Changes of albumin secondary structure after palmitic acid binding : FT-IR spectroscopic study

Purpose: Albumin is an universal transport protein. Plasma pool of free fatty acids arising from triglyceride hydrolysis, critical in energy metabolism and etiology of metabolic disorders is transported by albumin. According to various studies albumin has from seven to nine binding sites with diverse affinity to long chain fatty acids. X-ray diffraction crystallography measurements have provided data only for pure human serum albumin or albumin with fully saturated binding sites. These results have shown that amount of -helices is higher after fatty acids binding. Molecular mechanics simulations suggest that binding of fatty acids in two high-affinity sites leads to major conformational changes in albumin structure. The aim of this research was to investigate albumin secondary structure upon gradually increasing fatty acids to protein mole ratio. Methods: Fourier transform infrared spectroscopy was applied to study changes of bovine serum albumin (as an analogue of human serum albumin) -helical structures after binding palmitic acid in a range of 0–20 palmitic acid: albumin molar ratios representing pure protein, partial, full saturation and excess binding sites capacity. Results: Amount of -helices was increasing along with the amount of palmitic acid: bovine serum albumin molar ratio and reached maximum value around 2 mol/mol. Conclusions: Our studies confirmed molecular mechanics simulations and crystallographic studies. Palmitic acid binding in two high-affinity sites leads to major structural changes, filling another sites only slightly influenced bovine serum albumin secondary structure. The systematic study of fatty acids and albumin interactions, using an experimental model mimicking metabolic disorders, may results in new tools for personalized nanopharmacotherapy

Mechanobiology of soft tissues: FT-Raman spectroscopic studies

FT-Raman spectroscopy was used to investigate microstructural changes in the secondary protein structure of soft tissues subjected to increasing levels of macroscopic strain. Main protein bands at 938 cm-1 assigned as v(Cα–C), 1668 cm-1 — amide I and 1268 cm-1 — amide III are sensitive to applied strain and undergo wavenumber shifting. Other main vibrational modes at 1004 cm-1 assigned to the phenyl ring breathing mode and 2940 cm-1 (n(CH3,CH2)) remain unaltered. Spectroscopic results were compared with the mechanical relations obtained from the standard protocol of uniaxial tensile tests carried out in a testing machine. A clear correlation between Raman band shifting and the level of mechanical stress was established. Initially the load is transferred through elastin and then gradually also by collagen. It was proved that transferring loads by soft tissues involves changes in structural protein conformation. This process was described in detail for a tendon. It was also confirmed that mechanical properties of soft tissues depend on collagen and elastin fiers orientation

Human skin properties determined by mechanical tests and Raman spectroscopy

Celem przeprowadzonych badań było porównanie charakterystyk otrzymanych: w testach wytrzymałościowych i z pomiarów spektroskopowych. W pracy wyznaczono podstawowe parametry mechaniczne skóry, które są zdeterminowane ułożeniem włókien kolagenowych. Następnie zarejestrowano widma ramanowskie badanej tkanki, zidentyfikowano pasma charakterystyczne dla białka kolagenowego. Na podstawie analizy uzyskanych wyników, dla kolejnych etapów rozciągnięcia skóry, zaobserwowano między innymi różnice w położeniu maksimum pasma amidu I (1658cm-1) w zależności od kierunku rozciągania próbki. Porównanie, w obu metodach charakterystycznych zakresów, zachodzących zmian wykazało możliwość stosowania Spektroskopii Ramana w celu wyznaczenia kierunku ułożenia włókien kolagenowych w trakcie rozciągania co jest istotne z punktu widzenia przeszczepów skóry.The aim of the investigations was to compare the characteristics obtained from strength tests and spectroscopic measurements. The basic skin parameters dependent on the arrangement of collagen fibres were determined. Then Raman spectra of the investigated tissue were recorded and bands characteristic of collagen protein were identified. An analysis of the results for the successive stages of skin stretching showed, among other things, differences in the location of the amid I band maximum (1658cm-1) depending on the direction of specimen stretching. A comparison of the characteristic ranges of change determined by the two methods showed that Raman spectroscopy can be used to ascertain the orientation of collagen fibres in the course of stretching, which information is essential for skin transplantation

Variability of Amyloid Propensity in Imperfect Repeats of CsgA Protein of Salmonella enterica and Escherichia coli

CsgA is an aggregating protein from bacterial biofilms, representing a class of functional amyloids. Its amyloid propensity is defined by five fragments (R1–R5) of the sequence, representing non-perfect repeats. Gate-keeper amino acid residues, specific to each fragment, define the fragment’s propensity for self-aggregation and aggregating characteristics of the whole protein. We study the self-aggregation and secondary structures of the repeat fragments of Salmonella enterica and Escherichia coli and comparatively analyze their potential effects on these proteins in a bacterial biofilm. Using bioinformatics predictors, ATR-FTIR and FT-Raman spectroscopy techniques, circular dichroism, and transmission electron microscopy, we confirmed self-aggregation of R1, R3, R5 fragments, as previously reported for Escherichia coli, however, with different temporal characteristics for each species. We also observed aggregation propensities of R4 fragment of Salmonella enterica that is different than that of Escherichia coli. Our studies showed that amyloid structures of CsgA repeats are more easily formed and more durable in Salmonella enterica than those in Escherichia coli