Fast Relaxation Imaging Research Articles

Connecting simulation and experiment of protein folding inside cells with fast relaxation imaging

Connecting simulation and experiment of protein folding inside cells with fast relaxation imaging

Protein Folding Stability and Kinetics in Alginate Hydrogels.

Proteins are commonly encapsulated in alginate gels for drug delivery and tissue-engineering applications. However, there is limited knowledge of how encapsulation impacts intrinsic protein properties such as folding stability or unfolding kinetics. Here, we use fast relaxation imaging (FReI) to image protein unfolding in situ in alginate hydrogels after applying a temperature jump. Based on changes in the Förster resonance energy transfer (FRET) response of FRET-labeled phosphoglycerate kinase (PGK), we report the quantitative impact of multiple alginate hydrogel concentrations on protein stability and folding dynamics. The gels stabilize PGK by increasing its melting temperature up to 18.4 °C, and the stabilization follows a nonmonotonic dependence on the alginate density. In situ kinetic measurements also reveal that PGK deviates more from two-state folding behavior in denser gels and that the gel decreases the unfolding rate and accelerates the folding rate of PGK, compared to buffer. Phi-value analysis suggests that the folding transition state of an encapsulated protein is structurally similar to that of folded protein. This work reveals both beneficial and negative impacts of gel encapsulation on protein folding, as well as potential mechanisms contributing to altered stability.

Understanding hepatitis B viral capsid diffusion, dissociation and toxicity in live hepatocytes.

Hepatitis B virus (HBV) is an enveloped DNA virus that infects human liver cells. HBV has infected more than 250 million people globally, despite the availability of a vaccine. Once HBV enters the cell, its life cycle involves disassembly of the capsid into individual capsid proteins, to release the genome into the host nucleus for repair and protein expression, followed by capsid-envelope protein interactions that lead either to nuclear import and possibly disassembly at the nuclear envelope, or secretion from the host cell. However, a quantitative model of all the steps involved is still lacking. Here we quantify HBV capsid diffusion, disassembly into core (capsid) protein, and capsid-envelope interactions in live hepatocytes, using Fast Relaxation Imaging (FReI), Widefield Intensified Fluctuation Imaging (WIFI), and single molecule tracking by Minflux to obtain a sub-cellular and as well as molecular-level understanding. Our observations show signs of liver-cell specific adaptations of HBV. We look at multiple cell lines of liver and non-liver origins to further explore this specificity, which may provide implications on the co-evolution and host-cell specificity of HBV even beyond the point of cell entry.

An in Vitro Cytomimetic of In-Cell RNA Folding.

To discover the cytomimetic that accounts for cytoplasmic crowding and sticking on RNA stability, we conducted a two-dimensional scan of mixtures of artificial crowding and sticking agents, PEG10k and M-PERTM . As our model RNA, we investigate the fourU RNA thermometer motif of Salmonella, a hairpin-structured RNA that regulates translation by unfolding and exposing its ribosome binding site (RBS) in response to temperature perturbations. We found that the addition of artificial crowding and sticking agents leads to a stabilization and destabilization of RNA folding, respectively, through the excluded volume effect and surface interactions. FRET-labels were added to the fourU RNA and Fast Relaxation Imaging (FReI), fluorescence microscopy coupled to temperature-jump spectroscopy, probed differences between folding stability of RNA inside single living cells and in vitro. Our results suggest that the cytoplasmic environment affecting RNA folding is comparable to a combination of 20 % v/v M-PERTM and 150 g/L PEG10k.

Cellular ATP Levels Determine the Stability of a Nucleotide Kinase.

