Spliceosomal SL1 RNA binding to U1-70K: the role of the extended RRM.

The RNA recognition motif (RRM) occurs widely in RNA-binding proteins, but does not always by itself support full binding. For example, it is known that binding of SL1 RNA to the protein U1-70K in the U1 spliceosomal particle is reduced when a region flanking the RRM is truncated. How the RRM flanking regions that together with the RRM make up an 'extended RRM' (eRRM) contribute to complex stability and structural organization is unknown. We study the U1-70K eRRM bound to SL1 RNA by thermal dissociation and laser temperature jump kinetics; long-time molecular dynamics simulations interpret the experiments with atomistic resolution. Truncation of the helix flanking the RRM on its N-terminal side, 'N-helix,' strongly reduces overall binding, which is further weakened under higher salt and temperature conditions. Truncating the disordered region flanking the RRM on the C-terminal side, 'C-IDR', affects the local binding site. Surprisingly, all-atom simulations show that protein truncation enhances base stacking interactions in the binding site and leaves the overall number of hydrogen bonds intact. Instead, the flanking regions of the eRRM act in a distributed fashion via collective interactions with the RNA when external stresses such as temperature or high salt mimicking osmotic imbalance are applied.

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.

Cytoskeletal Drugs Modulate Off-Target Protein Folding Landscapes Inside Cells.

The dynamic cytoskeletal network of microtubules and actin filaments can be disassembled by drugs. Cytoskeletal drugs work by perturbing the monomer-polymer equilibrium, thus changing the size and number of macromolecular crowders inside cells. Changes in both crowding and nonspecific surface interactions ("sticking") following cytoskeleton disassembly can affect the protein stability, structure, and function directly or indirectly by changing the fluidity of the cytoplasm and altering the crowding and sticking of other macromolecules in the cytoplasm. The effect of cytoskeleton disassembly on protein energy landscapes inside cells has yet to be observed. Here we have measured the effect of several cytoskeletal drugs on the folding energy landscape of two FRET-labeled proteins with different in vitro sensitivities to macromolecular crowding. Phosphoglycerate kinase (PGK) was previously shown to be more sensitive to crowding, whereas variable major protein-like sequence expressed (VlsE) was previously shown to be more sensitive to sticking. The in-cell effects of drugs that depolymerize either actin filaments (cytochalasin D and latrunculin B) or microtubules (nocodazole and vinblastine) were compared. The crowding sensor protein CrH2-FRET verified that cytoskeletal drugs decrease the extent of crowding inside cells despite also reducing the overall cell volume. The decreased compactness and folding stability of PGK could be explained by the decreased extent of crowding induced by these drugs. VlsE's opposite response to the drugs shows that depolymerization of the cytoskeleton also changes sticking in the cellular milieu. Our results demonstrate that perturbation of the monomer-polymer cytoskeletal equilibrium, for example, during natural cell migration or stresses from drug treatment, has off-target effects on the energy landscapes of proteins in the cell.

Labeling for Quantitative Comparison of Imaging Measurements in Vitro and in Cells.

Qualitative imaging of biomolecular localization and distribution inside cells has revolutionized cell biology. Most of these powerful techniques require modifications to the target biomolecule. Over the past 10 years, these techniques have been extended to quantitative measurements, from in-cell protein folding rates to complex dissociation constants to RNA lifetimes. Such measurements can be affected even when a target molecule is just mildly perturbed by its labels. Here, the impact of labeling on protein (and RNA) structure, stability, and function in cells is discussed via practical examples from the recent literature. General guidelines for selecting and validating modification sites are provided to bring the best from cell biology and imaging to quantitative biophysical experiments inside cells.

Binding, folding and insertion of a ß-hairpin peptide at a lipid bilayer surface: Influence of electrostatics and lipid tail packing.

Antimicrobial peptides (AMPs) act as host defenses against microbial pathogens. Here we investigate the interactions of SVS-1 (KVKVKVKVdPlPTKVKVKVK), an engineered AMP and anti-cancer ß-hairpin peptide, with lipid bilayers using spectroscopic studies and atomistic molecular dynamics simulations. In agreement with literature reports, simulation and experiment show preferential binding of SVS-1 peptides to anionic over neutral bilayers. Fluorescence and circular dichroism studies of a Trp-substituted SVS-1 analogue indicate, however, that it will bind to a zwitterionic DPPC bilayer under high-curvature conditions and folds into a hairpin. In bilayers formed from a 1:1 mixture of DPPC and anionic DPPG lipids, curvature and lipid fluidity are also observed to promote deeper insertion of the fluorescent peptide. Simulations using the CHARMM C36m force field offer complementary insight into timescales and mechanisms of folding and insertion. SVS-1 simulated at an anionic mixed POPC/POPG bilayer folded into a hairpin over a microsecond, the final stage in folding coinciding with the establishment of contact between the peptide's valine sidechains and the lipid tails through a "flip and dip" mechanism. Partial, transient folding and superficial bilayer contact are seen in simulation of the peptide at a zwitterionic POPC bilayer. Only when external surface tension is applied does the peptide establish lasting contact with the POPC bilayer. Our findings reveal the influence of disruption to lipid headgroup packing (via curvature or surface tension) on the pathway of binding and insertion, highlighting the collaborative effort of electrostatic and hydrophobic interactions on interaction of SVS-1 with lipid bilayers.

