Coherent X-ray Scattering Reveals Nanoscale Fluctuations in Hydrated Proteins

Hydrated proteins undergo a transition in the deeply supercooled regime, which is attributed to rapid changes in hydration water and protein structural dynamics. Here, we investigate the nanoscale stress relaxation in hydrated lysozyme proteins stimulated and probed by X-ray Photon Correlation Spectroscopy (XPCS). This approach allows us to access the nanoscale dynamic response in the deeply supercooled regime (T = 180 K) which is typically not accessible through equilibrium methods. The relaxation time constants exhibit Arrhenius temperature dependence upon cooling with a minimum in the Kohlrausch-Williams-Watts exponent at T = 227 K. The observed minimum is attributed to an increase in dynamical heterogeneity, which coincides with enhanced fluctuations observed in the two-time correlation functions and a maximum in the dynamic susceptibility quantified by the normalised variance $\chi_T$. Our study provides new insights into X-ray stimulated stress relaxation and the underlying mechanisms behind spatio-temporal fluctuations in biological granular materials

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

The Role of Molecular Heterogeneity in the Structural Dynamics of Aqueous Solutions

The liquid-liquid critical point hypothesis suggests that liquid water exists in two liquid states with different local structures, so-called high- and low-density liquid (HDL, LDL). At ambient pressure water locally fluctuates between these two states, with the fluctuations becoming more pronounced as the liquid is supercooled. In this thesis, we explore the role of molecular heterogeneity in the structural dynamics of aqueous solutions, specifically investigating the interplay of different solutes in water with the hypothesized HDL-LDL fluctuations. In our experimental approach, we utilize coherent light and X-ray scattering techniques, including small- and wide-angle X-ray scattering (SAXS, WAXS), as well as correlation methods, such as dynamic light scattering (DLS) and X-ray photon correlation spectroscopy (XPCS), that enable us to probe structural dynamics at a broad range of length and time scales.  Using DLS, we measure the diffusive dynamic behaviour of differently sized nanomolecular probes in supercooled water, finding that it is effectively similar and independent of probe size down to molecular scales of ≈1 nm. In contrast to single water molecules, these probes experience a similar dynamic environment, which coincides with the bulk viscosity. These results could suggest that anomalous influence from the hypothesized water fluctuations becomes apparent first on sub-nm length scales. Furthermore, we explore how the presence of small polar-organic solutes modulates the water phase diagram, utilizing glycerol-water solutions as a model system. By outrunning freezing with the rapid evaporative cooling technique, combined with ultrafast X-ray scattering at X-ray free-electron lasers (XFELs), we are able to probe the liquid structure in deeply supercooled dilute glycerol-water solutions. Our findings indicate the existence of HDL- and LDL-like fluctuations upon supercooling, with a Widom line shifted to slightly lower temperatures compared to pure water. Further experiments on deeply supercooled glycerol-water solutions at intermediate glycerol concentrations, combining WAXS and SAXS/XPCS, provide additional insights. These results reveal a first-order-like liquid-liquid transition involving discontinuous changes in the inter-atomic liquid structure and nanoscale liquid dynamics, which precedes ice crystallization.  Lastly, with the aim of developing powerful tools for resolving dynamics within spatially heterogeneous systems, including aqueous solutions, we combine the spatial resolution of nanofocused coherent X-ray beams with dynamic measurements by XPCS. Here, we successfully demonstrate a first proof-of-concept experiment of so-called nanofocused XPCS at MAX IV synchrotron radiation facility. In future experiments, we plan to go beyond standard XPCS at synchrotrons, towards accessing ultrafast atomic-scale liquid dynamics by X-ray speckle visibility spectroscopy (XSVS) at XFELs

