Suren Tatulian - Academia.edu (original) (raw)

Papers by Suren Tatulian

Journal of Natural Products, Dec 22, 2008

Guggulsterone (7) and cembranoids (8-12) from Commiphora mukul stem bark resin guggul were shown ... more Guggulsterone (7) and cembranoids (8-12) from Commiphora mukul stem bark resin guggul were shown to be specific modulators of two independent sites that are also modulated by bile salts (1-6) to control cholesterol absorption and catabolism. Guggulsterone (7) antagonized the chenodeoxycholic acid (3)-activated nuclear farnesoid X receptor (FXR), which regulates cholesterol metabolism in the liver. The cembranoids did not show a noticeable effect on FXR, but lowered the cholate (1)-activated rate of human pancreatic IB phospholipase A2 (hPLA2), which controls gastrointestinal absorption of fat and cholesterol. Analysis of the data using a kinetic model has suggested an allosteric mechanism for the rate increase of hPLA2 by cholate and also for the rate-lowering effect by certain bile salts or cembranoids on the cholate-activated hPLA2 hydrolysis of phosphatidylcholine vesicles. The allosteric inhibition of PLA2 by certain bile salts and cembranoids showed some structural specificity. Biophysical studies also showed specific interaction of the bile salts with the interface-bound cholate-activated PLA2. Since cholesterol homeostasis in mammals is regulated by FXR in the liver for metabolism and by PLA2 in the intestine for absorption, modulation of PLA2 and FXR by bile acids and selected guggul components suggests novel possibilities for hypolipidemic and hypocholesterolemic therapies.

Journal of Molecular Biology, May 1, 1997

Activation of phospholipase A 2 (PLA 2) upon binding to phospholipid assemblies is poorly underst... more Activation of phospholipase A 2 (PLA 2) upon binding to phospholipid assemblies is poorly understood. X-ray crystallography revealed little structural change in the enzyme upon binding of monomeric substrate analogs, whereas small conformational changes in PLA 2 complexed with substrate micelles and an inhibitor were found by NMR. The structure of PLA 2 bound to phospholipid bilayers is not known. Here we uncover by FTIR spectroscopy a splitting in the a-helical region of the amide I absorbance band of PLA 2 upon binding to lipid bilayers. We provide evidence that a higher frequency component, which is only observed in the membrane-bound enzyme, is a property of more¯exible helices. Formation of exible helices upon interaction with the membrane is likely to contribute to PLA 2 activation.

Biochemistry, Sep 26, 2003

Biophysical Journal, Feb 1, 2017

Biophysical Journal, Feb 1, 2016

Proteasome is the essential multicatalytic protease from the ubiquitinproteasome pathway, respons... more Proteasome is the essential multicatalytic protease from the ubiquitinproteasome pathway, responsible for the regulated degradation of the bulk of intracellular proteins. Cancer cells are more susceptible than healthy cells to blocking of the proteasome catalytic activity. Consequently, proteasome inhibitors are used as successful drugs against blood cancers. Still, many patients do not respond to the treatment or develop resistance. Since all the currently used proteasome-targeting drugs are competitive inhibitors blocking the active sites, we turned our attention to novel noncompetitive regulators affecting proteinprotein interactions (PPI), especially attractive for the multisubunit and modular enzyme like the proteasome. Testing effects of competitive inhibitors requires only monitoring of peptidase activity of the proteasome catalytic core. The PPI-targeting compounds often perform disappointingly weak in such tests. Here we show that biophysical methods provide straightforward tools to follow the impact of PPI disruption on proteasome. We assessed smallmolecule compounds that distinctly affect assembly and activity of the proteasome: (i) analogues of the rapamycin binding domain, (ii) Tat peptide mimetics modeled after the proteasome-binding domain of HIV-1Tat protein, (iii) derivatives of Pro and Arg-rich fragment of cathelicidin PR39, and (iv) TCH imidazoline derivatives. Probing topography of the catalytic core with AFM identified the gate status and alpha face dynamics as an excellent sensor of a type of the compounds proteasome interactions. Intrinsic fluorescence of tryptophans revealed divergent communication pathways affected by the compounds. AFM based analysis of proteasomal super-assemblies demonstrated distinct modes of compounds-induced disruption of PPIs. Finally, Peak Force QNM AFM demonstrated profound changes in cell elasticity, adhesiveness and roughness of cultured cells treated with the compounds. Based on these findings we attempted to build a model that can help predict the effects of small molecules on proteasome and cell physiology.

