Fourier Transform Infrared Spectroscopy in the Study of Hydrated Biological Macromolecules

The interaction between biological macromolecules (proteins, nucleic acids, lipids and other biomolecules in the cell) and environmental water is an important determining factor in their conformational properties, stability and function. The hydration processes of biopolymers have been extensively studied in the past 20 years with reference to a considerable variety of models and concepts. In all recent works, a distinction is made between intracellular water that maintains the ordinary liquid state (bulk water) and water ordered in extended hydrogen‐bonded lattices at the surface and structured in the internal grooves of macromolecules (hydration water) in dependence on the chemical properties of the macromolecule surface. FTIR spectroscopy has been implemented in this field both for the sensitivity in the conformational analysis of biological macromolecules and the reliability in the investigation of the water network. A perturbation technique such as dehydration‐rehydration treatment modifies the macromolecule structure and water distribution. It was applied to two structurally different proteins: lysozyme, a globular (α + β) protein and collagen, a fibrous protein characterized by the triple helix structure. Submitted to the treatment both of them display irreversible conformational changes

Hydration structure analysis of lysozyme amyloid fibrils by thermally stimulated depolarization currents (TSDC) technique

Thermally stimulated depolarization currents technique has been employed to investigate the conformation of hen egg white lysozyme in native and amyloid form, in the state of powder at very low hydration level. The technique, able to detect the current generated by thermally activated reorientation of water dipoles previously oriented by an electric field, exploits H2O dipoles, belonging to the solvation shell, as a probe to gain information on the protein conformation.Large differences are detected between the TSDC spectra related to the two different protein conformations, for what concerns the number and position of the main peaks, the native form displaying two peaks, atTM=175 K and atTM=297 K, and the amyloid one, only one at intermediate temperature (TM=235 K). The spectra have been compared with those monitored for poly-L-lysine (MW 80400), as received and prepared in different ways, i.e.α-helix,β-sheet, and coil conformation, respectively. The poly-L-lysine spectra show specific features that can be attributed to water texture around the secondary structure adopted by the macromolecule: the results stress how TSDC technique is a tool of great potential value in the conformational analysis of proteins

Insight on collagen self-assembly mechanisms by coupling molecular dynamics and UV spectroscopy techniques

Self-assembly of rat tail collagen type I was investigated by means of turbidity measurements and molecular dynamics simulations. Turbidity curves collected at different pH values show that the rate of aggregation was not linear in dependence from pH, with the fastest kinetics at pH 5.0 and the lowest at neutral pH. MD simulations were carried out on two regions with different hydropathicity, monitoring the aggregation of up to four staggered tropocollagen fragments at different ionic strength. At physiological conditions, association of lowly charged regions occurs more easily than for highly charged ones, the latter seeming to aggregate in a sequential way. The first contacts indicate for both regions that the driving force is hydrophobic, the electrostatic contribution becoming relevant at short distance. The direct inter-tropocollagen H-bonds confirm that fibrillogenesis is driven by loss of surface water from the monomers and involves in large percentage hydroxyproline residues. Low ionic strength dynamics leads to the formation of incorrect assemblies, driven by not shielded pairwise charge interactions

Hydration structure analysis of lysozyme amyloid fibrils by thermally stimulated depolarization currents (TSDC) technique

Abstract. Thermally stimulated depolarization currents technique has been employed to investigate the conformation of hen egg white lysozyme in native and amyloid form, in the state of powder at very low hydration level. The technique, able to detect the current generated by thermally activated reorientation of water dipoles previously oriented by an electric field, exploits H 2 O dipoles, belonging to the solvation shell, as a probe to gain information on the protein conformation. Large differences are detected between the TSDC spectra related to the two different protein conformations, for what concerns the number and position of the main peaks, the native form displaying two peaks, at T M = 175 K and at T M = 297 K, and the amyloid one, only one at intermediate temperature (T M = 235 K). The spectra have been compared with those monitored for poly-L-lysine (MW 80400), as received and prepared in different ways, i.e. α-helix, β-sheet, and coil conformation, respectively. The poly-L-lysine spectra show specific features that can be attributed to water texture around the secondary structure adopted by the macromolecule: the results stress how TSDC technique is a tool of great potential value in the conformational analysis of proteins