Non-Steric Interactions Predict the Trend and Steric Interactions the Offset of Protein Stability in Cells.

Although biomolecules evolved to function in the cell, most biochemical assays are carried out in vitro. In-cell studies highlight how steric and non-steric interactions modulate protein folding and interactions. VlsE and PGK present two extremes of chemical behavior in the cell: the extracellular protein VlsE is destabilized in eukaryotic cells, whereas the cytoplasmic protein PGK is stabilized. VlsE and PGK are benchmarks in a systematic series of solvation environments to distinguish contributions from non-steric and steric interactions to protein stability, compactness, and folding rate by comparing cell lysate, a crowding agent, ionic buffer and lysate buffer with in-cell results. As anticipated, crowding stabilizes proteins, causes compaction, and can speed folding. Protein flexibility determines its sensitivity to steric interactions or crowding. Non-steric interactions alone predict in-cell stability trends, while crowding provides an offset towards greater stabilization. We suggest that a simple combination of lysis buffer and Ficoll is an effective new in vitro mimic of the intracellular environment on protein folding and stability.

Soluble Zwitterionic Poly(sulfobetaine) Destabilizes Proteins.

The widespread interest in neutral, water-soluble polymers such as poly(ethylene glycol) (PEG) and poly(zwitterions) such as poly(sulfobetaine) (pSB) for biomedical applications is due to their widely assumed low protein binding. Here we demonstrate that pSB chains in solution can interact with proteins directly. Moreover, pSB can reduce the thermal stability and increase the protein folding cooperativity relative to proteins in buffer or in PEG solutions. Polymer-dependent changes in the tryptophan fluorescence spectra of three structurally-distinct proteins reveal that soluble, 100 kDa pSB interacts directly with all three proteins and changes both the local polarity near tryptophan residues and the protein conformation. Thermal denaturation studies show that the protein melting temperatures decrease by as much as ∼1.9 °C per weight percent of polymer and that protein folding cooperativity increases by as much as ∼130 J mol-1 K-1 per weight percent of polymer. The exact extent of the changes is protein-dependent, as some proteins exhibit increased stability, whereas others experience decreased stability at high soluble pSB concentrations. These results suggest that pSB is not universally protein-repellent and that its efficacy in biotechnological applications will depend on the specific proteins used.

End-Stage Renal Disease Increases Rates of Adverse Glucose Events When Treating Diabetic Ketoacidosis or Hyperosmolar Hyperglycemic State.

IN BRIEF Treatment guidelines for diabetic emergencies are well described in patients with normal to moderately impaired kidney function. However, management of patients with end-stage renal disease (ESRD) is an ongoing challenge. This article describes a retrospective study comparing the rates of adverse glucose events (defined as hypoglycemia or a decrease in glucose >200 mg/dL/h) between patients with ESRD and those with normal kidney function who were admitted with diabetic ketoacidosis (DKA) or hyperosmolar hyperglycemic state (HHS). These results indicate that current treatment approaches to DKA or HHS in patients with ESRD are suboptimal and require further evaluation.

The Role of Electrostatic Interactions in Folding of ß-Proteins.

Atomic-level molecular dynamic simulations are capable of fully folding structurally diverse proteins; however, they are limited in their ability to accurately represent electrostatic interactions. Here we have experimentally tested the role of charged residues on stability and folding kinetics of one of the most widely simulated ß-proteins, the WW domain. The folding of wild type Pin1 WW domain, which has two positively charged residues in the first turn, was compared to the fast folding mutant FiP35 Pin1, which introduces a negative charge into the first turn. A combination of FTIR spectroscopy and laser-induced temperature-jump coupled with infrared spectroscopy was used to probe changes in the amide I region. The relaxation dynamics of the peptide backbone, ß-sheets and ß-turns, and negatively charged aspartic acid side chain of FiP35 were measured independently by probing the corresponding bands assigned in the amide I region. Folding is initiated in the turns and the ß-sheets form last. While the global folding mechanism is in good agreement with simulation predictions, we observe changes in the protonation state of aspartic acid during folding that have not been captured by simulation methods. The protonation state of aspartic acid is coupled to protein folding; the apparent pKa of aspartic acid in the folded protein is 6.4. The dynamics of the aspartic acid follow the dynamics of the intermediate phase, supporting assignment of this phase to formation of the first hairpin. These results demonstrate the importance of electrostatic interactions in turn stability and formation of extended ß-sheet structures.

