| 1. |
- Karjalainen, Eeva-Liisa, 1980-
(författare)
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The choreography of protein vibrations : Improved methods of observing and simulating the infrared absorption of proteins
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The work presented in this thesis has striven toward improving the capability to study proteins using infrared (IR) spectroscopy. This includes development of new and improved experimental and theoretical methods to selectively observe and simulate protein vibrations.A new experimental method of utilising adenylate kinase and apyrase as helper enzymes to alter the nucleotide composition and to perform isotope exchange in IR samples was developed. This method enhances the capability of IR spectroscopy by enabling increased duration of measurement time, making experiments more repeatable and allowing investigation of partial reactions and selected frequencies otherwise difficult to observe. The helper enzyme mediated isotope exchange allowed selective observation of the vibrations of the catalytically important phosphate group in a nucleotide dependent protein such as the sarcoplasmic reticulum Ca2+-ATPase. This important and representative member of P-type ATPases was further investigated in a different study, where a pathway for the protons countertransported in the Ca2+-ATPase reaction cycle was proposed based on theoretical considerations. The transport mechanism was suggested to involve separate pathways for the ions and the protons.Simulation of the IR amide I band of proteins enables and supports structure-spectra correlations. The characteristic stacking of beta-sheets observed in amyloid structures was shown to induce a band shift in IR spectra based on simulations of the amide I band. The challenge of simulating protein spectra in aqueous medium was also addressed in a novel approach where optimisation of simulated spectra of a large set of protein structures to their corresponding experimental spectra was performed. Thereby, parameters describing the most important effects on the amide I band for proteins could be determined. The protein spectra predicted using the optimised parameters were found to be well in agreement with experiment.
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| 2. |
- Rudbeck, Maria, 1979-
(författare)
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The Beauty of the Bitter Devils : A Theoretical Study on Phosphate Molecules
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Phosphate transfer reactions are catalyzed by a large number of enzymes comprising kinases, mutases and phosphatases. These enzymes play a fundamental role in controlling numerous life processes and it is therefore important to understand the origin of their potent catalytic power. An example is the Ca2+-ATPase. In the E2P-state, this enzyme hydrolyses the phosphorylated amino acid, Asp351, 106 to 107 fold faster than when the model compound, acetyl phosphate, is hydrolyzed in in water.This thesis explores the catalytic power of Ca2+-ATPase using theoretical method based on quantum mechanics. The studies of this protein were made by performing quantum chemical calculations on models of phosphoric monoesters as well as on the explicit reaction pathway of the hydrolysis. The studies show the importance of electrostatic interactions as well as the role of the specific active site residue Glu183, a residue that acts as a base in the catalytic pathway. Furthermore, based on the calculations, the interpretation of the experimental infrared spectrum of the E2P-state of Ca2+-ATPase, could be further elucidated as well as modified.The experimental infrared spectrum of phosphoenol pyruvate in water has also been elucidated through calculations. This molecule is converted into pyruvate in the last step of the glycolytic pathway, a reaction that is catalyzed by pyruvate kinase (PK). These results further enabled the interpretation of the experimental spectrum of the PK's catalytic reaction.These two processes, the transport of Ca2+ into the sarcoplasmatic reticulum against a concentration gradient and the glycolysis, are two important actions of a muscle cell.
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| 3. |
- Vosough, Faraz, 1986-
(författare)
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Structure and dynamics of amyloid-beta oligomers
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Alzheimer's disease (AD) is the most common cause of dementia, affects tens of millions of people all over the world and inflicts huge socioeconomic costs on the societies. AD is a neurodegenerative disease; it progresses over time and is highly debilitating at the advanced stages. Biochemical and genetic studies in the last decades have revealed that amyloid-beta (Aβ) peptides play underlying roles in the molecular pathology of AD. Aβ peptides are small, aggregation-prone polypeptides produced in the neural tissue. Soluble aggregates of Aβ peptides, known as Aβ oligomers are regarded as the major neurotoxic species in AD brain.In this thesis, in vitro studies were performed on Aβ oligomers with a number of spectroscopy and biochemical methods. The main technique used in this thesis is infrared (IR) spectroscopy, a variety of vibrational spectroscopy methods. IR spectroscopy measures the absorption of IR radiation by the vibrational transitions of oscillating dipoles within chemical structures and provides structural information on chemical bonds and functional groups in molecules. The method can provide valuable data on the secondary structure of proteins and therefore is a powerful tool for studying protein self-assembly and aggregation.Different samples of homogeneous and heterogeneous Aβ42 oligomers were prepared and studied with IR spectroscopy and biochemical methods to establish correlations between oligomers' physical properties (size and homogeneity) and IR parameters. Additionally, the effects of lithium and nickel ions on the formation of homogeneous Aβ42 oligomers were studied. Separately, isotope-edited IR spectroscopy was used to study the molecular structure of homogeneous and heterogeneous Aβ42 oligomers. The obtained data can be helpful to establish IR spectroscopy for characterization of Aβ oligomers, as well as studies on their dynamics and interactions with each other and other biomolecules or inorganic materials. Moreover, the findings in this thesis add to the available knowledge on the molecular structure of Aβ oligomers and help to develop relevant molecular models. Such information can be helpful for the development of diagnostics and therapeutic strategies for AD.
