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Ramamoorthy, A.

Publications and source records attributed to Ramamoorthy, A..

At least 37 records · Page 2Linked to original sources
bioRxiv2023

Factors influencing the detergent-free membrane protein isolation using synthetic nanodisc-forming polymers

The detergent-free isolation of membrane proteins using synthetic polymers is becoming the desired approach for functional and structural studies of membrane proteins. Since the expression levels for many membrane proteins are low and a high yield of functionalized reconstituted membrane proteins is essential for in vitro studies, it is crucial to optimize the experimental conditions for a given polymer to effectively solubilize target membranes/proteins. The factors that affect membrane solubilization and subsequently the isolation of a target membrane protein include polymer concentration, polymer charge, temperature, pH, and concentration of divalent metal ions. Therefore, it is important to have knowledge about the efficacy of different types of polymers in solubilizing cell membranes. In this study, we evaluate the efficacy of inulin-based non-ionic polymers in solubilizing E. coli membranes enriched with rat flavin mononucleotide binding-domain (FBD) of cytochrome-P450-reductase (CPR) and rabbit cytochrome-b5 (Cyt-b5) under various solubilization conditions. Our results show that a 1:1 (w/w) membrane:polymer ratio, low temperature, high pH and sub-millimolar concentration of metal ions favor the solubilization of E. coli membrane enriched with FBD or Cyt-b5. Conversely, the presence of excess divalent metal ions affected the final protein levels in the polymer-solubilized samples. We believe that the results from this study provides knowledge to assess and plan the use of non-ionic polymers in membrane protein studies.

biochemistry↗
bioRxiv2022

Ganglioside GM1 produces Aβ40 cytotoxic protofibril structure

Monosialoganglioside GM1-bound amyloid {beta}-peptides have been found in patients brains exhibiting early pathological changes of Alzheimer s disease. Herein, we report the ability of non-micellar GM1 to modulate A{beta}40 aggregation resulting in the formation of stable, short, rod-like, and cytotoxic A{beta}40 protofibrils with the ability to potentiate both A{beta}40 and A{beta}42 aggregation.

biophysics↗
bioRxiv2022

Combining Stem Cell Rejuvenation and Senescence Targeting to Synergistically Extend Lifespan

Why biological age is a major risk factor for many of the most important human diseases remains mysterious. We know that as organisms age, stem cell pools are exhausted while senescent cells progressively accumulate. Independently, induction of pluripotency via expression of Yamanaka factors (Oct4, Klf4, Sox2, c-Myc; OKSM) and clearance of senescent cells have each been shown to ameliorate cellular and physiological aspects of aging, suggesting that both processes are drivers of organismal aging. However, stem cell exhaustion and cellular senescence likely interact in the etiology and progression of age-dependent diseases because both undermine tissue and organ homeostasis in different if not complementary ways. Here, we combine transient cellular reprogramming (stem cell rejuvenation) with targeted removal of senescent cells to test the hypothesis that simultaneously targeting both cell-fate based aging mechanisms will maximize life and health span benefits. We show that these interventions protect the intestinal stem cell pool, lower inflammation, activate pro-stem cell signaling pathways, and synergistically improve health and lifespan. Our findings suggest that a combination therapy, simultaneously replacing lost stem cells and removing senescent cells, shows synergistic potential for anti-aging treatments. Our finding that transient expression of both is the most effective suggests that drug-based treatments in non-genetically tractable organisms will likely be the most translatable.

developmental biology↗
bioRxiv2021

Synthesis, Characterization, and Nanodisc formation of Non-ionic Polymers

Despite lipid-nanodiscs are increasingly used in the structural studies of membrane proteins, drug delivery and other applications, the interaction between the nanodisc-belt and the protein to be reconstituted is a major limitation. To overcome this limitation and to further broaden the scope of nanodiscs, a family of non-ionic amphiphilic polymers synthesized by hydrophobic functionalization of fructo-oligosaccharides/inulin is reported. We show the stability of lipid-nanodiscs formed by these polymers against pH and divalent metal ions, and their magnetic-alignment properties. The reported results also demonstrate that the non-ionic polymers extract membrane proteins with unprecedented efficiency.