Omformning av molekylära potentialer via den dynamiska Starkeffekten

The dynamic (ac) Stark effect refers to the energy shifting of electronic states induced by an oscillating electric field. Conveniently, the magnitude of the ac Stark shift scales with the square of the electric field amplitude, i.e. with light intensity. Using this fundamental effect to reshape molecular potentials, and steer the course of chemical reactions, is known as dynamic Stark control. The aim of this study was to investigate the dynamic Stark effect on the photodissociation of molecular oxygen (O2) in the Schumann-Runge continuum, SRC (130–175 nm). Absorption in the SRC leads to dissociation via the so-called B state, yielding O(1D) + O(3P), or the J state, forming O(3P) + O(3P). Both of these dissociative excited states may be well-described in terms of mixed valence and Rydberg state character, in which each of the two states are strongly coupled to a Rydberg state of similar symmetry. Due to the mixed character of the B and J states, simulations predict that dynamic Stark shifting of the coupled Rydberg states leads to a dramatic change in dissociation channel branching ratio, as well as a red-shift of the absorption spectrum. This study aimed at experimentally testing this theoretical prediction. A 400-nm femtosecond laser pulse was employed as a combined pump and control field, simultaneously inducing a three-photon transition into the SRC and ac Stark shifting the potentials. A detection scheme to detect the changes in absorption of the B channel with pump pulse intensity was devised and implemented. The chosen detection scheme, in which emission at 762 nm from the O2(b−X) transition is measured, in principle monitors O(1D) from the B channel via an energy transfer reaction. The experimental results overall show consistency between simulations and experiment. The measured 762-nm emission exhibited a pump pulse intensity-dependence that likely reflects the dynamic Stark reshaping of the excited state potentials. However, saturation is clearly present in the data, complicating data interpretation. Furthermore, deviations between experiment and simulations are large at high pulse intensities, indicating that O(1D) is additionally generated by absorption into higher excited states. Finally, structured features that deviate from the simulations at low pulse intensities may possibly be assigned to vibrational resonances to high-lying Rydberg states by four-photon absorption. Den dynamiska (ac) Starkeffekten beskriver energiskiftet för elektroniska tillstånd som induceras av ett oscillerande elektriskt fält. Storleken på detta skift ökar med kvadraten av den elektriska fältstyrkan, det vill säga med ljusintensitet. Tillämpningen av denna fundamentala effekt i syfte att omforma molekylära potentialer, och därmed styra kemiska reaktioner, kallas för dynamisk Starkkontroll. Syftet med denna studie var att undersöka hur den dynamiska Starkeffekten påverkar den fotoinducerade dissociationen av molekylärt syre (O2) inom Schumann-Runge kontinuumet, SRC (130–175 nm). Absorption i SRC resulterar i dissociation via det så kallade B-tillståndet, som bildar O  (1D) + O(3P), eller via J-tillståndet, som leder till bildandet av O(3P) + O(3P). Båda dessa dissociativa tillstånd har en karaktär som kan beskrivas som en blandning av ett valenstillstånd och ett Rydbergstillstånd.  Simuleringar antyder att, till följd av valens- och Rydbergskaraktären hos B och J-tillståndet, leder dynamisk Starkskiftning av de kopplade Rydbergstillstånden till en dramatisk ändring i det relativa utbytet för de två dissociationskanalerna, samt till ett röd- skift av absorptionsspektrumet. Denna studie hade som ändamål att experimentellt testa denna teoretiska förutsägelse. En femtosekundslaser vid 400 nm användes som kombinerat excitations- och kontrollfält, vilket parallellt inducerar en trefoton-övergång in i SRC och ac Starkskiftar potentialerna. En detektionsmetod som mäter variationer i absorptionen för B-kanalen som funktion av pulsintensitet designades och implementerades. I den valda metoden detekteras emission vid 762 nm från O2(b − X)-övergången, vilket i sin tur ger en mätning av O(1D) som genereras från B- kanalen via en energiöverföringsreaktion. De experimentella resultaten stämmer relativt väl överens med simuleringarna. Den uppmätta emissionen vid 762 nm uppvisar ett intensitetsberoende som i stora drag reflekterar ac Stark- skiftningen av potentialerna. Utöver detta finns dock ett stort bidrag från mättnad, vilket försvårar tolkningen av datan. Vi-dare avviker den experimentella datan betydligt vid höga pulsintensiteter, vilket sannolikt tyder på att O(1D) även genereras genom absorption till högre exciterade tillstånd. Slutligen ob-serveras mindre, men tydliga avvikelser vid låga pulsintensiteter. Dessa kan möjligen tillordnas vibrationsresonanser med högre Rydbergstillstånd genom fyrfoton-absorption. 