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| 4. |
- Baronio, Cesare Michele, 1987-
(författare)
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Computational infrared spectroscopy : Calculation of the amide I absorption of proteins
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Infrared spectroscopy is an important technique that allows to retrieve structural information from the analysis of absorption spectra. The main application of infrared spectroscopy within life science is the study of the amide I band, which is correlated with protein backbone conformation and, consequently, with the secondary structure of proteins. However, band assignment and interpretation of the infrared spectra is not straightforward.Therefore, several simulation methods were developed to guide the interpretation of experimental amide I spectra. In this thesis, one of these methods is a normal mode analysis, which is based on the evaluation of the intrinsic vibration of the amide groups and the interactions between them. The calculation considers several effects: transition dipole coupling, nearest neighbor interaction, the local environment effect and the effect of hydrogen bond. From the normal mode analysis, it is possible to obtain the simulated infrared spectrum and the contribution of each amide group to a specific spectral range of the spectrum.The aim of this thesis and of the included publications is to explain this approach, to improve it and to show its potential. Results from simulations were compared with experimental data for different proteins of interest: amyloid-β oligomers and β-helix proteins. Simulated and experimental infrared spectra showed similar bands. Simulations also provided additional conclusions: they confirmed the random mixing of amyloid-β peptides in oligomers; they suggested that amyloid-β peptides contribute at least two strands in the structure of the oligomers; they revealed that the high wavenumber band, typical of antiparallel β-sheets, can be caused by other secondary structures, but not by parallel β-sheets. In addition, to verify and to improve the accuracy of this approach, simulation results were also put in a direct comparison with results from density functional theory calculations. From this comparison, a new optimal set of parameters for the calculations is suggested.
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| 5. |
- Eremina, Nadejda, 1985-
(författare)
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Infrared spectroscopic studies : from small molecules to large
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Infrared light (IR) was first discovered by Friedrich Wilhelm Herschel in 1800. However, until 1940’s, molecular IR studies involved only water and small organic molecules, because of the long measurement times. Development Fourier transform infrared spectroscopy (FTIR) has minimized the time required to obtain data, making it possible to investigate bigger biological systems, e.g. proteins and nucleic acids.This thesis concentrates on the applications of different IR spectroscopic techniques to a variety of biological systems and development of new approaches to study complicated biological events.The first paper in this work concerns using so-called caged compounds to study the aggregation of Alzheimer’s Aβ-peptide which is linked to the formation of neurotoxic fibrils in the brain. By adding caged-sulfate to the Aβ samples we were able to change the pH of the sample, while recording IR data and study fibril formation in a time-resolved manner. Then we used caged–ADP to study the production of ATP and creatine, mediated by creatine kinase (CK). Using CK as a helper enzyme we studied the effects of the phosphate binding on the secondary structure of SR Ca2+ATPse and determined the structural differences between two similar states Ca2E1ADP and Ca2E1ATP.In the second part of the thesis we used ATR-FTIR spectroscopy and a specially designed dialysis setup, to develop a general method to detect ligand binding events by observing the IR absorbance changes in the water hydration shell around the molecules. The same method was used to determine the binding of DNA to the transcription factors of the E2F family. E2F proteins play main part in the gene regulatory networks that control cell development. However how they recognize their DNA-binding sites and the mechanism of binding is not well understood. By using ATR-FTIR, we observed the changes in the secondary structure of the proteins, as well as the distortions to the DNA upon E2F-DNA complex formation.
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| 6. |
- Helmfors, Henrik
(författare)
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Cell-penetrating peptides : an uptake mechanism & a new endosomolytic peptide
- 2013
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Peptide-based drugs have slowly begun migrating from laboratories into pharmacies and now there are several on the market. However, currently only one gene based therapy that is relies on a viral delivery vector has been approved. The long-term goal of our research is to leverage the cell-penetrating peptide (CPP) technology into a potent, safe and simple delivery vector for oligonucleotide (ON) based therapies.Cell-penetrating peptides have been actively researched for more than 20 years, and many CPPs have been discovered. However, it is not fully understood how the peptides are able to enter cells. In this thesis we present a novel receptor for CPP:ON complexes. Pharmacological inhibition and siRNA knockdown of the class A scavenger receptors (SCARAs) demonstrate that these receptors are the main pathway by which CPP:ON complexes are taken up. As the intracellular fate of particles taken up by (receptor mediated) endocytosis is entrapment in endosomes this thesis also presents a new peptide for ON delivery that has endosomolytic properties. Additionally this new peptide (PepFect 15) is also taken up via receptor-mediated endocytosis by the SCARAs.