biochemistry↗
bioRxiv2020

Natural-abundance 17O NMR spectroscopy of magnetically aligned lipid nanodiscs

Natural-abundance 17O NMR experiments are used to investigate the hydrated water in magnetically alligned synthetic polymer based lipid-nanodiscs. Residual quadrupole couplings (RQCs) measured from the observed five 17O (central and satellite) transitions, and molecular dynamics simulations, are used to probe the ordering of water moecules across the lipid bilayer.

biophysics↗
bioRxiv2020

Conformational Tuning of Amylin by Charged SMA Copolymers

Human amylin forms structurally heterogeneous amyloids that have been linked to type-2 diabetes. Thus, understanding the molecular interactions governing amylin aggregation can provide mechanistic insights in its pathogenic formation. Here, we demonstrate that fibril formation of amylin is altered by synthetic amphipathic copolymer derivatives of the styrene- maleic-acid (SMAQA and SMAEA). High-speed AFM is used to follow the real-time aggregation of amylin by observing the rapid formation of de novo globular oligomers and arrestment of fibrillation by the positively-charged SMAQA. We also observed an accelerated fibril formation in the presence of the negatively-charged SMAEA. These findings were further validated by fluorescence, SOFAST-HMQC, DOSY and STD NMR experiments. Conformational analysis by CD and FT-IR revealed that the SMA copolymers modulate the conformation of amylin aggregates. While the species formed with SMAQA are -helical, the ones formed with SMAEA are rich in {beta}-sheet structure. The interacting interfaces between SMAEA or SMAQA and amylin are mapped by NMR and microseconds all-atom MD simulation. SMAEA displayed {pi}-{pi} interaction with Phe23, electrostatic {pi}-cation interaction with His18 and hydrophobic packing with Ala13 and Val17; whereas SMAQA showed a selective interaction with amylins C-terminus (residues 31-37) that belongs to one of the two {beta}-sheet regions (residues 14-19 and 31-36) involved in amylin fibrillation. Toxicity analysis showed both SMA copolymers to be non-toxic in vitro and the amylin species formed with the copolymers showed minimal deformity to zebrafish embryos. Together, this study demonstrates that chemical tools, such as copolymers, can be used to modulate amylin aggregation, alter the conformation of species.

biophysics↗
bioRxiv2020

Degradation of Alzheimers Amyloid-beta by a Catalytically Inactive Insulin Degrading Enzyme

It is known that insulin-degrading-enzyme (IDE) plays a crucial role in the clearance of Alzheimers amyloid-{beta} (A{beta}). The cysteine-free IDE mutant (cf-E111Q-IDE) is catalytically inactive against insulin, but its effect on A{beta} degradation is unknown that would help in the allosteric modulation of the enzyme activity. Herein, the degradation of A{beta}(1-40) by cf-E111Q-IDE via a non-chaperone mechanism is demonstrated by NMR and LC-MS, and the aggregation of fragmented peptides is characterized using fluorescence and electron microscopy. cf-E111Q-IDE presented a reduced effect on the aggregation kinetics of A{beta}(1-40) when compared with the wild-type IDE. Whereas LC-MS and diffusion ordered NMR spectroscopy revealed the generation of A{beta} fragments by both wild-type and cf-E111Q-IDE. The aggregation propensities and the difference in the morphological phenotype of the full-length A{beta}(1-40) and its fragments are explained using multi-microseconds molecular dynamics simulations. Notably, our results reveal that zinc binding to A{beta}(1-40) inactivates cf-E111Q-IDEs catalytic function, whereas zinc removal restores its function as evidenced from high-speed AFM, electron microscopy, chromatography, and NMR results. These findings emphasize the catalytic role of cf-E111Q-IDE on A{beta} degradation and urge the development of zinc chelators as an alternative therapeutic strategy that switches on/off IDEs function.