Fast helix formation in the B domain of protein A revealed by site-specific infrared probes.

Comparison of experimental and computational protein folding studies can be difficult because of differences in structural resolution. Isotope-edited infrared spectroscopy offers a direct measure of structural changes involved in protein folding at the single-residue level. Here we demonstrate the increased resolution of site-specific infrared probes to the peptide backbone in the B domain of staphylococcal protein A (BdpA). (13)C═(18)O-labeled methionine was incorporated into each of the helices using recombinant protein expression. Laser-induced temperature jumps coupled with infrared spectroscopy were used to probe changes in the peptide backbone on the submillisecond time scale. The relaxation kinetics of the buried helices, solvated helices, and labeled positions were measured independently by probing the corresponding bands assigned in the amide I region. Using these wavelength-dependent measurements, we observe a fast nanosecond phase and slower microsecond phase at each position. We find at least partial formation of helices 1-3 in the fast intermediate state that precedes the transition state. These measurements provide direct, time-resolved experimental evidence of the early formation of partial helical structure in helices 1 and 3, supporting folding models proposed by computer simulations.

WW domain folding complexity revealed by infrared spectroscopy.

Although the intrinsic tryptophan fluorescence of proteins offers a convenient probe of protein folding, interpretation of the fluorescence spectrum is often difficult because it is sensitive to both global and local changes. Infrared (IR) spectroscopy offers a complementary measure of structural changes involved in protein folding, because it probes changes in the secondary structure of the protein backbone. Here we demonstrate the advantages of using multiple probes, infrared and fluorescence spectroscopy, to study the folding of the FBP28 WW domain. Laser-induced temperature jumps coupled with fluorescence or infrared spectroscopy have been used to probe changes in the peptide backbone on the submillisecond time scale. The relaxation dynamics of the ß-sheets and ß-turn were measured independently by probing the corresponding IR bands assigned in the amide I region. Using these wavelength-dependent measurements, we observe three kinetics phases, with the fastest process corresponding to the relaxation kinetics of the turns. In contrast, fluorescence measurements of the wild-type WW domain and tryptophan mutants exhibit single-exponential kinetics with a lifetime that corresponds to the slowest phase observed by infrared spectroscopy. Mutant sequences provide evidence of an intermediate dry molten globule state. The slowest step in the folding of this WW domain is the tight packing of the side chains in the transition from the dry molten globule intermediate to the native structure. This study demonstrates that using multiple complementary probes enhances the interpretation of protein folding dynamics.

Oleic acid differentially affects lipid droplet storage of de novo synthesized lipids in hepatocytes and adipocytes.

Lipogenesis is a vital but often dysregulated metabolic pathway. Here we use optical photothermal infrared imaging to quantify lipogenesis rates of isotopically labelled oleic acid and glucose concomitantly in live cells. In hepatocytes, but not adipocytes, we find that oleic acid feeding at 60 µM increases the number and size of lipid droplets (LDs) while simultaneously inhibiting storage of de novo synthesized lipids in LDs. Our results demonstrate alternate regulation of lipogenesis between cell types.

Identifying the minimal sets of distance restraints for FRET-assisted protein structural modeling.

Proteins naturally occur in crowded cellular environments and interact with other proteins, nucleic acids, and organelles. Since most previous experimental protein structure determination techniques require that proteins occur in idealized, non-physiological environments, the effects of realistic cellular environments on protein structure are largely unexplored. Recently, Förster resonance energy transfer (FRET) has been shown to be an effective experimental method for investigating protein structure in vivo. Inter-residue distances measured in vivo can be incorporated as restraints in molecular dynamics (MD) simulations to model protein structural dynamics in vivo. Since most FRET studies only obtain inter-residue separations for a small number of amino acid pairs, it is important to determine the minimum number of restraints in the MD simulations that are required to achieve a given root-mean-square deviation (RMSD) from the experimental structural ensemble. Further, what is the optimal method for selecting these inter-residue restraints? Here, we implement several methods for selecting the most important FRET pairs and determine the number of pairs Nr that are needed to induce conformational changes in proteins between two experimentally determined structures. We find that enforcing only a small fraction of restraints, Nr/N≲0.08, where N is the number of amino acids, can induce the conformational changes. These results establish the efficacy of FRET-assisted MD simulations for atomic scale structural modeling of proteins in vivo. Significance: Determining protein structure in vivo is essential for understanding protein function. Most protein structures have been studied in non-physiological conditions using x-ray crystallography, NMR spectroscopy, and cryo-electron microscopy. Thus, we do not know whether the cellular environment significantly affects protein structure. We emphasize the benefits of FRET-assisted molecular dynamics simulations in characterizing protein structure in vivo at the atomic scale. We identify the minimum number of FRET pairs that can induce conformational changes in several proteins, including one that has been characterized using in-cell NMR.