The energy currency of the cell ATP, is used by kinases to drive key cellular processes. However, the connection of cellular ATP abundance and protein stability is still under investigation. Using Fast Relaxation Imaging paired with alanine scanning and ATP depletion experiments, we study the nucleotide kinase (APSK) domain of 3′-phosphoadenosine-5′-phosphosulfate (PAPS) synthase, a marginally stable protein. Here, we show that the in-cell stability of the APSK is determined by ligand binding and directly connected to cellular ATP levels. The observed protein stability change for different ligand-bound states or under ATP-depleted conditions ranges from ΔGf 0 = -10.7 to +13.8 kJ/mol, which is remarkable since it exceeds changes measured previously, for example upon osmotic pressure, cellular stress or differentiation. The results have implications for protein stability during the catalytic cycle of APS kinase and suggest that the cellular ATP level functions as a global regulator of kinase activity.

Stabilization and Kinetics of an Adsorbed Protein Depends on the Poly(N-isopropylacrylamide) Grafting Density.

The solubility transition at the lower critical solution temperature (LCST, 32 °C) of poly(N-isopropylacrylamide) (PNIPAM) is widely used as a thermal switch to rapidly and reversibly capture and release proteins and cells. It is generally assumed that proteins adsorbed to PNIPAM above the LCST are unaffected by polymer interactions. Here we show that the folding stability of the enzyme phosphoglycerate kinase (PGK) is increased by interactions with end-grafted PNIPAM films above the LCST. We systematically compare two protein mutants with different stabilities. The stabilization mirrors the degree of protein adsorption under grafting conditions studied previously. Maximum stabilization occurs when proteins adsorb to low density, collapsed polymer "mushrooms". In the denser polymer "brush" regime, protein stabilization decreases back to a value indistinguishable from the bulk solution, consistent with low protein adsorption on dense, collapsed brushes. The temperature-dependent kinetics measured by Fast Relaxation Imaging reveals that PNIPAM does not affect the overall folding/unfolding mechanism. Based on the different stabilizations of two mutants and the relaxation kinetics, we hypothesize that the polymer acts mainly by increasing the conformational entropy of the folded protein by interacting with the protein surface and less by crowding the unfolded state of PGK.

Folding Stability and Self‐Association of a Triplet‐Repeat (CAG)20 RNA Hairpin in Cytomimetic Media

AbstractRNA molecules with trinucleotide repeat expansions are involved in the pathology of various neurodegenerative diseases, such as Huntington's disease. A central research objective is to understand how trinucleotide repeat RNAs fold and function in disease progression. Studies that investigate the sequence structure, stability and self‐assembly of such RNAs mainly focused on in vitro methods in dilute aqueous solution. Functional studies in cells reveal further aspects of transport, protein association or assembly in phase separated compartments. Our study aims to understand governing factors for folding stability of trinucleotide repeats on the cellular level by assessing the influence of key components of the cellular medium in vitro. We investigate (CAG)20 RNA in complex and cytomimetic media using Fast Relaxation Imaging. Using distinct cosolutes we analyze the different enthalpic and entropic contributions that determine folding stability. We find a remarkable destabilization and aggregation of the RNA in cellular lysate that can be attributed to specific and non‐specific interactions with the unfolded state.

In-Cell Titration of Small Solutes Controls Protein Stability and Aggregation.

The components of the intracellular environment vary widely in size: from large multiprotein complexes to atomic ions. Besides water, low-molecular-weight solutes (<1 kDa) such as electrolytes, metabolites, and carbohydrates are by far the most abundant of these components. Small solutes are thus key contributors to the solvation environment in the cell. Small solutes have been known for decades to alter protein structure or activity in vitro, through their interactions with protein surfaces or hydration shells. Here we use the cell itself as our test tube, by titrating its hydration, ion, or carbohydrate composition systematically. We trigger the selective uptake of specific solutes by exposing cells to hyperosmotic media. We then measure protein structure, stability, unfolding kinetics, and aggregation in these different intracellular environments by using fast relaxation imaging. We compare these results with controls where solutes cannot enter the cell and only hydration is altered. Protein structure, thermal stability, and aggregation onset all depend on the concentration and chemical nature of the solute titrated into the cell. Our work highlights the important contributions of small solutes in defining how proteins interact within the cell and suggests that intracellular variation of the solute composition could be an important regulator of protein function.