Biophysical Journal, 2010

We describe here the expression, purification, solid state NMR sample preparation, and initial st... more We describe here the expression, purification, solid state NMR sample preparation, and initial structural and functional data for three membrane proteins from Mycobacterium tuberculosis (Mtb). The three proteins are FtsX, Rv0008c and Rv1861. Solid state NMR is uniquely able to characterize protein structure in a liquid crystalline lipid bilayer environment. We have used N terminal His tag for protein purification. Nickel-NTA chromatography was performed using a semi automated FPLC instrument. Purified 15N labeled proteins were eluted into 0.2% (Rv0008c and Rv1861) and 0.4% (FtsX) solution of dodecylphosphocholine (DPC) detergent. The approximate protein yield were 45mg/l (Rv0008c), 50mg/l (FtsX) and 25mg/l (Rv1861) respectively. Samples for solid state NMR were prepared by removing the detergent from the purified protein by exhaustive dialysis against 10mM Tris-HCl (pH-8.0) coincident with reconstitution into lipid bilayers. To prepare aligned samples, pelleted liposomes were layered on to thin glass slides and stacked. 30-35 glass slides were hydrated in a process called 'wet stacking'followed by sealing them into a rectangular glass cell. 400 and 600 MHz magnets were used to determine the 1D and 2D spectra of these aligned samples such that the bilayer is parallel to the applied magnetic field direction. FtsX is an ABC transporter containing 4 transmembrane helices (TMH) and its interaction with FtsZ participate in cell division. Rv1861 has 3 TMH and is known to hydrolyze ATP. It forms a stable octameric structure that is presumably facilitated by the GxxxG, GxxxA, and AxxxA sequences in the trasmembrane stretches. Rv0008c is a Mtb membrane protein and participates in cell division. It has been found previously in our laboratory that Rv0008c interacts with Rv0011c and this interaction along with other membrane proteins can facilitate the Mtb cell division process.

Biophysical Journal, 2014

Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) where the cataly... more Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) where the catalytic CTA1 subunit separates from the holotoxin and unfolds due to its intrinsic instability. Unfolded CTA1 then moves through an ER translocon pore to reach its cytosolic target. Due to the instability of CTA1, it must be actively refolded in the cytosol to achieve the proper conformation for modification of its G protein target. The cytosolic heat shock protein Hsp90 is involved with the ER-to-cytosol translocation of CTA1, yet the mechanistic role of Hsp90 in CTA1 translocation remains unknown. Potential post-translocation roles for Hsp90 in modulating the activity of cytosolic CTA1 are also unknown. Here, we show by isotope-edited Fourier transform infrared spectroscopy that Hsp90 induces a gain-of-function in disordered CTA1 at physiological temperature. Only the ATP-bound form of Hsp90 interacts with disordered CTA1, and its refolding of CTA1 is dependent upon ATP hydrolysis. Surface plasmon resonance experiments found that Hsp90 does not release CTA1, even after ATP hydrolysis and the return of CTA1 to a folded conformation. The interaction with Hsp90 allowed disordered CTA1 to attain an active state and did not prevent further stimulation of toxin activity by ADP-ribosylation factor 6, a host cofactor for CTA1. Our studies suggest CTA1 translocation involves a ratchet mechanism which couples the Hsp90-mediated refolding of CTA1 with CTA1 extraction from the ER. Continued association of Hsp90 with refolded CTA1 allows the cytosolic toxin to attain an active conformation but does not protect it from proteasomal degradation.

Biophysical Journal, Feb 1, 2009

Activation of human pancreatic phospholipase A 2 (PLA 2) in the presence of DPPC/DPPG (7:3) vesic... more Activation of human pancreatic phospholipase A 2 (PLA 2) in the presence of DPPC/DPPG (7:3) vesicles was induced by a temperature shift from 4 to 38 o C. PLA 2 activity was monitored by changes in fluorescence of bis-Pyrene-PC (2.5 mol % in the membranes), while simultaneous far-and near-UV circular dichroism (CD) spectra identified changes in the secondary and tertiary structures of the protein in real time. The 4-to-38 o C temperature shift caused dramatic changes in both bis-Pyrene-PC fluorescence and the protein CD spectra. The monomer fluorescence signal of bis-Pyrene-PC rapidly increased and the excimer signal decreased, demonstrating PLA 2 activation. Drastic weakening in the a-helical CD signal of the protein, i.e., a 20% decrease in the n-p* transition intensity at 222 nm, was detected upon enzyme activation. The a-helical signal exhibited a significantly smaller change upon a similar temperature shift under non-catalytic conditions (1 mM EGTA), while little changes were detected in the absence of lipid. Strong changes in the tertiary structure during PLA 2 activation were also identified. Initially, at 4 o C, the near-UV CD spectra showed a weak negative band around 280 nm. Upon a shift to 38 o C, strong positive CD bands rapidly developed around 250 and 280 nm, implying significant changes in the conformation and/or the microenvironment of Tyr and Trp side chains of PLA 2 , possibly accompanied with a global tertiary structure perturbation associated with deformation of the abundant disulfide bonds in the protein. These experiments provide new information on the structure-function relationship of PLA 2 by near-simultaneous measurements of PLA 2 activity and its secondary and tertiary structures.

