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. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 5: Manuscript.</p>

Radiation damage limits the resolution in imaging experiments. Damage is caused by energy deposited into the sample during exposure. Ultrashort and extremely bright X-ray pulses from free-electron lasers (FELs) offer the possibility to outrun key damage processes, and temporarily improve radiation tolerance. Theoretical models indicate that high detail-resolutions could be realized on non-crystalline samples with very short pulses, before plasma expansion. Studies presented here describe the interaction of a very intense and ultrashort X-ray pulse with material, and investigate boundary conditions for flash diffractive imaging both theoretically and experimentally. In the hard X-ray regime, predictions are based on particle simulations with a continuum formulation that accounts for screening from free electrons. First experimental results from the first soft X-ray free-electron laser, the FLASH facility in Hamburg, confirm the principle of flash imaging, and provide the first validation of our theoretical models. Specifically, experiments on nano-fabricated test objects show that an interpretable image can be obtained to high resolution before the sample is vaporized. Radiation intensity in these experiments reached 10^14 W/cm^2, and the temperature of the sample rose to 60000 Kelvin after the 25 femtosecond pulse left the sample. Further experiments with time-delay X-ray holography follow the explosion dynamics over some picoseconds after illumination. Finally, this thesis presents results from biological flash-imaging studies on living cells. The model is based on plasma calculations and fluid-like motions of the sample, supported by the time-delay measurements. This study provides an estimate for the achievable resolutions as function of wavelength and pulse length. The technique was demonstrated by our team in an experiment where living cells were exposed to a single shot from the FLASH soft X-ray laser.

Structural biology has become an important part of researching various diseases and drug development. In this thesis, I provide details on how I worked with approaches to structural characterization of a heteromeric GABA(A)R. These pentameric ligand gated ion channels take part in regulating inhibition of action potentials in nerve cells by allowing the passage of Cl- ions when bound by gamma-aminobutyric acid (GABA). They are formed by the assembly of five subunits which can be of various different types, denoted by greek letters and a number. Much is still unknown about how GABA and several other ligands bind to these ion channels and how that impacts function. Obtaining a structure of these proteins can aid in closing those knowledge gaps. It is reasonable to screen the proteins you have before you study their structures by Cryo-EM in order to get the best result, a methodology for which is described here. I have followed this methodology to screen two heteromeric GABA$_A$R that we wish to determine the structure of, alpha 5 beta 3 and rho 1 gamma 2. Neither of the combinations of genes we used to express these proteins proved to produce the desired fully assembled heteromeric protein. In the case of alpha 5 beta 3, we only witnessed building blocks, with no fully assembled channels. In rho 1 gamma 2, we instead only witnessed fully formed homomers of the rho 1 subunit. These findings then exclude the gene constructs used from further structural study, and the methodology described will inform the next steps to be taken. / Strukturbiologi har blivit en viktig del av forskningen kring många sjukdomar samt utveckling av läkemedel. I denna uppsats delger jag hur jag arbetat med metoder för strukturell karakterisering av en heteromerisk GABA(A)R. Dessa pentameriska ligandstyrda jonkanaler deltar i regleringen av hämning av aktionspotentialer i nervceller genom att tillåta passagen av Cl- joner när gamma-aminosmörsyra (GABA) binder. Dessa består av fem subenheter som kan vara en av flera olika typer, vilka anges med en grekisk bokstav och en siffra. Mycket om hur GABA och andra ligander binder till dessa jonkanaler och hur det påverkar dess funktion är fortfarande okänt. Att hitta en struktur av dessa proteiner kan hjälpa oss att stänga kunskapsgapen. Det är klokt att undersöka om genen man ska använda för att uttrycka det sökta proteinet ger det man söker innan man sen börjar studera strukturen. Jag har beskrivit en metodologi för detta och följt den för två heteromeriska proteiner, alpha 5 beta 3 och rho 1 gamma 2. Ingen av kombinationerna av gener vi använt för att uttrycka dessa proteiner har producerat de sökta, fullt ihoppbyggda proteinerna. I fallet för alpha 5 \beta 3 så ser vi endast byggstenar och inga kompletta proteiner, och för rho 1 gamma 2 så ser vi endast homomeriska proteiner av rho 1. Dessa slutsatser exkluderar de genkonstruktioner vi använt från vidare strukturella studier, och stegen som bör tas härnäst beskrivs av den använda metodologin.

X-ray lasers bring us into a new world in photon science by delivering extraordinarily intense beams of x-rays in very short bursts that can be more than ten billion times brighter than pulses from other x-ray sources. These lasers find applications in sciences ranging from astrophysics to structural biology, and could allow us to obtain images of single macromolecules when these are injected into the x-ray beam. A macromolecule injected into vacuum in a microdroplet will be affected by evaporation and by the dynamics of the carrier liquid before being hit by the x-ray pulse. Simulations of neutral and charged water droplets were performed to predict structural changes and changes of temperature due to evaporation. The results are discussed in the aspect of single molecule imaging. Further studies show ionization caused by the intense x-ray radiation. These simulations reveal the development of secondary electron cascades in water. Other studies show the development of these cascades in KI and CsI where experimental data exist. The results are in agreement with observation, and show the temporal, spatial and energetic evolution of secondary electron cascades in the sample. X-ray diffraction is sensitive to structural changes on the length scale of chemical bonds. Using a short infrared pump pulse to trigger structural changes, and a short x-ray pulse for probing it, these changes can be studied with a temporal resolution similar to the pulse lengths. Time resolved diffraction experiments were performed on a phase transition during resolidification of a non-thermally molten InSb crystal. The experiment reveals the dynamics of crystal regrowth. Computer simulations were performed on the infrared laser-induced melting of bulk ice, giving a comprehension of the dynamics and the wavelength dependence of melting. These studies form a basis for planning experiments with x-ray lasers.