biophysics↗
bioRxiv2019

High Throughput Screening at the Membrane Interface Reveals New Inhibitors of Amyloid-β

Amyloid-{beta} aggregation at the cell-membrane of neruonal cells is implicated as a source of toxicity for Alzheimers disease. Small molecules have been studied for their ability to supress amyloid aggregation and toxicity, but the presence of membranes negate their activity. Here, we have identified 5 small molecules that are active at the membrane interface.

biochemistry↗
bioRxiv2019

Small Molecule Induced Toxic Human-IAPP Species Characterized by NMR

In this study, the effect of CurDAc, a water-soluble curcumin derivative, on the formation and stability of amyloid fibers is revealed. CurDAc interaction with amyloid is structurally selective which is reflected in a strong interference with hIAPP aggregation, while showing weaker interactions with human-calcitonin and Amyloid-{beta}1-40 in comparison. Remarkably, CurDAc also exhibited potent fiber disaggregation for hIAPP generating a toxic oligomeric species.

biophysics↗
bioRxiv2019

Expression, Purification, and Functional Reconstitution of 19F-labeled Cytochrome b5 in Peptide Nanodiscs for NMR Studies

Microsomal cytochrome b5 (cytb5) is a membrane-bound protein capable of donating the second electron to cytochrome P450s (CYP P450s) in the CYP P450s monooxygenase reactions. Recent studies have demonstrated the importance of the transmembrane domain of cytb5 in the interaction with cytP450 by stabilizing its monomeric structure. While recent NMR studies have provided high-resolution insights into the structural interactions between the soluble domains of ~16-kDa cytb5 and ~57-kDa cytP450 in a membrane environment, there is need for studies to probe the residues in the transmembrane region as well as to obtain intermolecular distance constraints to better understand the very large size cytb5-cytP450 complex structure in a near native membrane environment. In this study, we report the expression, purification, functional reconstitution of 19F-labeled full-length rabbit cytb5 in peptide based nanodiscs for structural studies using NMR spectroscopy. Size exclusion chromatography, dynamic light scattering, transmission electron microscopy, and NMR experiments show a stable reconstitution of cytb5 in 4F peptide-based lipid-nanodiscs. Our results demonstrate that the use of peptide-nanodiscs containing cytb5 19F-labeled with 5-fluorotryptophan (5FW) enables, for the first time, the detection of residues from the transmembrane domain in different lipid compositions by solution 19F NMR experiments. 19F NMR results revealing the interaction of the transmembrane domain of cytb5 with the full-length rabbit Cytochrome P450 2B4 (CYP2B4) are also presented. We expect the results presented in this study to be useful to devise approaches to probe the structure, dynamics and functional roles of transmembrane domains of a membrane protein, and also to measure intermolecular 19F-19F distance constraints to determine the structural interactions between the transmembrane domains.

biochemistry↗
bioRxiv2019

Magnetic-Alignment of Polymer Nanodiscs Probed by Solid-State NMR Spectroscopy

The ability of amphipathic polymers to self-assemble with lipids and form nanodiscs has been a boon for the field of functional reconstitution of membrane proteins. In a field dominated by detergent micelles, a unique feature of polymer nanodiscs is their much-desired ability to align in the presence of an external magnetic field. Magnetic alignment facilitates the application of solid-state NMR spectroscopy and aids in the measurement of residual dipolar couplings (RDCs) via well-established solution NMR spectroscopy. In this study, we comprehensively investigate the magnetic-alignment properties of SMA-QA polymer based nanodiscs by using 31P and 14N solid-state NMR experiments under static conditions. The results reported herein demonstrate the spontaneous magnetic-alignment of large-size ([≥] 20 nm diameter) SMA-QA nanodiscs (also called as macro-nanodiscs) with the lipid-bilayer-normal perpendicular to the magnetic field direction. Consequently, the orientation of macro-nanodiscs are further shown to flip their alignment axis parallel to the magnetic field direction upon the addition of a paramagnetic lanthanide salt. These results demonstrate the use of SMA-QA polymer nanodiscs for solid-state NMR applications including structural studies on membrane proteins.