Fourier transform infrared conformational investigation of type I collagen aged by in vitro induced dehydration and non-enzymatic glycation treatments

The two main ageing-inducing events in the collagenous tissues are the water loss and the formation of intermolecular crosslinks based on the reaction of collagen with matrix carbohydrates, following a mechanism known as non-enzymatic-glycation. With the aim to mimic the two deleterious processes for the protein structure, rat-tail collagen was submitted to hydration changes and allowed to interact with two sugars characterized by different reducing properties, D-glucose and D-ribose. Fourier transform infrared (FTIR) spectroscopy was employed to investigate the conformational changes induced in the protein by the two treatments by analyzing the subsequent spectra modifications. FTIR spectra monitored: i) the amplitude and position changes of the two characteristic absorption bands OH stretching and Amide I, in dependence on the humidity level: a significant hysteresis effect in the ν(OH) band (ν~3400 cm–1) amplitude of the protein dehydrated and then rehydrated to the initial relative humidity (aw=0.92- 0.06) may be related to the enhancement of the β-sheet fraction in the protein structure as revealed by the parallel modification in the Amide I band (ν~1650 cm–1); ii) the area of the carbohydrate double band peaking at 1080 cm–1 and 1031 cm–1, associated to the accumulation of the glycation products, depending on the sugar concentration and incubation time. The association of both sugars to collagen only minimally affects the protein secondary structure as revealed by Amide I band Gaussian analysis. The whole set of results suggests hints to hypothesize a self-assembly model for collagen molecules induced by ageing

Study of skin degradation in ancient Egyptian mummies: complementarity of Fourier transform infrared spectroscopy and histological analysis

Some dynastic and pre-dynastic Egyptian mummies from the Giovanni Marro Museum of Anthropology and Ethnography, University of Turin, Italy have been studied by means of the combined approach of both Fourier transform infrared spectroscopy and histological analysis, with the aim of investigating the preservation state of the skin of ancient archaeological remains, as a consequence of the differences between the two kinds of mummification processes, i.e. natural and by means of embalming substances. The results suggest that the balms used in the dynastic mummies embalming process really could have played an important role in the prevention of corpse deterioration

Ft-Ir spectroscopy and microspectroscopy of ancient egyptian embalmed heads from the Museum of Anthropology and Ethnography of the University of Turin

Fourier Transform Infrared (FT-IR) spectroscopy and microspectroscopy were used to gain insight into the state of preservation of the skin of two Egyptian dynastic embalmed heads (VI-XI Dynasty) and one Predynastic mummy. The mummies came from the necropoles of Asiut and Gebelein (Upper Egypt), and are curated at the Museum of Anthropology and Ethnography of the University of Turin. The state of preservation was inferred from the biochemical composition of the skin, in particular from the secondary structure of the proteins. The secondary structure of the skin protein was investigated by studying the position and shape of the Amide I band, analyzed by means of self-deconvolution techniques. The increase in the b-sheet relative amount in the protein conformation of the mummified tissues with respect to the modern skin, was correlated with aging processes of collagen and keratins, the most abundant proteins in the skin. The steps of the degradation processes are hypothesized and described, and the differences in the recorded state of degradation were ascribed to the diverse mummification procedures undergone by the studied human remains. Other non-skin-derived features in the IR micro spectra of the embalmed specimens were detected and identified either as embalming materials or microbial attack traces

Melanin-Containing Films: Growth from Dopamine Solutions versus Layer-by-Layer Deposition

Films formed by oxidation of dopamine are of interest for functionalisation of solid–liquid interfaces owing to their versatility. However, the ability to modulate the properties of such films, for example, permeability to ionic species and the absorption coefficient, is urgently needed. Indeed, melanin films produced by oxidation of dopamine absorb strongly over the whole UV/Vis part of the electromagnetic spectrum and are impermeable to anions even for a film thickness as low as a few nanometers. Herein we combine oxidation of dopamine to produce a solution containing dopamine–melanin particles and their alternating deposition with poly(diallyldimethylammonium chloride) to produce films which have nearly the same morphology as pure dopamine–melanin films but are less compact, more transparent and more permeable to ferrocyanide anions.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78219/1/3299_ftp.pd