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| 7. |
- Kumar, Saroj, 1976-
(författare)
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Infrared studies : Method development and binding of ligands to pyruvate kinase
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Infrared spectroscopy is a valuable technique for the study of ligand induce change in biomolecules. Our development of a dialysis accessory to attenuated total reflection infrared spectroscopy makes this technique more universal for ligand binding studies. We use this method to understand the binding of phosphoenolpyruvate (PEP) and Mg2+ to pyruvate kinase (PK), where conformational changes of PK were revealed upon binding of PEP and Mg2+. To investigate the effect of the protein environment on the bound PEP, we used labeled PEP, which helped assign and evaluate the infrared absorption bands. The effects of different divalent and monovalent ions on PEP binding to PK were also studied. We could demonstrate that the β-sheets were perturbed differently with Na+ as compared to the other monovalent ions. The pattern of structural changes does not correlate with the activity profiles of the monovalent ions. Thus, it seems unlikely that the ion effects on activity are due to the ion effects on the structure of the PEP:PK complex. Comparing different divalent ions, a particularly large conformational change and a more homogeneous binding mode was observed with Mn2+ and attributed to a more closed conformation of the complex. The allosteric effect of fructose 1, 6 bisphosphate (FBP) on PEP binding to PK in presence of various ions (Mg2+, Mn2+, K+, Na+) was studied. The experiments indicated that the conformational change of PEP binding to PK:Mg2+:K+ in the presence of FBP was about twice as large as in its absence, which is tentatively ascribed to a higher occupancy of the closed state of PK. The affinity for PEP increased in presence of Mg2+ and K+. No allosteric effects were observed with the other ion combinations Mn2+/K+ and Mg2+/Na+. A method of ligand binding by observing a change in water absorption was developed and established with four different proteins. The results suggest that the decrease of water absorption is due to the release of bound water into the bulk during the ligand binding process, which can be a used as label-free indicator of ligand-protein binding.
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| 8. |
- Li, Chenge, 1988-
(författare)
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Infrared spectroscopy : a tool for protein characterization
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Infrared (IR) spectroscopy, which belongs to vibrational spectroscopy, detects the vibrations of molecules, for example, proteins. The absorption of the peptide group gives rise to 9 characteristic bands in the infrared region, named A, B, I-VII, with a decreasing energy or wavenumber (cm-1). Among the 9 bands, amide I, which is mainly caused by C=O stretching vibration, is most sensitive to backbone structure and environment, and therefore can be used for structural analysis. In this thesis, a membrane protein sarcoplasmic reticulum Ca2+-ATPase (SERCA1a) and a self-assembling peptide was studied with IR spectroscopy. In the first two papers, IR spectroscopy was used to assess the quality of a recombinant SERCA1a. A yeast-based expression system was applied to express recombinant SERCA1a, and the reaction cycle as well as the structure was analysed with IR spectroscopy. Different reaction intermediates were accumulated under different buffer conditions upon the release of ATP. The results showed that the recombinant protein shared similar IR features compared to the native protein. However, two SERCA1a preparations showed a difference around 1640 cm-1 in the amide I region. Using curve fitting, the band was assigned to β structure, and further investigation indicated that the difference in this region originates from protein aggregation. In the third paper, a co-fitting approach was tested and showed to be a more reliable method for structural analysis, and it can be applied in the biological IR spectroscopy. In the fourth paper, a peptide was computational designed and was predicted to self-assemble to amyloid fibrils, the formation of the fibril was confirmed with both electron microscopy and X-ray diffraction. IR spectroscopy was used to analyze further the structural details and the results support our structural predication.
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| 9. |
- De Oliveira, Danilo Hirabae, et al.
(författare)
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Structural conversion of the spidroin C-terminal domain during assembly of spider silk fibers
- 2024
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 15:1
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Tidskriftsartikel (refereegranskat)abstract
- The major ampullate Spidroin 1 (MaSp1) is the main protein of the dragline spider silk. The C-terminal (CT) domain of MaSp1 is crucial for the self-assembly into fibers but the details of how it contributes to the fiber formation remain unsolved. Here we exploit the fact that the CT domain can form silk-like fibers by itself to gain knowledge about this transition. Structural investigations of fibers from recombinantly produced CT domain from E. australis MaSp1 reveal an α-helix to β-sheet transition upon fiber formation and highlight the helix No4 segment as most likely to initiate the structural conversion. This prediction is corroborated by the finding that a peptide corresponding to helix No4 has the ability of pH-induced conversion into β-sheets and self-assembly into nanofibrils. Our results provide structural information about the CT domain in fiber form and clues about its role in triggering the structural conversion of spidroins during fiber assembly.
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| 10. |
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