Direct Imaging of Protein Stability and Folding Kinetics in Hydrogels.

We apply fast relaxation imaging (FReI) as a novel technique for investigating the folding stability and dynamics of proteins within polyacrylamide hydrogels, which have diverse and widespread uses in biotechnology. FReI detects protein unfolding in situ by imaging changes in fluorescence resonance energy transfer (FRET) after temperature jump perturbations. Unlike bulk measurements, diffraction-limited epifluorescence imaging combined with fast temperature perturbations reveals the impact of local environment effects on protein-biomaterial compatibility. Our experiments investigated a crowding sensor protein (CrH2) and phosphoglycerate kinase (PGK), which undergoes cooperative unfolding. The crowding sensor quantifies the confinement effect of the cross-linked hydrogel: the 4% polyacrylamide hydrogel is similar to aqueous solution (no confinement), while the 10% hydrogel is strongly confining. FRAP measurements and protein concentration gradients in the 4% and 10% hydrogels further support this observation. PGK reveals that noncovalent interactions of the protein with the polymer surface are more important than confinement for determining protein properties in the gel: the mere presence of hydrogel increases protein stability, speeds up folding relaxation, and promotes irreversible binding to the polymer even at the solution-gel interface, whereas the difference between the 4% and the 10% hydrogels is negligible despite their large difference in confinement. The imaging capabilities of FReI, demonstrated to be diffraction limited, further revealed spatially homogeneous protein unfolding across the hydrogels at 500 nm length scales and revealed differences in protein properties at the gel-solution boundary.

Spliceosomal U1A Protein-SL2 RNA Binding Affinity Decreases in Cells

While extensive biochemical and biophysical studies have been carried out to elucidate protein-RNA binding mechanisms and dynamics in vitro, most of these studies do not take into consideration the effect of the cellular environment. Here we have experimentally tested the role of the cellular environment on protein transport and binding affinity in one of the most widely studied RNA recognition motifs, the spliceosomal protein U1A, and its binding partner, stem loop 2 (SL2) of the U1 small nuclear RNA. U1A-SL2 localization, stability and binding kinetics were monitored in live U2OS cells by fast relaxation imaging (FreI), which combines a temperature jump with fluorescence microscopy of the FRET (Fluorescence resonance energy transfer)-labeled RNA and protein. U1A protein alone was found to diffuse across the cell, whereas SL2 RNA and the U1A-SL2 complex localized in the nucleus. This demonstrates that SL2 RNA mediates transport of the U1A protein to the nucleus. The binding affinity in live cells was reduced compared to in vitro. The dissociation rate was unchanged in cells; however, the association rate was an order of magnitude lower, resulting in a two order of magnitude decrease in the dissociation constant. Introduction of a macromolecular crowder, Ficoll 70, in vitro further stabilized the complex. This suggests that differences between binding affinities as measured in vitro and in live cells cannot be explained by crowding alone. Instead, high binding affinities as measured in vitro may be necessary for selectivity in vivo, where competition exists between multiple binding partners.

PAPS-Synthase: Dissecting Folding of a Large and Naturally Fragile Protein In Vitro and In Cellulo

PAPS synthases are bifunctional enzymes providing the cell with the sulfate donor PAPS (3'-phosphoadenosine-5′-phosphosulfate), which is further used by sulfotransferases for modification of several biomolecules (e.g. steroids). Isoform PAPS synthase 2 (PAPSS2) has been shown to be fragile within in vitro experiments, but is stabilized by binding of its endogenous ligands. Mutations affecting its activity lead to disease states like bone and cartilage malformation as well as metabolic diseases. Compared to in vitro conditions, the cellular milieu is crowded by large biopolymers resulting in intermolecular interactions and excluded-volume effects affecting each biopolymer inside the cell.To understand the stability of this large enzyme (70 kDa), we conducted biophysical studies on the individual domains, APS kinase and ATP sulfurylase, as well as the full length protein. Using Fast Relaxation Imaging we studied disease-relevant mutants of PAPSS2 directly within the cell. The data provide novel thermodynamic insights into PAPSS2 stability inside the cellular milieu and its influence on the naturally destabilized protein. These results also unravel new insights into disease mechanisms of PAPSS2 mutations.