Omformning av molekylära potentialer via den dynamiska Starkeffekten

The dynamic (ac) Stark effect refers to the energy shifting of electronic states induced by an oscillating electric field. Conveniently, the magnitude of the ac Stark shift scales with the square of the electric field amplitude, i.e. with light intensity. Using this fundamental effect to reshape molecular potentials, and steer the course of chemical reactions, is known as dynamic Stark control. The aim of this study was to investigate the dynamic Stark effect on the photodissociation of molecular oxygen (O2) in the Schumann-Runge continuum, SRC (130–175 nm). Absorption in the SRC leads to dissociation via the so-called B state, yielding O(1D) + O(3P), or the J state, forming O(3P) + O(3P). Both of these dissociative excited states may be well-described in terms of mixed valence and Rydberg state character, in which each of the two states are strongly coupled to a Rydberg state of similar symmetry. Due to the mixed character of the B and J states, simulations predict that dynamic Stark shifting of the coupled Rydberg states leads to a dramatic change in dissociation channel branching ratio, as well as a red-shift of the absorption spectrum. This study aimed at experimentally testing this theoretical prediction. A 400-nm femtosecond laser pulse was employed as a combined pump and control field, simultaneously inducing a three-photon transition into the SRC and ac Stark shifting the potentials. A detection scheme to detect the changes in absorption of the B channel with pump pulse intensity was devised and implemented. The chosen detection scheme, in which emission at 762 nm from the O2(b−X) transition is measured, in principle monitors O(1D) from the B channel via an energy transfer reaction. The experimental results overall show consistency between simulations and experiment. The measured 762-nm emission exhibited a pump pulse intensity-dependence that likely reflects the dynamic Stark reshaping of the excited state potentials. However, saturation is clearly present in the data, complicating data interpretation. Furthermore, deviations between experiment and simulations are large at high pulse intensities, indicating that O(1D) is additionally generated by absorption into higher excited states. Finally, structured features that deviate from the simulations at low pulse intensities may possibly be assigned to vibrational resonances to high-lying Rydberg states by four-photon absorption. Den dynamiska (ac) Starkeffekten beskriver energiskiftet för elektroniska tillstånd som induceras av ett oscillerande elektriskt fält. Storleken på detta skift ökar med kvadraten av den elektriska fältstyrkan, det vill säga med ljusintensitet. Tillämpningen av denna fundamentala effekt i syfte att omforma molekylära potentialer, och därmed styra kemiska reaktioner, kallas för dynamisk Starkkontroll. Syftet med denna studie var att undersöka hur den dynamiska Starkeffekten påverkar den fotoinducerade dissociationen av molekylärt syre (O2) inom Schumann-Runge kontinuumet, SRC (130–175 nm). Absorption i SRC resulterar i dissociation via det så kallade B-tillståndet, som bildar O  (1D) + O(3P), eller via J-tillståndet, som leder till bildandet av O(3P) + O(3P). Båda dessa dissociativa tillstånd har en karaktär som kan beskrivas som en blandning av ett valenstillstånd och ett Rydbergstillstånd.  Simuleringar antyder att, till följd av valens- och Rydbergskaraktären hos B och J-tillståndet, leder dynamisk Starkskiftning av de kopplade Rydbergstillstånden till en dramatisk ändring i det relativa utbytet för de två dissociationskanalerna, samt till ett röd- skift av absorptionsspektrumet. Denna studie hade som ändamål att experimentellt testa denna teoretiska förutsägelse. En femtosekundslaser vid 400 nm användes som kombinerat excitations- och kontrollfält, vilket parallellt inducerar en trefoton-övergång in i SRC och ac Starkskiftar potentialerna. En detektionsmetod som mäter variationer i absorptionen för B-kanalen som funktion av pulsintensitet designades och implementerades. I den valda metoden detekteras emission vid 762 nm från O2(b − X)-övergången, vilket i sin tur ger en mätning av O(1D) som genereras från B- kanalen via en energiöverföringsreaktion. De experimentella resultaten stämmer relativt väl överens med simuleringarna. Den uppmätta emissionen vid 762 nm uppvisar ett intensitetsberoende som i stora drag reflekterar ac Stark- skiftningen av potentialerna. Utöver detta finns dock ett stort bidrag från mättnad, vilket försvårar tolkningen av datan. Vi-dare avviker den experimentella datan betydligt vid höga pulsintensiteter, vilket sannolikt tyder på att O(1D) även genereras genom absorption till högre exciterade tillstånd. Slutligen ob-serveras mindre, men tydliga avvikelser vid låga pulsintensiteter. Dessa kan möjligen tillordnas vibrationsresonanser med högre Rydbergstillstånd genom fyrfoton-absorption. 