Biophysical Journal, 2013

CRC Press eBooks, Feb 16, 1999

Journal of Physical Chemistry B, Jul 31, 2002

ABSTRACT Key enzymes involved in transmembrane signaling and lipid metabolism (e.g., protein kina... more ABSTRACT Key enzymes involved in transmembrane signaling and lipid metabolism (e.g., protein kinase C and phospholipases A2, C, and D) are activated by binding to cellular membranes. Elucidation of the molecular mechanisms of these peripheral membrane proteins requires detailed characterization of their interactions with membrane lipids. Previously, EPR studies on protein−membrane interactions have been analyzed using a formalism for integral membrane proteins, permitting determination of the lipid-to-protein stoichiometry (N) and the relative affinity of the labeled versus unlabeled lipids for the protein (Kr). Here, a formalism is developed that permits a comprehensive description of the membrane binding of peripheral proteins. The interaction of an interfacially activated enzyme, secretory phospholipase A2 (PLA2), with membranes containing spin-labeled lipids is studied by EPR spectroscopy. Under noncatalytic conditions, binding of PLA2 to fluid membranes (order parameter Szz ≈ 0.24) causes the formation of a second, immobilized lipid component with Szz ≈ 0.80. Under catalytic conditions, a third, more mobile component is observed that is evidently generated by the lipid hydrolysis product, lysophospholipid. In addition to N and Kr, the new theory allows the determination of the following parameters: the fraction of protein-accessible lipids (f), the membrane-binding constant of PLA2 (K), the fraction of the labeled lipids associated with a membrane-bound protein (nr), and the microscopic Gibbs free energies of protein binding of labeled (ΔGlab) and unlabeled lipids (ΔGunlab). The experimental and theoretical approaches described in this work expand the limits of characterization of protein−lipid interactions by EPR spectroscopy.

Biochemistry, Aug 8, 2019

Protein disulfide isomerase (PDI) is a redox-dependent protein with oxidoreductase and chaperone ... more Protein disulfide isomerase (PDI) is a redox-dependent protein with oxidoreductase and chaperone activities. It is a U-shaped protein with an abb’xa’ structural organization where the a and a' domains have CGHC active sites, the b and b' domains are involved with substrate binding, and x is a flexible linker. PDI exhibits substantial flexibility and undergoes cycles of unfolding and refolding in its interaction with cholera toxin, suggesting PDI can regain a folded, functional conformation after exposure to stress conditions. To determine whether this unfolding-refolding cycle is a substrate-induced process or an intrinsic physical property of PDI, we used circular dichroism to examine the structural properties of PDI subjected to thermal denaturation. PDI exhibited remarkable conformational resilience that is linked to its redox status. In the reduced state, PDI exhibited a 54°C unfolding transition temperature (Tm) and regained 85% of its native structure after nearly complete thermal denaturation. Oxidized PDI had a lower Tm of 48-50°C and regained 70% of its native conformation after 75% denaturation. Both reduced and oxidized PDI were functional after refolding from these denatured states. Additional studies documented increased stability of a PDI construct lacking the a’ domain and decreased thermal stability of a construct lacking the a domain. Furthermore, oxidation of the a domain limited the ability of PDI to refold. The stability and conformational resilience of PDI are thus linked to both redox-dependent and domain-specific effects. These findings document previously unrecognized properties of PDI and provide insight into the physical foundation of its biological function.

