REPOSITORY > RESULTS

Abstract

The process of protein folding describes the transition by which an unordered polypeptide chain attains its functional native three-dimensional structure. A detailed understanding of the principles which govern the protein folding, such as conformational preferences of amino acid residues, the neighbour residue effect and the solvent effects, remains an important unsolved challenge that encodes the folding energy landscape of a protein. Knowing the factors that contribute to the conformational stability of amino acid residues would provide the insight not only into the molecular basis of unfolded states, but also into the earliest events that occur during the protein folding and misfolding. To study the conformational preferences of peptide backbone in unfolded state, the short alanine peptides were examined. By changing the peptide’s environment, the competition between the intra- and intermolecular hydrogen bonding was challenged. We applied IR, Raman, VCD, NMR and UV-CD spectroscopy to determine the distribution of conformations in aqueous and non-aqueous solution. We have observed high sensitivity of amide III spectral region where each conformation has the characteristic band frequency and shape in the case of VCD. In water, the PII conformation of alanine dipeptide was found to be stabilized by formation of directionally oriented water bridges between C=O and NH peptide groups using DFT calculations. The agreement between the experimental and calculated spectra improved significantly upon the addition of explicit solvent molecules to the computational model in the case of polar solvents. Additionally, we found a good correlation between the conformational stabilization of short alanine peptides by the solvent features and the electrostatic screening model, where conformations strongly depend on the local electrostatic energy and its screening by the solvent. With increasing the peptide chain the neighbour effect of alanine residue was observed that resulted in decreasing of PII population and strengthening the favourable backbone electrostatic interactions in short alanine peptides.
Protein misfolding can lead to the protein self-assembly and furthermore to the formation of amyloid fibrils that give rise to serious lethal diseases. The mechanism of misfolding and aggregation of proteins composing the amyloid fibrils are poorly understood. Spectroscopic investigations of the conformational changes during fibrillation require the elucidation of the relationship between spectral features and the protein folding reaction coordinate(s). By applying infrared spectroscopy, we have gained the insights into peptide secondary structures through assignation of the amide bands for different conformations of the model peptide poly-L-lysine (PLL). In water at low pH values, PLL mainly possessed the PII and β structures, while at higher pH values and low temperatures, characteristic band for the α-helical conformation was found. The increase in temperature induced the formation of β structures that are components of amyloid fibrils. To determine the assignment of the infrared bands of individual conformations, different solvents were used that selectively stabilize one type of conformation. Among all the solvents, only ethylene glycol promoted the formation of a uniform PII-helix, suggesting that the PII structure is not limited to the presence of water molecules or charged side chains, as previously assumed. Knowing the spectral assignment, we could follow the fibrillation mechanism of PLL by applying the difference spectroscopy that provide us with information about the structural changes during temperature induced PLL fibrillation. Spectral changes below transition temperature were ascribed mainly to the melting of α-helces. The band assigned to PII-helix starts to lose the intensity just before the fibrillation of PLL started, thus indicating its role as intermediate structure in fibrillation process. In order to affect the kinetic of PLL fibrillation, we performed a series of measurements of PLL with promotor of PLL fibrillation, such as salt NaClO4, or inhibitor of PLL fibrillation, such as DPPA+DPPC vesicles. The salt NaClO4 lowered the transition temperature from the α-helical structure to the β-sheet fibrils of PLL for 10 °C, where the mechanism of its action was accounted to the stabilization of the PII-helix. With the addition of the DPPA+DPPC vesicles to the PLL, the stabilization of α- and PII-helices was observed and, simultaneously, the destabilization of those vesicles by this interaction. However, above the melting temperature of the vesicles, the band characteristic for the β-strand appeared, followed by the increase of intensity of the band that is characteristic for the aggregated β-sheets. We proposed a mechanism for PLL amyloid fibril formation in which the α-helical PLL melts into PII-helix, followed by the formation of -strands that stack into β-sheets. We showed that difference infrared spectroscopy is a very convenient method to obtain structural properties of the intermediate in the fibrillation process.
The therapeutic importance of gaining a detailed knowledge on insulin fibrillation in relation to type I diabetes has led to intensive studies focusing on its fibrillation kinetics and structural characteristics. Insulin fibrils feature the characteristics that are common to all amyloid fibrils, such as an elongated, unbranched morphology, characteristic cross-β diffraction pattern and Thioflavin T fluorescence. A full understanding of the fibrillation process requires structural elucidation of every species and determination of the kinetics of the interconversion between species on the reaction pathway. Therefore, we focused on the kinetic of insulin fibrillation that was measured by analysing the intensities of the bands in amide I region of IR spectra that are characteristic of β-sheets. The kinetic parameters were comparable to those obtained with Thioflavin T fluorescent measurements. Additionally, the concentrational measurements suggest that the rate of elongation of fibrils is consistent with a first-order reaction. Infrared spectroscopy allows monitoring the structural changes that occur during fibrillation and consequently, the individual structural component present in evolving equilibrium can be quantified. We observed the melting of α-helix and PII conformation of native insulin with the formation of β-sheets. Two different low-frequency bands in amide I region characteristic of β-sheet provide the insight into the nature of the intermolecular contacts in the insulin fibrils and consequently into the morphology of the fibrils.