility is restricted. We demonstrate the absence of intercalating water molecules that cause the electrostatic screening (shielding) of hydrogen bonds in bulk water as the critical element for the enhanced hydrogen bonding around a hydrophobic solute. Our results confirm the classical view of hydrophobic hydration.
COBISS.SI-ID: 6068250
The influence of a stepwise hydration on the secondary structure of Concanavalin A, a 273 amino acid residues long lectin protein, was monitored by infrared spectroscopy. An analysis of Amide I and Amide III bands, assignment of model bands and determination of the populations of secondary structure elements using computed integral intensity of particular amino bands, was used to determine the proportion of b-sheet in protein films recorded under various steps of hydration and solution in water. In dry protein film 53% of amino acid residues are in b-sheet conformation. The hydration increases a population of b-sheet to 57% determined in fully hydrated film which finally reached 61% in water solution. On the basis of characteristic differential spectra calculated from the various hydration levels, we established that in the initial stage of hydration water molecules bind through hydrogen bonds directly to the main and side chains of protein. Hydration of side chains mainly occupies COO and COOH groups. The in-crease of b-sheet population induced by hydration rearranged the water molecules attached to COOH side chain groups. A parallel analysis of Amide III bands shows that the Amid III region provides more complete information regarding the protein structure. Contemporary analysis of this region is very supportive, because it offers additional structural parameters which significantly contribute to reliability of secondary structure analysis by applying Amide I mode. Moreover, besides the comparable information about the population of secondary structure elements, the analysis of the Amide III area provides also the distribution of conformations of amino acids which are found in unstructured parts of protein.
COBISS.SI-ID: 6089242
During drug development, it is important to have a suitable crystalline form of the active pharmaceutical ingredient (API). Mostly, the basic options originate in the form of free base, acid, or salt. Substances that are stable only within a certain pH range are a challenge for the formulation. For the prazoles, which are known to be sensitive to degradation in an acid environment, the formulation is stabilized with alkaline additives or with the application of API formulated as basic salts. Therefore, preparation and characterization of basic salts are needed to monitor any possible salinization of free molecules. We synthesized salts of omeprazole from the group of alkali metals (Li, Na, and K) and alkaline earth metals (Mg, Ca). The purpose of the presented work is to demonstrate the applicability of vibrational spectroscopy to discriminate between the OMP and OMP-salt molecules. For this reason, the physicochemical properties of 5 salts were probed using infrared and Raman spectroscopy, NMR, TG, DSC, and theoretical calculation of vibrational frequencies. We found out that vibrational spectroscopy serves as an applicable spectroscopic tool which enables an accurate, quick, and nondestructive way to determine the characteristic of OMP and its salts.
COBISS.SI-ID: 27436087
Proteolipid protein (PLP) is one of the main proteins of myelin sheath that are destroyed during the progress of multiple sclerosis (MS). The immunodominant PLP139–151 epitope is known to induce experimental autoimmune encephalomyelitis (EAE, animal model of MS), wherein residues 144 and 147 are recognized by T cell receptor (TCR) during the formation of trimolecular complex with peptide-antigen and major histocompability complex. The conformational behavior of linear and cyclic peptide analogues of PLP, namely PLP139–151 and cyclic (139–151) (L144, R147) PLP139–151, have been studied in solution by means of nuclear magnetic resonance (NMR) methods in combination with unrestrained molecular dynamics simulations. The results indicate that the side chains of mutated amino acids in the cyclic analogue have different spatial orientation compared with the corresponding side chains of the linear analogue, which can lead to reduced affinity to TCR. NMR experiments combined with theoretical calculations pave the way for the design and synthesis of potent restricted peptides of immunodominant PLP139–151 epitope as well as non-peptide mimetics that rises as an ultimate goal.
COBISS.SI-ID: 6208026
Cholesterol is essential for development, growth, and maintenance of organisms. Mutations in cholesterol biosynthetic genes are embryonic lethal and few polymorphisms have been so far associated with pathologies in humans. Previous analyses show that lanosterol 14[alpha]-demethylase (CYP51A1) from the late part of cholesterol biosynthesis has only a few missense mutations with low minor allele frequencies and low association with pathologies in humans. The aim of this study is to evaluate the role of amino acid changes in the natural missense mutations of the hCYP51A1 protein. We searched SNP databases for existing polymorphisms of CYP51A1 and evaluated their effect on protein function. We found rare variants causing detrimental missense mutations of CYP51A1. Some missense variants were also associated with a phenotype in humans. Two missense variants have been prepared for testing enzymatic activity in vitro but failed to produce a P450 spectrum. We performed molecular modeling of three selected missense variants to evaluate the effect of the amino acid substitution on potential interaction with its substrate and the obligatory redox partner POR. We show that two of the variants, R277L and especially D152G, have possibly lower binding potential toward obligatory redox partner POR. D152G and R431H have also potentially lower affinity toward the substrate lanosterol. We evaluated the potential effect of damaging variants also using data from other in vitro CYP51A1 mutants. In conclusion, we propose to include damaging CYP51A1 variants into personalized diagnostics to improve genetic counseling for certain rare disease phenotypes.
COBISS.SI-ID: 33314265