PLOS ONE, Aug 22, 2011

AB toxins such as ricin and cholera toxin (CT) consist of an enzymatic A domain and a receptor-bi... more AB toxins such as ricin and cholera toxin (CT) consist of an enzymatic A domain and a receptor-binding B domain. After endocytosis of the surface-bound toxin, both ricin and CT are transported by vesicle carriers to the endoplasmic reticulum (ER). The A subunit then dissociates from its holotoxin, unfolds, and crosses the ER membrane to reach its cytosolic target. Since protein unfolding at physiological temperature and neutral pH allows the dissociated A chain to attain a translocationcompetent state for export to the cytosol, the underlying regulatory mechanisms of toxin unfolding are of paramount biological interest. Here we report a biophysical analysis of the effects of anionic phospholipid membranes and two chemical chaperones, 4-phenylbutyric acid (PBA) and glycerol, on the thermal stabilities and the toxic potencies of ricin toxin A chain (RTA) and CT A1 chain (CTA1). Phospholipid vesicles that mimic the ER membrane dramatically decreased the thermal stability of RTA but not CTA1. PBA and glycerol both inhibited the thermal disordering of RTA, but only glycerol could reverse the destabilizing effect of anionic phospholipids. In contrast, PBA was able to increase the thermal stability of CTA1 in the presence of anionic phospholipids. PBA inhibits cellular intoxication by CT but not ricin, which is explained by its ability to stabilize CTA1 and its inability to reverse the destabilizing effect of membranes on RTA. Our data highlight the toxin-specific intracellular events underlying ER-to-cytosol translocation of the toxin A chain and identify a potential means to supplement the long-term stabilization of toxin vaccines.

Computational Biology and Chemistry, Oct 1, 2008

FEBS Journal, Apr 10, 2017

Identification and characterization of the most cytotoxic Ab species is necessary for advancement... more Identification and characterization of the most cytotoxic Ab species is necessary for advancement in AD diagnostics and therapeutics. While in brain tissue multiple Ab species act in combination, structure/toxicity studies and immunotherapy trials have been focused on individual forms of Ab. As a result, the molecular composition and the structural features of "toxic Ab oligomers" have remained unresolved. Here, we have used a novel approach, hydration from gas phase coupled with isotope-edited Fourier transform infrared (FTIR) spectroscopy, to identify the prefibrillar assemblies formed by Ab and AbpE and to resolve the structures of both peptides in combination. The peptides form unusual b-sheet oligomers stabilized by intramolecular H-bonding as opposed to intermolecular Hbonding in the fibrils. Time-dependent morphological changes in peptide assemblies have been visualized by atomic force microscopy. Ab/AbpE hetero-oligomers exert unsurpassed cytotoxic effect on PC12 cells as compared to oligomers of individual peptides or fibrils. These findings lead to a novel concept that Ab/AbpE hetero-oligomers, not just Ab or AbpE oligomers, constitute the main neurotoxic conformation. The hetero-oligomers thus present a new biomarker that may be targeted for development of more efficient diagnostic and immunotherapeutic strategies to combat AD.

Journal of Biological Chemistry, Apr 1, 1995

Methods in molecular biology, 2019

Fourier transform infrared (FTIR) spectroscopy has become one of the major techniques of structur... more Fourier transform infrared (FTIR) spectroscopy has become one of the major techniques of structural characterization of proteins, peptides, and protein-membrane interactions. While the method does not have the capability of providing the precise, atomic-resolution molecular structure, it is exquisitely sensitive to conformational changes occurring in proteins upon functional transitions or intermolecular interactions. The sensitivity of vibrational frequencies to atomic masses has led to development of "isotope-edited" FTIR spectroscopy, where structural effects in two proteins, one unlabeled and the other labeled with a heavier stable isotope, such as 13C, are resolved simultaneously based on spectral downshift (separation) of the amide I band of the labeled protein. The same isotope effect is used to identify site-specific conformational changes in proteins by site-directed or segmental isotope labeling. Negligible light scattering in the infrared region provides an opportunity to study intermolecular interactions between large protein complexes, interactions of proteins and peptides with lipid vesicles, or protein-nucleic acid interactions without light scattering problems often encountered in ultraviolet spectroscopy. Attenuated total reflection FTIR (ATR-FTIR) is a surface-sensitive version of infrared spectroscopy that has proved useful in studying membrane proteins and lipids, protein-membrane interactions, mechanisms of interfacial enzymes, the structural features of membrane pore forming proteins and peptides, and much more. The purpose of this chapter was to provide a practical guide to analyze protein structure and protein-membrane interactions by FTIR and ATR-FTIR techniques, including procedures of sample preparation, measurements, and data analysis. Basic background information on FTIR spectroscopy, as well as some relatively new developments in structural and functional characterization of proteins and peptides in lipid membranes, is also presented.