Towards Three-Color Live-Cell Imaging of Spliceosome Assembly

The process whereby introns are removed from pre-mRNAs occurs in a complex macromolecular machine known as the spliceosome. The spliceosome is dynamic, assembling each of its five small ribonucleoprotein particles (snRNPs) and hundreds of associated proteins prior to each splicing event. This complexity has made characterization of splicing incredibly challenging. While extensive biochemical and biophysical studies have been carried out to elucidate the binding mechanism and dynamics, these studies do not take into consideration the effect of the cellular environment on the kinetics of protein and RNA interactions. We recently demonstrated that fast relaxation imaging (FReI), which combines fluorescence microscopy and temperature jumps, provides a powerful method to study protein-RNA interactions inside a living cell (Guzman Dissertation 2015). That study provided insight into binding kinetics and RNA-mediated localization of the U1A-SL2 complex, components of the U1 snRNP subunit of the spliceosome. We are now developing a three-color FReI that enables the correlation of multiple events and determination of their order and timing. By monitoring the FRET between successively larger complexes, we will be able to determine the association kinetics, dissociation kinetics and coupling within and between snRNP and associated proteins during spliceosome assembly in the native cellular environment.

In-Cell Protein Folding - PAPS Synthases

Here, we study the stabilities of different naturally fragile mutants of 3' phosphoadenosine 5' phosphosulfate (PAPS) synthases. The goal is to establish a relationship between the stability of the mutants in the cellular environment and their enzymatic activity. In vitro stability measurements have suggested that protein stability is a major factor for cellular PAPS availability [1]. We aim to specifically analyze different cellular factors for in-cell PAPSS stability. One crucial factor is the macromolecular crowding effect. The cell is filled up to a volume of 40 % with macromolecules. Often, artificial macromolecular crowding agents are used to mimic these conditions. We previously studied macromolecular crowding effects via a thermodynamic analysis of the thermal unfolding of ubiquitin [2]. We observed enthalpic stabilization and entropic destabilization forces for all tested crowders. Further, we tested how such artificial cosolutes reflect the physicochemical properties of the complex cellular environment. Therefore, we developed a FRET-based macromolecular crowding sensor to study the crowding effect in living cells [3]. We found that the in-cell crowding effect is distributed heterogeneously and can change significantly upon osmotic stress. In comparison, we now used Fast Relaxation Imaging [4] to study the stability of PAPSS within the cellular environment and compare the results to in vitro crowding studies.

Protein folding dynamics in the cell.

Protein folding is a remarkably fast unimolecular reaction, spanning microseconds to hours at room temperature. Thus, free energy differences and activation barriers on the free energy landscape of proteins are rather small. This opens up the possibility of living cells modulating their protein's landscapes, providing cells another way to control the function of their proteomes after transcriptional control, translational control, and post-translational modification. In this Feature Article, we discuss advances in physicochemical studies of protein stability and folding inside living cells. We focus in particular on our studies using fast relaxation imaging (FREI). Although the effect of the cell on protein free energy landscapes is only a few kT, the strong cooperativity of many folding and binding processes allows small modulation of the energy and entropy to produce a large population modulation. Lastly, we discuss some biomolecular processes that are particularly likely to be affected by in-cell modulation of the proteome, and thus of interest for quantitative physical chemistry studies.