biophysics↗
bioRxiv2019

Apolipoprotein A-I Mimetic 4F Peptide Generates Amyloid Cytotoxins by Forming Hetero-oligomers with β-amyloid

Apolipoproteins are involved in pathological conditions of Alzheimers disease (AD), truncated apolipoprotein fragments and {beta}-amyloid (A{beta}) peptides coexist as neurotoxic heteromers within the plaques. Therefore, it is important to investigate these complexes at the molecular level to better understand their properties and roles in the pathology of AD. Here, we present a mechanistic insight into such heteromerization using a structurally homologue apolipoprotein fragment of apoA-I (4F) complexed with A{beta}(M1-42) and characterize their toxicity. The 4F peptide slows down the aggregation kinetics of A{beta}(M1-42) by constraining its structural plasticity. NMR and CD experiments identified 4F-A{beta}(M1-42) heteromers as being comprised of unstructured A{beta}(M1-42) and helical 4F. A uniform {approx}2-fold reduction in A{beta}42 15N/1H NMR signal intensities with no observable chemical shift perturbation indicated the formation of a large complex, which was further confirmed by diffusion NMR experiments. Microsecond scale atomistic molecular dynamics simulations showed that 4F interaction with A{beta}(M1-42) is electrostatically driven and induces unfolding of A{beta}(M1-42). Neurotoxicity profiling of A{beta}(M1-42) complexed with 4F confirms a significant reduction in cell-viability and neurite growth. The molecular architecture of heteromerization between 4F and A{beta}(M1-42) discovered in this study provides evidence towards our understanding of the role of apolipoproteins or their truncated fragments in exacerbating AD pathology.

biophysics↗
bioRxiv2019

Exploiting Heterogeneous Time Scale of Dynamics toEnhance 2D HETCOR Solid-State NMR Sensitivity

Multidimensional solid-state NMR spectroscopy plays a significant role in offering atomic-level insights into molecular systems. In particular, heteronuclear chemical shift correlation (HETCOR) experiments could provide local chemical and structural information in terms of spatial heteronuclear proximity and through-bond connectivity. In solid state, the transfer of magnetization between heteronuclei, a key step in HETCOR experiments, is usually achieved using cross-polarization (CP) or INEPT (insensitive nuclei enhanced by polarization transfer) depending on the sample characteristics and magic-angle-spinning (MAS) frequency. But, for a multiphase system constituting molecular components that differ in their time scales of mobilities, CP efficiency is pretty low for mobile components because of the averaging of heteronuclear dipolar couplings whereas INEPT is inefficient due to the short T2 of immobile components and can be non-selective due to strong proton spin diffusion for immobile components especially under moderate spinning speeds. Herein, in this study we present two 2D pulse sequences that enable the sequential acquisition of 13C/1H HETCOR NMR spectra for the rigid and mobile components by taking full advantage of the abundant proton magnetization in a single experiment with barely increasing the overall experimental time. In particular, the 13C-detected HETCOR experiment could be applied under slow MAS conditions, where a multiple-pulse sequence is typically employed to enhance 1H spectral resolution in the indirect dimension. In contrast, the 1H-detected HETCOR experiment should be applied under ultrafast MAS, where CP and transient heteronuclear nuclear Overhauser effect (NOE) polarization transfer are combined to enhance 13C signal intensities for mobile components. These pulse sequences are experimentally demonstrated on two model systems to obtain 2D 13C/1H chemical shift correlation spectra of rigid and mobile components independently and separately. These pulse sequences can be used for dynamics difference based spectral editing and resonance assignments. Therefore, we believe the proposed 2D HETCOR NMR pulse sequences will be beneficial for the structural studies of heterogeneous systems containing molecular components that differ in their time scale of motions for understanding the interplay of structures and properties.

