RESUMEN
Gram-negative bacteria possess a complex structural cell envelope that constitutes a barrier for antimicrobial peptides that neutralize the microbes by disrupting their cell membranes. Computational and experimental approaches were used to study a model outer membrane interaction with an antimicrobial peptide, melittin. The investigated membrane included di[3-deoxy-d-manno-octulosonyl]-lipid A (KLA) in the outer leaflet and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) in the inner leaflet. Molecular dynamics simulations revealed that the positively charged helical C-terminus of melittin anchors rapidly into the hydrophilic headgroup region of KLA, while the flexible N-terminus makes contacts with the phosphate groups of KLA, supporting melittin penetration into the boundary between the hydrophilic and hydrophobic regions of the lipids. Electrochemical techniques confirmed the binding of melittin to the model membrane. To probe the peptide conformation and orientation during interaction with the membrane, polarization modulation infrared reflection absorption spectroscopy was used. The measurements revealed conformational changes in the peptide, accompanied by reorientation and translocation of the peptide at the membrane surface. The study suggests that melittin insertion into the outer membrane affects its permeability and capacitance but does not disturb the membrane's bilayer structure, indicating a distinct mechanism of the peptide action on the outer membrane of Gram-negative bacteria.
Asunto(s)
Péptidos Antimicrobianos , Lipopolisacáridos , Lipopolisacáridos/química , Meliteno/química , Péptidos/química , Bacterias Gramnegativas/metabolismoRESUMEN
Piptoporus betulinus is a fungus known for its medicinal properties. It possesses antimicrobial, anti-inflammatory, and anti-cancer activity. In this study, several tests were performed to evaluate the cytotoxic effect of the ethanolic extract of Piptoporus betulinus on two melanoma human cell lines, WM115 primary and A375 metastatic cell lines, as well as Hs27 human skin fibroblasts. The extract proved to affect cancer cells in a dose-dependent manner, and at the same time showed a low cytotoxicity towards the normal cells. The total phenolic content (TPC) was determined spectrophotometrically by the Folin-Ciocalteu method (F-C), and the potential antioxidant activity was measured by ferric-reducing antioxidant power (FRAP) assay. One of the active compounds in the extract is betulin. It was isolated and then its cytotoxic activity was compared to the results obtained from the Piptoporus betulinus extract. To further understand the mechanism of action of the extract's anticancer activity, tests on model cell membranes were conducted. A model membrane of a melanoma cell was designed and consisted of 1,2-dimyristoyl-sn-glycero-3-phosphocholine, disialoganglioside-GD1a and cholesterol: DMPC:GD1a:chol (5:2:3 mole ratio). Changes in a Langmuir monolayer were observed and described based on Π-Amol isotherm and compressibility modulus changes. LB lipid bilayers were deposited on a hydrophilic gold substrate and analyzed by IR and X-ray photoelectron spectroscopy. Our study provides new data on the effect of Piptoporus betulinus extract on melanoma cells and its impact on the model of melanoma plasma membranes.
Asunto(s)
Etanol , Melanoma , Humanos , Membrana Celular , Antioxidantes/farmacología , Extractos Vegetales/farmacología , Melanoma/tratamiento farmacológico , Proliferación CelularRESUMEN
The structural complexity of the cell envelope of Gram-negative bacteria limits the fabrication of realistic models of bacterial cell membranes. A vertical Langmuir-Blodgett withdrawing was used to deposit a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) monolayer on the Au(111) surface. The second leaflet composed of di[3-deoxy-D-manno-octulosonyl]-lipid A (KLA) was deposited using Langmuir-Schaefer transfer. The use of an electrode material as a support for the POPE-KLA bilayer allowed electrochemical control of the membrane's stability, compactness, and structure. Capacitance-potential curves showed a typical pattern for the supported lipid bilayers electrochemical characteristic. The minimum membrane capacitance was â¼4 µF cm-2 and did not change in the following desorption-adsorption cycles, indicating the presence of a stable bilayer structure with an asymmetric composition of both leaflets. However, at a molecular scale, as elucidated in spectroelectrochemical experiments, large differences in the response of both leaflets to electric potentials were observed. The acyl chains in POPE and KLA existed in a liquid state. The quantitative analysis of the CH stretching modes indicated potential-driven reorientations in the hydrophobic fragment of the bilayer, already in the adsorbed state. To assign observed rearrangements to POPE and KLA lipids in both leaflets, per-deuterated d31-POPE was transferred into the inner leaflet. Since no potential-dependent changes of the CD2 stretching modes in the d31-POPE-KLA bilayer were observed, reorientations in the acyl chain region were assigned to the KLA molecules. Mg2+ ions were bound to the polar head groups of KLA. The strength of electrostatic interactions in the polar head group region of KLA was dependent on the direction of the electric field. At negative electric potentials, the binding of divalent cations weakened, which gave the KLA molecules increased orientational flexibility. This behavior in electric fields is peculiar for the outer membrane and indicates that the microbial cell membranes have different electrochemical properties than phospholipid bilayers.