Utvärdering av Amyloidfibriller som Stödmaterial för Photon Upconversion via Sensitized Triplet-Triplet Annihilation

In the face of global warming and shrinking resources of fossil fuels the interest in solar energy has increased in recent years. However, the low energy and cost efficiency of current solar cells has up to this date hindered solar energy from playing a major role on the energy market. Photon upconversion is the process in which light of low energy is converted to high energy photons. Lately, this phenomenon has attracted renewed interest and ongoing research in this field mainly focuses on solar energy applications, solar cells in particular. The aim of this study was to investigate and evaluate amyloid fibrils as nanotemplates for an upconversion system based on the dyes platinum octaetylporphyrin (PtOEP) and 9,10- diphenylanthracene (DPA). This well-known pair of organic dyes upconverts light in the visible spectrum through a mechanism known as sensitized triplet-triplet annihilation. Amyloid fibrils are β-sheet rich protein fibril structures, formed by self-assembly of peptides. Amyloid fibrils were prepared from whey protein isolate using heat and acidic solutions. Dyes were incorporated according to a wellestablished technique, in which dyes are grinded together with the protein in solid state prior to fibrillization. Photophysical properties of pure fibrils and dye-incorporated fibrils were studied using UV-VIS spectroscopy and fluorescence spectroscopy. Atomic force microscopy was further employed to confirm the presence of amyloid fibrils as well as to study fibril structure. Results indicate that amyloid fibrils may not be the optimal host material for the upconversion system PtOEP/DPA. It was found that the absorption and emission spectra of this system overlap to a great deal with that of the fibrils. Though no upconverted emission clearly generated by the dye system was recorded, anti-Stokes emission was indeed observed. Interestingly, this emission appears to be strongly enhanced by the presence of dyes. It is suggested that this emission may be attributed to the protein residues rather than the amyloid structure. Future studies are encouraged to further investigate these remarkable findings.Intresset för solceller har ökat under de senaste åren, till stor del tillföljd av den globala uppvärmningen och de sinande oljeresurserna. Dagens solceller har dock problem med låg energi- och kostnadseffektivitet, vilket gör att solenergin än så länge har svårt att hävda sig på energimarknaden. Photon upconversion är ett fotofysikaliskt fenomen där fotoner med låg energi omvandlas till fotoner med hög energi. Den senaste tiden har denna process fått förnyat intresse och forskningen inom området har ökat, inte minst med sikte på att integrera processen i solceller och därmed öka dess effektivitet. Målet med denna studie var att undersöka huruvida amyloidfibriller kan användas som stomme för ett photon upconversion-system baserat på platinum-oktaetylporfyrin (PtOEP) och 9,10-difenylantracen (DPA). Dessa två organiska färgämnen är ett välkänt par som konverterar synligt ljus med låg frekvens till mer hög frekvent ljus i det synliga spektrumet, via en mekanism som kallas sensitized triplet-triplet annihilation. Amyloidfibriller är proteinbaserade fiberstrukturer med hög andel β-flak, vilka bildas genom självassociation av peptider. I denna studie skapades amyloidfibriller av vassleprotein genom upphettning i sur lösning. Färgämnena inkorporerades enligt en välbeprövad metod där proteinet mortlas tillsammans med färgämnena i fast tillstånd, innan fibrilleringsprocessen påbörjas. De fotofysikaliska egenskaperna hos fibriller med och utan färgämnen analyserade med UV-VIS samt fluorescensspektroskopi. Atomkraftsmikroskopi användes för att bekräfta att fibriller fanns i proven, samt för att studera dess struktur. De erhållna resultaten antyder att amyloidfibriller inte är ett optimalt material för systemet PtOEP/DPA, delvis på grund av att absorptions- och emissionsspektrumet för systemet överlappar med fibrillernas egna spektrum. Anti-Stokes emission detekterades, men denna är med stor sannolikhet inte orsakad av färgämnena. Dock noterades, intressant nog, att denna emission ökar betydligt i närvaro av färgämnena. En möjlighet är att denna emission är kopplad till monomerer i proteinet snarare än till fibrillstrukturen, eftersom emission observerades hos både nativt och fibrillerat protein. Framtida studier uppmuntras att vidare undersöka dessa effekter