Scientific Reports, Feb 25, 2019

the amyloid β (Aβ) peptide and its shorter variants, including a highly cytotoxic Aβ 25-35 peptid... more the amyloid β (Aβ) peptide and its shorter variants, including a highly cytotoxic Aβ 25-35 peptide, exert their neurotoxic effect during Alzheimer's disease by various mechanisms, including cellular membrane permeabilization. The intrinsic polymorphism of Aβ has prevented the identification of the molecular basis of Aβ pore formation by direct structural methods, and computational studies have led to highly divergent pore models. Here, we have employed a set of biophysical techniques to directly monitor Ca 2+-transporting Aβ 25-35 pores in lipid membranes, to quantitatively characterize pore formation, and to identify the key structural features of the pore. Moreover, the effect of membrane cholesterol on pore formation and the structure of Aβ 25-35 has been elucidated. the data suggest that the membraneembedded peptide forms 6-or 8-stranded β-barrel like structures. The 8-stranded barrels may conduct Ca 2+ ions through an inner cavity, whereas the tightly packed 6-stranded barrels need to assemble into supramolecular structures to form a central pore. Cholesterol affects Aβ 25-35 pore formation by a dual mechanism, i.e., by direct interaction with the peptide and by affecting membrane structure. Collectively, our data illuminate the molecular basis of Aβ membrane pore formation, which should advance both basic and clinical research on Alzheimer's disease and membrane-associated pathologies in general. Proteolytic cleavage of the amyloid precursor protein (APP) produces the amyloid β (Aβ) peptide, which forms extracellular fibrillar deposits in cross β-sheet conformation 1-3. Identification of amyloid plaques in the brains of Alzheimer's patients led to the amyloid cascade hypothesis, directly relating Aβ deposits with the etiology of the disease 4-6. Further evidence identified the prefibrillar soluble oligomers of Aβ as the most cytotoxic entities causing neuronal cell dysfunction and death 7-10. Aβ peptide occurs in brain tissue in various forms. The 40-and 42-amino acid residue peptides, Aβ 1-40 and Aβ 1-42 , are the dominant species, with the latter being less abundant but more toxic 8. Shorter variants of Aβ, resulting from truncation by various proteases, are also found in human brain. Among these, the 11amino acid residue Aβ 25-35 peptide (GSNKGAIIGLM) is highly cytotoxic and has been the subject of extensive research on the mechanism of action of Aβ and modulation of its toxicity 11-15. There is strong evidence that the 25-35 segment of Aβ plays an important role in the aggregation properties and cytotoxicity of the peptide 11,16,17. Moreover, similar cytotoxic effects of Aβ 25-35 and Aβ 1-42 , involving DNA damage, transcription dysregulation, and apoptosis, have been reported 18-20 , suggesting these peptides may share common mechanism of toxicity. Aβ 25-35 cytotoxicity has been shown to involve mitochondrial membrane permeabilization through activation of expression of mitochondrial permeability transition pore protein(s) 21,22. In addition, studies on direct Aβ 25-35-membrane interactions suggested membrane binding, insertion, and ion-conducting pore formation by the peptide 17,23-29. Aβ 25-35 binds to anionic membranes, promoted by its excess positive charge due to Lys 28 , as well as to zwitterionic phosphatidylcholine (PC) membranes, although less efficiently 28-30. Inhibition of membrane

Journal of Physical Chemistry B, Nov 1, 2017

Amyloid β (Aβ) peptide contributes to Alzheimer's disease by a yet unidentified mechanism. In bra... more Amyloid β (Aβ) peptide contributes to Alzheimer's disease by a yet unidentified mechanism. In brain tissue, Aβ occurs in various forms, including an undecapeptide Aβ 25-35 , which exerts neurotoxic effect through mitochondrial dysfunction and/or Ca 2+-permeable pore formation in cell membranes. This work was aimed at biophysical characterization of membrane binding and pore formation by Aβ 25-35. Interaction of Aβ 25-35 with anionic and zwitterionic membranes was analyzed by microelectrophoresis. In pore formation experiments, Aβ 25-35 was incubated in aqueous buffer to form oligomers and added to Quin-2-loaded vesicles. Gradual increase in Quin-2 fluorescence was interpreted in terms of membrane pore formation by the peptide, Ca 2+ influx and binding to intravesicular Quin-2. The kinetics and magnitude of this process were used to evaluate the rate constant of pore formation, peptide-peptide association constants, and the oligomeric state of the pores. Decrease in membrane anionic charge and high ionic strength conditions significantly suppressed membrane binding and pore formation, indicating the importance of electrostatic interactions in these events. Circular dichroism spectroscopy showed that Aβ 25-35 forms the most efficient pores in β-sheet conformation. The data are consistent with an oligo-oligomeric pore model composed of up to 8 peptide units, each containing 6 to 8 monomers.