Oxygen-induced chromophore degradation in the photoswitchable red fluorescent protein rsCherry.

Reversibly switchable monomeric Cherry (rsCherry) is a photoswitchable variant of the red fluorescent protein mCherry. We report that this protein gradually and irreversibly loses its red fluorescence in the dark over a period of months at 4 °C and a few days at 37 °C. We also find that its ancestor, mCherry, undergoes a similar fluorescence loss but at a slower rate. X-ray crystallography and mass spectrometry reveal that this is caused by the cleavage of the p-hydroxyphenyl ring from the chromophore and the formation of two novel types of cyclic structures at the remaining chromophore moiety. Overall, our work sheds light on a new process occurring within fluorescent proteins, further adding to the chemical diversity and versatility of these molecules.

Probing Universal Protein Dynamics Using Hydrogen-Deuterium Exchange Mass Spectrometry-Derived Residue-Level Gibbs Free Energy.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful technique to monitor protein intrinsic dynamics. The technique provides high-resolution information on how protein intrinsic dynamics are altered in response to biological signals, such as ligand binding, oligomerization, or allosteric networks. However, identification, interpretation, and visualization of such events from HDX-MS data sets is challenging as these data sets consist of many individual data points collected across peptides, time points, and experimental conditions. Here, we present PyHDX, an open-source Python package and webserver, that allows the user to batch extract the universal quantity Gibbs free energy at residue levels over multiple protein conditions and homologues. The output is directly visualized on a linear map or 3D structures or is exported as .csv files or PyMOL scripts.

Structural Dynamics of the Functional Nonameric Type III Translocase Export Gate.

Type III protein secretion is widespread in Gram-negative pathogens. It comprises the injectisome with a surface-exposed needle and an inner membrane translocase. The translocase contains the SctRSTU export channel enveloped by the export gate subunit SctV that binds chaperone/exported clients and forms a putative ante-chamber. We probed the assembly, function, structure and dynamics of SctV from enteropathogenic E. coli (EPEC). In both EPEC and E. coli lab strains, SctV forms peripheral oligomeric clusters that are detergent-extracted as homo-nonamers. Membrane-embedded SctV9 is necessary and sufficient to act as a receptor for different chaperone/exported protein pairs with distinct C-domain binding sites that are essential for secretion. Negative staining electron microscopy revealed that peptidisc-reconstituted His-SctV9 forms a tripartite particle of ∼22 nm with a N-terminal domain connected by a short linker to a C-domain ring structure with a ∼5 nm-wide inner opening. The isolated C-domain ring was resolved with cryo-EM at 3.1 Å and structurally compared to other SctV homologues. Its four sub-domains undergo a three-stage "pinching" motion. Hydrogen-deuterium exchange mass spectrometry revealed this to involve dynamic and rigid hinges and a hyper-flexible sub-domain that flips out of the ring periphery and binds chaperones on and between adjacent protomers. These motions are coincident with local conformational changes at the pore surface and ring entry mouth that may also be modulated by the ATPase inner stalk. We propose that the intrinsic dynamics of the SctV protomer are modulated by chaperones and the ATPase and could affect allosterically the other subunits of the nonameric ring during secretion.

Effect of point mutation on structure-function correlation of hemoglobin variants, HbE and HbD Punjab.

Hemoglobinopathies are examples of autosomal recessive disorders of human hemoglobin. Hemoglobin E (HbE) and Hemoglobin D Punjab (HbD Punjab) are two of the most common hemoglobin variants geographically spread across Asian continent. These two variants differ from normal human hemoglobin (HbA) at a single amino acid residue caused by the point mutation of ß globin gene. The presence of the mutated amino acid residue causes perturbation in the function of both variants. However, the structure-function correlation of these variants has not been established yet. In the present study, we analyzed the conformational changes associated with oxygenation of hemoglobin variants using hydrogen/deuterium exchange-based mass spectrometry of backbone amide hydrogens of α and ß globin chains in the tetrameric hemoglobin molecule. We also performed the functional assay of these variants using oxygen dissociation equilibrium curve. Compared to HbA, both variants showed reduced oxygen affinity, as reported earlier. The functional perturbations exhibited by these variants were correlated well with their structural alterations with respect to the reported changes in the residue level interactions upon oxygenation of normal hemoglobin, monitored through the hydrogen/deuterium exchange kinetics of several peptic peptides originated from the isotopically exchanged oxy and deoxy forms of HbE and HbD Punjab.