Asunto(s)
Membrana Dobles de Lípidos , Fosfatidiletanolaminas , Bacterias Gramnegativas , Potenciales de la Membrana , FosfolípidosRESUMEN
Forisomes are giant self-assembling mechanoproteins that undergo reversible structural changes in response to Ca2+ and various other stimuli. Artificial forisomes assembled from the monomer MtSEO-F1 can be used as smart biomaterials, but the molecular basis of their functionality is not understood. To determine the role of protein polymerization in forisome activity, we tested the Ca2+ association of MtSEO-F1 dimers (the basic polymerization unit) by circular dichroism spectroscopy and microscale thermophoresis. We found that soluble MtSEO-F1 dimers neither associate with Ca2+ nor undergo structural changes. However, polarization modulation infrared reflection absorption spectroscopy revealed that aggregated MtSEO-F1 dimers and fully-assembled forisomes associate with Ca2+ , allowing the hydration of poorly-hydrated protein areas. A change in the signal profile of complete forisomes indicated that Ca2+ interacts with negatively-charged regions in the protein complexes that only become available during aggregation. We conclude that aggregation is required to establish the Ca2+ response of forisome polymers.
Asunto(s)
Proteínas de Plantas , Proteínas de Plantas/química , PolimerizacionRESUMEN
The cell membrane of Gram-negative bacteria interacting with an antimicrobial peptide presents a complex supramolecular assembly. Fabrication of models of bacterial cell membranes remains a large experimental challenge. Langmuir-Blodgett and Langmuir-Schaefer (LS-LB) transfer makes possible the deposition of multicomponent asymmetric lipid bilayers onto a gold surface. Two lipids: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and di[3-deoxy-D-manno-octulosonyl]-lipid A (KLA) were used to deposit a model of the outer membrane of Gram-negative bacteria on the Au(111) substrate. The use of gold as the solid substrate enables control of the membrane potential. Molecular scale changes in the model membrane exposed to physiological electric fields and interacting with melittin antimicrobial peptide are discussed in this paper. The interaction of the outer membrane with melittin leads to an increase in the membrane capacitance and permeability to ions and water. The stability of the outer membrane with bound melittin decreases at positive membrane potentials. In situ polarization modulation infrared reflection absorption spectroscopy is used to investigate membrane potential-dependent changes in the structure of the outer membrane interacting with melittin. The hydration of the ester carbonyl groups is not affected by the interaction with melittin. However, the orientation and hydrogen bond network with the carboxylate groups in KLA changes drastically after POPE-KLA bilayer interacts with melittin. We propose that the positively charged groups in the amino acids present at the C-terminus of the peptide interact directly with the polar head group of KLA. Simultaneously, the packing order in hydrocarbon chains in the membrane with bound melittin increases. A hydrophobic match between the chains in the lipids and the peptide, which spans the membrane, seems to be responsible for the ordering of the hydrocarbon chains region of the bilayer. The N-terminus enters into the hydrophobic region of the membrane and forms a channel to the hydrophilic head groups in POPE.