Dynamics of an ultrafast folding subdomain in the context of a larger protein fold.

Small fast folding subdomains with low contact order have been postulated to facilitate the folding of larger proteins. We have tested this idea by determining how the fastest folding linear ß-hairpin, CLN025, which folds on the nanosecond time scale, folds within the context of a two-hairpin WW domain system, which folds on the microsecond time scale. The folding of the wild type FBP28 WW domain was compared to constructs in which each of the loops was replaced by CLN025. A combination of FTIR spectroscopy and laser-induced temperature-jump coupled with infrared spectroscopy was used to probe changes in the peptide backbone. The relaxation dynamics of the ß-sheets and ß-turn were measured independently by probing the corresponding bands assigned in the amide I region. The folding rate of the CLN025 ß-hairpin is unchanged within the larger protein. Insertion of the ß-hairpin into the second loop results in an overall stabilization of the WW domain and a relaxation lifetime five times faster than the parent WW domain. In both mutants, folding is initiated in the turns and the ß-sheets form last. These results demonstrate that fast folding subdomains can be used to speed the folding of more complex proteins, and that the folding dynamics of the subdomain is unchanged within the context of the larger protein.

Chemical interactions modulate λ6-85 stability in cells.

Because steric crowding is most effective when the crowding agent is similar in size to the molecule that it acts upon and the average macromolecule inside cells is much larger than a small protein or peptide, steric crowding is not predicted to affect their folding inside cells. On the other hand, chemical interactions should perturb in-cell structure and stability because they arise from interactions between the surface of the small protein or peptide and its environment. Indeed, previous in vitro measurements of the λ-repressor fragment, λ6-85 , in crowding matrices comprised of Ficoll or protein crowders support these predictions. Here, we directly quantify the in-cell stability of λ6-85 and distinguish the contribution of steric crowding and chemical interactions to its stability. Using a FRET-labeled λ6-85 construct, we find that the fragment is stabilized by 5°C in-cells compared to in vitro. We demonstrate that this stabilization cannot be explained by steric crowding because, as anticipated, Ficoll has no effect on λ6-85 stability. We find that the in-cell stabilization arises from chemical interactions, mimicked in vitro by mammalian protein extraction reagent (M-PER™). Comparison between FRET values in-cell and in Ficoll confirms that U-2 OS cytosolic crowding is reproduced at macromolecule concentrations of 15% w/v. Our measurements validate the cytomimetic of 15% Ficoll and 20% M-PER™ that we previously developed for protein and RNA folding studies. However, because the in-cell stability of λ6-85 is reproduced by 20% v/v M-PER™ alone, we predict that this simplified mixture could be a useful tool to predict the in-cell behaviors of other small proteins and peptides.

Spatiotemporal Heterogeneity of De Novo Lipogenesis in Fixed and Living Single Cells.

De novo lipogenesis (DNL) is a critical metabolic process that provides the majority of lipids for adipocyte and liver tissue. In cancer, obesity, type II diabetes, and nonalcoholic fatty liver disease DNL becomes dysregulated. A deeper understanding of the rates and of subcellular organization of DNL is necessary for identifying how this dysregulation occurs and varies across individuals and diseases. However, DNL is difficult to study inside the cell because labeling lipids and their precursors is not trivial. Existing techniques either can only measure parts of DNL, like glucose uptake, or do not provide spatiotemporal resolution. Here, we track DNL in space and time as isotopically labeled glucose is converted to lipids in adipocytes using optical photothermal infrared microscopy (OPTIR). OPTIR provides submicron resolution infrared imaging of the glucose metabolism in both living and fixed cells while also reporting on the identity of lipids and other biomolecules. We show significant incorporation of the labeled carbons into triglycerides in lipid droplets over the course of 72 h. Live cells had better preservation of lipid droplet morphology, but both showed similar DNL rates. Rates of DNL, as measured by the ratio of 13C-labeled lipid to 12C-labeled lipid, were heterogeneous, with differences within and between lipid droplets and from cell to cell. The high rates of DNL measured in adipocyte cells match upregulated rates of DNL previously reported in PANC1 pancreatic cancer cells. Taken together, our findings support a model where DNL is locally regulated to meet energy needs within cells.