Trigger factor is a bona fide secretory pathway chaperone that interacts with SecB and the translocase.

Bacterial secretory preproteins are translocated across the inner membrane post-translationally by the SecYEG-SecA translocase. Mature domain features and signal peptides maintain preproteins in kinetically trapped, largely soluble, folding intermediates. Some aggregation-prone preproteins require chaperones, like trigger factor (TF) and SecB, for solubility and/or targeting. TF antagonizes the contribution of SecB to secretion by an unknown molecular mechanism. We reconstituted this interaction in vitro and studied targeting and secretion of the model preprotein pro-OmpA. TF and SecB display distinct, unsuspected roles in secretion. Tightly associating TF:pro-OmpA targets the translocase at SecA, but TF prevents pro-OmpA secretion. In solution, SecB binds TF:pro-OmpA with high affinity. At the membrane, when bound to the SecA C-tail, SecB increases TF and TF:pro-OmpA affinities for the translocase and allows pro-OmpA to resume translocation. Our data reveal that TF, a main cytoplasmic folding pathway chaperone, is also a bona fide post-translational secretory chaperone that directly interacts with both SecB and the translocase to mediate regulated protein secretion. Thus, TF links the cytoplasmic folding and secretion chaperone networks.

Understanding molecular features of aggregation-resistant tau conformer using oxidized monomer.

BACKGROUND: Aggregation of tau into paired helical filament (PHF) is a hallmark of Alzheimer's disease (AD), and Cys-mediated disulfide bond formation plays a vital role in tau fibrillation. While intermolecular disulfide bond between Cys residues in microtubule-binding repeat (MTBR) region facilitates tau aggregation, intramolecular disulfide bond attenuates the same, though the molecular basis for such phenomenon remains obscure. Thus intramolecular disulfide-bonded tau monomer might be an excellent model to understand the unique features of aggregation-resistant tau conformer. METHODS: We synthesized the Cys cross-linked tau40 monomer by oxidation and characterized the altered conformational dynamics in the molecule by Hydrogen-deuterium exchange, limited proteolysis and fluorescence quenching. RESULTS: Deuterium exchange study showed that rigidity was imparted in the core PHF region of oxidized tau40 in MTBR segment, consisting of the fundamental PHF6 motif. Conformational rigidity was prominent in C-terminal tail region also. Limited proteolysis supported reduced accessibility of MTBR region in the molecule. CONCLUSIONS: PHF formation of oxidized tau40 might be attenuated either by induction of intramolecular H-bonding between the regions of high ß-structure propensity in second and third MTBR (R2, R3), thus preventing intermolecular interaction between them, or by imparted rigidity in R2-R3, preventing the formation of extended ß-structure preceding fibrillation. Data indicated plausible effect of conformational adaptation on the nucleation process of oxidized tau40 assembly. GENERAL SIGNIFICANCE: Our findings unravel the essential molecular features of aggregation-resistant tau conformer. Therapeutics stabilizing such conformers in vivo might be of high benefit in arresting tau assembly during AD and other tauopathies.

Effect of ascorbic acid rich, micro-nutrient fortified supplement on the iron bioavailability of ferric pyrophosphate from a milk based beverage in Indian school children.

BACKGROUND AND OBJECTIVES: Nutritional anemia is a significant public health issue with 50-80% prevalence in Indian children. Fortification of food, specifically milk, with iron is a potential approach to increase dietary iron intake. Ferric pyrophosphate [Fe4(P2O7)3] is organoleptically neutral and is less soluble in acid medium and, further, has low bioavailability in milk. However, since ascorbic acid is a potent enhancer of iron absorption, the coadministration of ascorbic acid with Fe4(P2O7)3 might enhance the absorption of iron. We evaluated the effect of ascorbic acid on iron absorption from a Fe4(P2O7)3 and an ascorbic acid fortified milk beverage with respect to milk fortified with Fe4(P2O7)3 alone. METHODS AND STUDY DESIGN: A double-blind, two-way crossover, randomized study was conducted in 25 mildly anemic children. The test group received milk fortified with beverage powder containing 7 mg isotopically labeled iron (57Fe/58Fe) as Fe4(P2O7)3, equimolar proportions of ascorbic acid and 200 mg of calcium whereas control group received milk fortified with energy, calcium and iron equivalent beverage powder. Fractional iron absorption was measured by erythrocyte incorporation of stable isotopes of iron (57Fe/58Fe) in both the groups. RESULTS: The fractional iron absorption from the control drink was 0.80% (95% CI: 0.57, 1.12). Fortifying the milk with an equimolar amount of ascorbic acid increased the fractional iron absorption almost 2-fold to 1.58% (95% CI: 1.13, 2.22). CONCLUSIONS: The presence of ascorbic acid in an equimolar ratio with that of iron from Fe4(P2O7)3 salt in milk as a fortificant enhanced iron absorption when compared to milk fortified with only Fe4(P2O7)3.