Asunto(s)
Membrana Dobles de Lípidos , Meliteno , Membrana Celular , Bacterias Gramnegativas , Espectrofotometría InfrarrojaRESUMEN
Adsorbed protein films consist of essential building blocks of many biotechnological and biomedical devices. The electrostatic potential may significantly modulate the protein behaviour on surfaces, affecting their structure and biological activity. In this study, lysozyme was used to investigate the effects of applied electric potentials on adsorption and the protein structure. The pH and the surface charge determine the amount and secondary structure of adsorbed lysozyme on a gold surface. In-situ measurements using polarization modulation infrared reflection absorption spectroscopy indicated that the concentration of both the adsorbed anions and the lysozyme led to conformational changes in the protein film, which was demonstrated by a greater amount of aggregated ß-sheets in films fabricated at net positive charges of the Au electrode (Eads > Epzc). The changes in secondary structure involved two parallel processes. One comprised changes in the hydration/hydrogen-bond network at helices, leading to diverse helical structures: α-, 310- and/or π-helices. In the second process ß-turns, ß-sheets, and random coils displayed an ability to form aggregated ß-sheet structures. The study illuminates the understanding of electrical potential-dependent changes involved in the protein misfolding process.
Asunto(s)
Oro/química , Muramidasa/química , Enlace de Hidrógeno , Pliegue de Proteína , Estructura Secundaria de Proteína , Electricidad Estática , Propiedades de SuperficieRESUMEN
Knowledge of the structure of the electrical double layer in ionic liquids (IL) is crucial for their applications in electrochemical technologies. We report the synthesis and applicability of an imidazolium-based amphiphilic ionic liquid with a perdeuterated alkyl chain for studies of electric potential-dependent rearrangements, and changes in the microenvironment in a monolayer on a Au(111) surface. Electrochemical measurements show two states of the organization of ions on the electrode surface. In situ IR spectroscopy shows that the alkyl chains in imidazolium cations change their orientation depending on the adsorption state. The methylene-d2 stretching modes in the perdeuterated IL display a reversible, potential-dependent appearance of a new band. The presence of this mode also depends on the anion in the IL. Supported by quantum chemical calculations, this new mode is assigned to a second νas (CD2 ) band in alkyl-chain fragments embedded in a polar environment of the anions/solvent present in the vicinity of the imidazolium cation and electrode. It is a measure of the potential-dependent segregation between polar and nonpolar environments in the layers of an IL closest to the electrode.
RESUMEN
A self-assembled monolayer (SAM) on gold was prepared from a diaminoterephthalate (DAT) derivative as functional molecule and 1-decanthiol as a backfiller. The DAT derivative is N-protected by a tert-butyloxycarbonyl (Boc) group and is anchored to the gold surface via a liponic acid as a stable anchor group. The terminal DAT moiety exhibits interesting effector properties such as fluorescence and electrochemical activity. Irreversible oxidation of the monolayer at 0.4 V (Hg|Hg2SO4) in 0.1 M HClO4 triggers deprotection of the DAT group and subsequent chemical reactions, during which 10% of the DAT groups of the original SAM are transformed to a new surface-bound, quasi-reversible redox couple with a formal potential of 0.0 V (Hg|Hg2SO4) and a standard rate constant of 8 s-1 in 0.1 M HClO4. Immersion of the mixed SAM in 0.1 M HClO4 at open circuit potential or oxidation in 0.1 M H2SO4 did not produce this surface-bound redox couple. The monolayers were thoroughly characterized by X-ray photoelectron spectroscopy (XPS) and polarization modulation infrared reflection absorption spectroscopy (PM IRRAS) after the different preparation steps indicating only minor changes in the overall composition of the monolayer, in particular, the preservation of the heteroatoms. The new redox couple is likely a diimine, in agreement with its ability to bind nucleophiles such as anilines by conjugate addition that could be followed by multicycle voltammetry and XPS. The DAT effector group is especially interesting because it can also report the binding reaction by changed electrochemical and fluorescence signals.