Oleic acid differentially affects de novo lipogenesis in adipocytes and hepatocytes.

Lipogenesis is a vital but often dysregulated metabolic pathway. We report super-resolution multiplexed vibrational imaging of lipogenesis rates and pathways using isotopically labelled oleic acid and glucose as probes in live adipocytes and hepatocytes. These findings suggest oleic acid inhibits de novo lipogenesis (DNL), but not total lipogenesis, in hepatocytes. No significant effect is seen in adipocytes. These differential effects may be due to alternate regulation of DNL between cell types and could help explain the complicated role oleic acid plays in metabolism.

Site-specific crosslinking reveals Phosphofructokinase-L inhibition drives self-assembly and attenuation of protein interactions.

Phosphofructokinase is the central enzyme in glycolysis and constitutes a highly regulated step. The liver isoform (PFKL) compartmentalizes during activation and inhibition in vitro and in vivo, respectively. Compartmentalized PFKL is hypothesized to modulate metabolic flux consistent with its central role as the rate limiting step in glycolysis. PFKL tetramers self-assemble at two interfaces in the monomer (interface 1 and 2), yet how these interfaces contribute to PFKL compartmentalization and drive protein interactions remains unclear. Here, we used site-specific incorporation of noncanonical photocrosslinking amino acids to identify PFKL interactors at interface 1, 2, and the active site. Tandem mass tag-based quantitative interactomics reveals interface 2 as a hotspot for PFKL interactions, particularly with cytoskeletal, glycolytic, and carbohydrate derivative metabolic proteins. Furthermore, PFKL compartmentalization into puncta was observed in human cells using citrate inhibition. Puncta formation attenuated crosslinked protein-protein interactions with the cytoskeleton at interface 2. This result suggests that PFKL compartmentalization sequesters interface 2, but not interface 1, and may modulate associated protein assemblies with the cytoskeleton.

Site-Specific Crosslinking Reveals Phosphofructokinase-L Inhibition Drives Self-Assembly and Attenuation of Protein Interactions.

Phosphofructokinase is the central enzyme in glycolysis and constitutes a highly regulated step. The liver isoform (PFKL) compartmentalizes during activation and inhibition in vitro and in vivo respectively. Compartmentalized PFKL is hypothesized to modulate metabolic flux consistent with its central role as the rate limiting step in glycolysis. PFKL tetramers self-assemble at two interfaces in the monomer (interface 1 and 2), yet how these interfaces contribute to PFKL compartmentalization and drive protein interactions remains unclear. Here, we used site-specific incorporation of noncanonical photocrosslinking amino acids to identify PFKL interactors at interface 1, 2, and the active site. Tandem mass tag-based quantitative interactomics reveals interface 2 as a hotspot for PFKL interactions, particularly with cytoskeletal, glycolytic, and carbohydrate derivative metabolic proteins. Furthermore, PFKL compartmentalization into puncta was observed in human cells using citrate inhibition. Puncta formation attenuated crosslinked protein-protein interactions with the cytoskeleton at interface 2. This result suggests that PFKL compartmentalization sequesters interface 2, but not interface 1, and may modulate associated protein assemblies with the cytoskeleton.

Raising the speed limit for ß-hairpin formation.

Understanding the folding of the ß-hairpin is a crucial step in studying how ß-rich proteins fold. We have studied CLN025, an optimized ten residue synthetic peptide, which adopts a compact, well-structured ß-hairpin conformation. Formation of the component ß-sheet and ß-turn structures of CLN025 was probed independently using a combination of equilibrium Fourier transform infrared spectroscopy and laser-induced temperature jump coupled with time-resolved infrared and fluorescence spectroscopies. We find that CLN025 is an ultrafast folder due to its small free energy barrier to folding and that it exceeds the predicted speed limit for ß-hairpin formation by an order of magnitude. We also find that the folding mechanism cannot be described by a simple two-state model, but rather is a heterogeneous process involving two independent parallel processes. Formation of stabilizing cross-strand hydrophobic interactions and turn alignment occur competitively, with relaxation lifetimes of 82 ± 10 and 124 ± 10 ns, respectively, at the highest probed temperature. The ultrafast and heterogeneous folding kinetics observed for CLN025 provide evidence for folding on a nearly barrierless free energy landscape, and recalibrate the speed limit for the formation of a ß-hairpin.