Deciphering Protein Stability in Cells

Deciphering Protein Stability in Cells

The Extracellular Protein VlsE Is Destabilized Inside Cells

We use U2OS cells as in vivo “test tubes” to study how the same cytoplasmic environment has opposite effects on the stability of two different proteins. Protein folding stability and kinetics were compared by fast relaxation imaging, which combines a temperature jump with fluorescence microscopy of FRET (Förster resonance energy transfer)-labeled proteins. While the stability of the cytoplasmic enzyme PGK (phosphoglycerate kinase) increases in cells, the stability of the cell surface antigen VlsE, which presumably did not evolve for stability inside cells, decreases. VlsE folding also slows down more than PGK folding in cells, relative to their respective aqueous buffer kinetics. Our FRET measurements provide evidence that VlsE is more compact inside cells than in aqueous buffer. Two kinetically distinct protein populations exist inside cells, making a connection with previous in vitro crowding studies. In addition, we confirm previous studies showing that VlsE is stabilized by 150mg/mL of the carbohydrate crowder Ficoll, even though it is destabilized in the cytoplasm relative to aqueous buffer. We propose two mechanisms for the observed destabilization of VlsE in U2OS cells: long-range interactions competing with crowding or shape-dependent crowding favoring more compact states inside the cell over the elongated aqueous buffer native state.

The Gold Nanorod-Biology Interface: From Proteins to Cells to Tissue

Gold nanorods absorb and scatter light strongly in the near-infrared portion of the electromagnetic spectrum, making them ideal tissue contrast agents for imaging techniques such as optical coherence tomography (OCT). Strong interactions occur at the nano-bio interface, such as proteins binding to gold nanorods forming a 'corona.' To fulfill the promise of nanorods for applications such as contrast agents, we must better understand the intrinsic interactions of these nanomaterials with biological systems at the molecular, cellular and tissue level. In this paper, we briefly review the nanorod-protein interface. We then present some new fast relaxation imaging (FReI) measurements of how the presence of strongly-absorbing gold nanorods affects protein binding and folding, taking into account inner filter effects and the strong quenching effect of nanorods on fluorescent-labeled proteins. Next we show that two-photon photoluminescence of the gold nanorods can be used to image the nanorods in tissue constructs, allowing us to independently study their tissue distribution so they can be used successfully as contrast agents in optical coherence microscopy.

Protein Folding across the Cell Cycle

The mammalian cell cycle is the biological pathway through which cells grow and divide. During this physically dramatic process the cell doubles in size, duplicates its genetic material, dissolves its nucleus, and ultimately divides into two genetically identical daughter cells. Due in part to the fundamental role of cell cycle abnormalities to cancer pathogenesis, cell cycle research remains a very active topic in the literature. Yet, there is a comparatively very little work exploring the influence of the cell cycle on protein biophysics. Considering the immense intracellular physical changes the cell undergoes during the cell cycle as well as the established sensitivity of protein folding to the intracellular environment, we aim to determine the in vivo protein folding trends as the cell progresses through the stages of the cell cycle. With Fast Relaxation Imaging (FReI), a fluorescence microscopy system for measuring in vivo protein folding, we explore the temperature induced folding thermodynamics and kinetics of a FRET-labeled model protein, yeast phosphoglycerate-kinase (PGK), in living U2OS cells arrested in the prometaphase stage of mitosis and the G1 stage of interphase. We see that PGK is more stable in mitotic cells compared to interphase cells, suggesting that the protein folding environment significantly changes as the cell progresses through the cell cycle.

Temperature Dependence of Protein Folding in Live Cell

Protein folding kinetics is known to be non-Arrhenius temperature dependent. We use Fast Relaxation Imaging (FReI) to measure stability and folding kinetics of FRET-labeled destabilized phosphoglycerate kinase (PGK). With modulated heating laser, we are able to measure the thermodynamics of PGK rapidly across the midpoint of transition of protein unfolding to minimize baseline shifts coming from photobleaching and protein aggregation.

The Ever Folding Protein Landscape

The Ever Folding Protein Landscape