RESUMEN
Compatible solutes accumulate in the cytoplasm of halophilic microorganisms, enabling their survival in a high-salinity environment. Ectoine is such a compatible solute. It is a zwitterionic molecule that strongly interacts with surrounding water molecules and changes the dynamics of the local hydration shell. Ectoine interacts with biomolecules such as lipids, proteins, and DNA. The molecular interaction between ectoine and biomolecules, in particular the interaction between ectoine and DNA, is far from being understood. In this paper, we describe molecular aspects of the interaction between ectoine and double-stranded DNA (dsDNA). Two 20 base pairs-long dsDNA fragments were immobilized on a gold surface via a thiol-tether. The interaction between the dsDNA monolayers with diluted and concentrated ectoine solutions was examined by means of X-ray photoelectron and polarization modulation infrared reflection absorption spectroscopies (PM IRRAS). Experimental results indicate that the ability of ectoine to bind water reduces the strength of hydrogen bonds formed to the ribose-phosphate backbone in the dsDNA. In diluted (0.1 M) ectoine solution, DNA interacts predominantly with water molecules. The sugar-phosphate backbone is involved in the formation of strong hydrogen bonds to water, which, over time, leads to a reorientation of the planes of nucleic acid bases. This reorientation destabilizes the strength of hydrogen bonds between the bases and leads to a partial dehybridization of the dsDNA. In concentrated ectoine solution (2.5 M), almost all water molecules interact with ectoine. Under this condition, ectoine is able to interact directly with DNA. Density functional theory (DFT) calculations demonstrate that the direct interaction involves the nitrogen atoms in ectoine and phosphate groups in the DNA molecule. The results of the quantum-chemical calculations show that rearrangements in the ribose-phosphate backbone, caused by a direct interaction with ectoine, facilitates contacts between the O atom in the phosphate group and H atoms in a nucleic acid base. In the PM IRRA spectra, an increase in the number of IR absorption modes in the base pair frequency region proves that the hydrogen bonds between bases become weaker. Thus, a sequence of reorientations caused by interaction with ectoine leads to a breakdown of hydrogen bonds between bases in the double helix.
Asunto(s)
Aminoácidos Diaminos , Biopolímeros , ADN , Enlace de HidrógenoRESUMEN
Planar asymmetric lipid bilayers composed of phosphatidylethanolamine and phosphatidylglycerol lipids are transferred onto a gold electrode surface. Lipids containing two saturated, one monounsaturated and two monounsaturated hydrocarbon chains compose the model membranes. Results of electrochemically controlled polarization modulation infrared reflection absorption spectroscopy and quartz crystal microbalance with energy dissipation studies reveal two different types of electric potential-dependent structural rearrangements in the bilayers. They are correlated with the geometry of the lipid molecule. Packing parameter correlates the cross-section area of the hydrophobic and hydrophilic parts of amphiphilic molecules. In bilayers composed of lipids with the packing parameter <1, the hydrocarbon chains are tilted with respect to the bilayer plane and the polar head groups are well hydrated. At a threshold potential an abrupt flow of water through the bilayer is connected with membrane dehydration and upward orientation of the chains. In bilayers composed of lipids with packing parameter ≥1, electric potentials have negligible effect on the membrane structure. A simple rule correlating the packing parameter with molecular scale changes occurring at electrified membranes has a large diagnostic implication for biomimetic studies and our understanding of molecular processes occurring in biological cell membranes.
Asunto(s)
Electrodos , Lípidos/química , Membranas Artificiales , Interacciones Hidrofóbicas e Hidrofílicas , Membrana Dobles de Lípidos/química , Espectrofotometría Infrarroja/métodosRESUMEN
Droplet-based bio-printing (DBB) techniques have been extensively accepted due to their simplicity, flexibility and cost performance. However, the applicability of inkjet printing for bioprinting techniques still faces challenges, such as a narrow range of available bio-ink materials, cell damage due to the pressure strike and high shear rate during the printing process. Here, a new droplet-based printing technique, pneumatic conveying printing (PCP), is described. This new technique is successfully adopted for cell-printing purposes. The cells present in the bio-ink are not exposed to any significant pressure and therefore the PCP technique is gentle to the cells. Furthermore, PCP allows the usage of inks with viscosities higher than 1000 mPa s, enabling the usage of bio-inks with high cell concentrations (several tens of millions per millilitre). As a proof of concept, two different cell types were printed with this novel technique. To achieve a printing resolution of 400 to 600 µm, cells were encapsulated into a hydrogel containing calcium alginate. Deposition of the bio-ink drop containing sodium alginate on a surface pre-treated in CaCl2 solution, ensures a fast cross-linking reaction and the formation of gel drops. Cells encapsulated in the alginate gel survive and proliferate. Our novel PCP technique is highly suitable for 2D and 3D cell bio-printing.