Nanocrystallites Modulate Intermolecular Interactions in Cryoprotected Protein Solutions

Studying protein interactions at low temperatures has important implications for optimizing cryostorage processes of biological tissue, food, and protein-based drugs. One of the major issues is related to the formation of ice nanocrystals, which can occur even in the presence of cryoprotectants and can lead to protein denaturation. The presence of ice nanocrystals in protein solutions poses several challenges since, contrary to microscopic ice crystals, they can be difficult to resolve and can complicate the interpretation of experimental data. Here, using a combination of small- and wide-angle X-ray scattering (SAXS and WAXS), we investigate the structural evolution of concentrated lysozyme solutions in a cryoprotected glycerol–water mixture from room temperature (T = 300 K) down to cryogenic temperatures (T = 195 K). Upon cooling, we observe a transition near the melting temperature of the solution (T ≈ 245 K), which manifests both in the temperature dependence of the scattering intensity peak position reflecting protein–protein length scales (SAXS) and the interatomic distances within the solvent (WAXS). Upon thermal cycling, a hysteresis is observed in the scattering intensity, which is attributed to the formation of nanocrystallites in the order of 10 nm. The experimental data are well described by the two-Yukawa model, which indicates temperature-dependent changes in the short-range attraction of the protein–protein interaction potential. Our results demonstrate that the nanocrystal growth yields effectively stronger protein–protein attraction and influences the protein pair distribution function beyond the first coordination shell

Nanofocused x-ray photon correlation spectroscopy

Here, we demonstrate an experimental proof of concept for nanofocused x-ray photon correlation spectroscopy, a technique sensitive to nanoscale fluctuations present in a broad range of systems. The experiment, performed at the NanoMAX beamline at MAX IV, uses a novel event-based x-ray detector to capture nanoparticle structural dynamics with microsecond resolution. By varying the nanobeam size from σ=88 nm to σ=2.5μm, we quantify the effect of the nanofocus on the small-angle scattering lineshape and on the diffusion coefficients obtained from nano-XPCS. We observe that the use of nanobeams leads to a multifold increase in speckle contrast, which greatly improves the experimental signal-to-noise ratio, quantified from the two-time intensity correlation functions. We conclude that it is possible to account for influence of the high beam divergence on the lineshape and measured dynamics by including a convolution with the nanobeam profile in the model

Resolving molecular diffusion and aggregation of antibody proteins with megahertz X-ray free-electron laser pulses

X-ray free-electron lasers (XFELs) with megahertz repetition rate can provide novel insights into structural dynamics of biological macromolecule solutions. However, very high dose rates can lead to beam-induced dynamics and structural changes due to radiation damage. Here, we probe the dynamics of dense antibody protein (Ig-PEG) solutions using megahertz X-ray photon correlation spectroscopy (MHz-XPCS) at the European XFEL. By varying the total dose and dose rate, we identify a regime for measuring the motion of proteins in their first coordination shell, quantify XFEL-induced effects such as driven motion, and map out the extent of agglomeration dynamics. The results indicate that for dose rates below $1.16\,\mathrm{kGy\mu s^{-1}}$ in a time window up to $10\,\mathrm{\mu s}$, it is possible to capture the protein dynamics before the onset of beam induced aggregation. We refer to this approach as "correlation before aggregation" and demonstrate that MHz-XPCS bridges an important spatio-temporal gap in measurement techniques for biological samples.Comment: 22 pages, 6 figure