bioengineering↗
bioRxiv2018

Self-assembly of polymer-encased lipid nanodiscs and membrane protein reconstitution

The absence of detergent and curvature makes nanodiscs to be excellent membrane mimetics. The lack of structural and mechanistic model of polymer-encapsulated lipid-nanodiscs limits their use to study the structure, dynamics and function of membrane proteins. In this study, we parametrized and optimized the coarse-graining (CG) bead-mapping for two differently charged and functionalized copolymers, namely styrene-maleic acid (SMAEA) and polymethacrylate (PMAQA), for the Martini force-field framework and showed nanodisc formation (< 8 nm diameter) on a time scale of tens of microseconds using molecular dynamics (MD) simulation. Structural models of ~ 2.0 or 4.8 kDa PMAQA and ~2.2 kDa SMAEA polymer based lipid-nanodiscs highlights the importance of polymer chemical structure, size and polymer:lipid molar ratio in the optimization of nanodisc structure. The ideal spatial arrangement of polymers in nanodisc, nanodisc size and thermal stability obtained from our MD simulation correlates well with the experimental observations. The polymer-nanodisc were tested for the reconstitution of single-pass or multi-pass transmembrane proteins. We expect this study to be useful in the development of novel polymer based lipid-nanodiscs and for the structural studies of membrane proteins. TOC GRAPHICS O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/476556v2_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@ae71c6org.highwire.dtl.DTLVardef@af961borg.highwire.dtl.DTLVardef@1a7e6c3org.highwire.dtl.DTLVardef@1f4a9a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗
bioRxiv2018

Alzheimer’s Amyloid-Beta Intermediates Generated by Polymer-Nanodiscs

Polymethacrylate-copolymer (PMA) encased lipid-nanodiscs (~10 nm) and macro-nanodiscs (>15 nm) are used to study A{beta}1-40 aggregation. We demonstrate that PMA-nanodiscs form a ternary association with A{beta} and regulate its aggregation kinetics by trapping intermediates. Results demonstrating reduced neurotoxicity of nanodisc-bound A{beta} oligomers are also reported.

biophysics↗
bioRxiv2018

Zinc Boosts EGCG’s hIAPP Amyloid Inhibition Both in Solution and Membrane

Amyloid aggregation of human islet amyloid polypeptide (hIAPP) is linked to insulin-producing islet cell death in type II diabetes. Previous studies have shown the amyloid inhibiting effects of zinc (Zn) and insulin that are co-present with hIAPP in islet cells, and the lipid membrane has been shown to significantly influence the aggregation kinetics. Increasing number of studies report the importance of developing small molecule inhibitors to suppress the hIAPPs toxicity. Particularly, the ability of epigallocatechin-gallate (EGCG) to inhibit amyloid aggregation of a variety of amyloid peptide/proteins including hIAPP initiated numerous studies including the development of compounds to potentially treat amyloid diseases. In this study, by using a combination of thioflavin-T fluorescence and transmission electron microscopy experiments, we demonstrate a significant enhancement in EGCGs efficiency, when mixed with Zn, to significantly suppress hIAPP amyloid aggregation both in presence and absence of lipid membrane. Circular dichroism experiments indicate the formation and stabilization of a helical structure of hIAPP in presence of EGCG:Zn complex. Our results also reveal the ability of EGCG or EGCG:Zn to suppress hIAPPs cellular toxicity and that the ability of EGCG to chelate with Zn suppresses zincs cellular toxicity. We suggest that the reported results would be useful to develop strategies to trap hIAPP intermediates for further biophysical and structural studies, and also to devise approaches to abolish amyloid aggregation and cellular toxicity.

biophysics↗