RESUMEN
Many cytoplasmic proteins contain a hydrophobic acyl chain, which facilitates protein binding to cell membranes. Hydrophobic interactions between the exposed acyl chain of the protein and hydrocarbon chains of lipids in the cell membrane are the driving force for this specific lipid-protein interaction. Recent studies point out that in addition to hydrophobic interactions the charge-charge and charge-dipole interactions between the polar head groups and basic amino acids contribute significantly to the binding process. Recoverin possesses a myristoyl chain at the N-terminus. In the presence of Ca2+ ions, the protein undergoes structural rearrangements, leading to the extrusion of the myristoyl chain, facilitating the protein binding to the membrane. In this work, we investigate the impact of interactions between the polar head group region of lipid molecules and recoverin which binds to the model membrane. The interaction with a planar lipid bilayer composed of phosphatidylcholine and cholesterol with myristoylated and nonmyristoylated recoverin is studied by in situ polarization modulation infrared reflection absorption spectroscopy. The binding of recoverin to the lipid bilayer depends on the transmembrane potential, indicating that the orientation of the permanent surface dipole in the supramolecular assembly of the lipid membrane influences the protein attachment to the membrane surface. Analysis of the amide I' mode indicates that the orientation of recoverin bound to the lipid bilayer is independent of the presence of myristoyl chain in the protein and of the folding of the protein into the tense or relaxed state. In contrast, it changes as a function of the membrane potential. At positive transmembrane potentials, the α-helical fragments of recoverin are oriented predominantly parallel to the bilayer surface. This orientation facilitates the insertion of the acyl chain of the protein into the hydrophobic region of the bilayer. At negative transmembrane potentials, the α-helical fragments of recoverin change their orientation with respect to the membrane surface, which is followed by the removal of the myristoyl chain from the membrane.
Asunto(s)
Membrana Celular/metabolismo , Ácido Mirístico/metabolismo , Recoverina/química , Recoverina/metabolismo , Membrana Dobles de Lípidos/metabolismo , Potenciales de la Membrana , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
The neuronal calcium sensor protein recoverin is expressed in retinal rod and cone cells and is involved in the calcium-dependent control of receptor (rhodopsin) phosphorylation and receptor inactivation. In its Ca2+-saturated form recoverin is attached to membranes by an exposed myristoyl group and responds to oscillating changes of intracellular Ca2+-concentration by performing a so-called Ca2+-myristoyl switch. In this work we analyze changes in a liquid lipid bilayer interacting with myristoylated and non-myristoylated recoverin by employing polarization modulation infrared reflection absorption spectroscopy (PM IRRAS) with electrochemical control. The lipid bilayer is transferred onto a polycrystalline gold electrode using Langmuir-Blodgett Langmuir-Schaefer transfer at the surface pressure πâ¯=â¯30â¯mNâ¯m-1 which ensures, necessary for the lipid-protein interaction, liquid state of the hydrocarbon chains of phospholipids. The model lipid bilayers are adsorbed directly on the Au electrode surface at transmembrane potentials -0.2â¯<â¯∆ÏM|Sâ¯<â¯0.25â¯V. The interaction with recoverin leads to a stabilization of the adsorbed state of the lipid bilayer at positive transmembrane potentials. The interaction leads to a decrease in the surface charge density that accumulates on the membrane covered electrode surface, indicating changes in the lateral interactions in the lipid membrane. In situ spectroelectrochemical studies confirm orientation changes in the hydrophobic environment of the model membrane. Insertion of the myristoyl group of recoverin into the membrane is connected with an increase in the tilt of the hydrocarbon chains with respect to the surface normal and decrease in the bilayer thickness. Potential-induced pore formation and desorption of the lipid bilayer from the membrane surface is accompanied by the removal of the acyl chains of recoverin from the membrane.