Effect of altered solution conditions on tau conformational dynamics: Plausible implication on order propensity and aggregation.

Intrinsically disordered protein tau plays a central role in maintaining neuronal network by stabilizing microtubules in axon. Tau reportedly possesses random coil architecture, which is largely inert to alteration in solution conditions. However, the presence of transient compact conformers and residual structure has been evident from previous reports. Also, during Alzheimer's disease, misfolded tau detaches from microtubule and forms ordered filaments, which is the hallmark of the disease. Despite its fundamental role in neuronal physiology and in pathological cascade of several fatal neurodegenerative diseases, tau conformational dynamics remains poorly understood. In the present study, we have explored the effect of ionic strength, temperature and solvent polarity on tau40 conformational preferences using ion mobility mass spectrometry. Investigation of collision cross section revealed that while low ionic strength, elevated temperature and reduced solvent polarity mostly induced partial collapse in tau40 conformers, higher ionic strength led to an expansion of the molecule. Limited proteolysis identified segments of tau40 projection domain and proline-rich region having high order propensity and a C-terminal region having vulnerability for further expansion at altered solution conditions. The high susceptibility for disorder-to-order transition in the above region of the protein might have crucial implication on its role as microtubule spacers, and in cellular signaling cascade. The conformational adaptation of tau40 did not enhance the heparin-induced aggregation proclivity of the protein. Nevertheless, the observed correlation of electrostatic interaction with fibrillation propensity of tau40 might indicate plausible link between hyperphosphorylation at diseased state with tau conformation and self-assembly.

Adsorption Induced Changes of Human Hemoglobin on Ferric Pyrophosphate Nanoparticle Surface Probed by Isotope Exchange Mass Spectrometry: An Implication on Structure-Function Correlation.

In general, proteins in the biological system interact with nanoparticles (NPs) via adsorption on the particle surface. Understanding the adsorption at the molecular level is crucial to explore NP-protein interactions. The increasing concerns about the risk to human health on NP exposure have been explored through the discovery of a handful protein biomarkers and biochemical analysis. However, detailed information on structural perturbation and associated functional changes of proteins on interaction with NPs is limited. Erythrocytes (red blood cells) are devoid of defense mechanism of protecting NP penetration through endocytosis. Therefore, it is important to investigate the interaction of erythrocyte proteins with NPs. Hemoglobin, the most abundant protein of human erythrocyte, is a tetrameric molecule consisting of α- and ß-globin chains in duplicate. In the present study, we have used hemoglobin as a model system to investigate NP-protein interaction with ferric pyrophosphate NPs [NP-Fe4(P2O7)3]. We report the formation of a bioconjugate of hemoglobin upon adsorption to NP-Fe4(P2O7)3 surface. Analysis of the bioconjugate indicated that Fe3+ ion of NP-Fe4(P2O7)3 contributed in the bioconjugate formation. Using hydrogen/deuterium exchange based mass spectrometry, it was observed that the amino termini of α- and ß-globin chains of hemoglobin were involved in the adsorption on NP surface whereas the carboxy termini of both chains became more flexible in its conformation compared to the respective regions of the normal hemoglobin. Circular dichroism spectra of desorbed hemoglobin indicated an adsorption induced localized structural change in the protein molecule. The formation of bioconjugate led to functional alteration of hemoglobin, as probed by oxygen binding assay. Thus, we hypothesize that the large amount of energy released upon adsorption of hemoglobin to NP surface might be the fundamental cause of structural perturbation of human hemoglobin and subsequent formation of the bioconjugate with an altered function.