Asunto(s)
Proteínas de la Membrana/metabolismo , Ácido Mirístico/metabolismo , Membrana Celular/metabolismo , Dimiristoilfosfatidilcolina/química , Dimiristoilfosfatidilcolina/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Membrana Dobles de Lípidos , Unión Proteica , Espectrofotometría Infrarroja/métodosRESUMEN
In this paper we analyze the changes in molecular orientation triggered by electrochemical reduction of an iron-containing surfactant in Langmuir-Blodgett films deposited onto gold electrodes. The metallosurfactant [Feiii(LN2O3)] (1) is an established molecular rectifier capable of unidirectional electron transfer between two electrodes. A gradual decrease in the activity is observed in sequential current vs. potential curves upon repeated cycles. Here we evaluate the redox response associated with the reduction of the Feiii/Feii couple in a single monolayer, as well as in a 5-layer LB film of 1. We use polarization modulation infrared reflection absorption spectroscopy (PM IRRAS) to follow structural and orientation changes associated with such applied potential scans. We observe that the reduction of the Fe center becomes increasingly irreversible because an Fe-Ophenolate bond is cleaved. This transformation is accompanied by an almost vertical change in the orientation of metallosurfactant molecules in LB films.
RESUMEN
Unique electronic and ligand recognition properties of the DNA double helix provide basis for DNA applications in biomolecular electronic and biosensor devices. However, the relation between the structure of DNA at electrified interfaces and its electronic properties is still not well understood. Here, potential-driven changes in the submolecular structure of DNA double helices composed of either adenine-thymine (dAdT)25 or cytosine-guanine (dGdC)20 base pairs tethered to the gold electrodes are for the first time analyzed by in situ polarization modulation infrared reflection absorption spectroscopy (PM IRRAS) performed under the electrochemical control. It is shown that the conformation of the DNA duplexes tethered to gold electrodes via the C6 alkanethiol linker strongly depends on the nucleic acid sequence composition. The tilt of purine and pyrimidine rings of the complementary base pairs (dAdT and dGdC) depends on the potential applied to the electrode. By contrast, neither the conformation nor orientation of the ionic in character phosphate-sugar backbone is affected by the electrode potentials. At potentials more positive than the potential of zero charge (pzc), a gradual tilting of the double helix is observed. In this tilted orientation, the planes of the complementary purine and pyrimidine rings lie ideally parallel to each other. These potentials do not affect the integral stability of the DNA double helix at the charged interface. At potentials more negative than the pzc, DNA helices adopt a vertical to the gold surface orientation. Tilt of the purine and pyrimidine rings depends on the composition of the double helix. In monolayers composed of (dAdT)25 molecules the rings of the complementary base pairs lie parallel to each other. By contrast, the tilt of purine and pyrimidine rings in (dGdC)20 helices depends on the potential applied to the electrode. Such potential-induced mobility of the complementary base pairs can destabilize the helix structure at a submolecular level. These pioneer results on the potential-driven changes in the submolecular structure of double stranded DNA adsorbed on conductive supports contribute to further understanding of the potential-driven sequence-specific electronic properties of surface-tethered oligonucleotides.
Asunto(s)
ADN/química , Electrodos , Fenómenos Electromagnéticos , Oro/química , Oligodesoxirribonucleótidos/química , Emparejamiento Base , Conformación de Ácido Nucleico , Espectrofotometría InfrarrojaRESUMEN
The polarization modulation infrared reflection-absorption spectra of collagen adsorbed on a titania surface and quantum chemical calculations are used to describe components of the amide I mode to the protein structure at a sub-molecular level. In this study, imino acid rich and poor fragments, representing the entire collagen molecule, are taken into account. The amide I mode of the collagen triple helix is composed of three absorption bands which involve: (i) (â¼1690cm(-1)) the CO stretching modes at unhydrated groups, (ii) (1655-1673cm(-1)) the CO stretching at carbonyl groups at imino acids and glycine forming intramolecular hydrogen bonds with H atoms at both NH2 and, unusual for proteins, CH2 groups at glycine at a neighbouring chain and (iii) (â¼1640cm(-1)) the CO stretching at carbonyl groups forming hydrogen bonds between two, often charged, amino acids as well as hydrogen bonds to water along the entire helix. The IR spectrum of films prepared from diluted solutions (c<50µgml(-1)) corresponds to solution spectra indicating that native collagen molecules interact with water adsorbed on the titania surface. In films prepared from solutions (c⩾50µgml(-1)) collagen multilayers are formed. The amide I mode is blue-shifted by 18cm(-1), indicating that intramolecular hydrogen bonds at imino acid rich fragments are weakened. Simultaneous red-shift of the amide A mode implies that the strength of hydrogen bonds at the imino acid poor fragments increases. Theoretically predicted distortion of the collagen structure upon adsorption on the titania surface is experimentally confirmed.