Mathivanan Chinnaraj | Saint Louis University (original) (raw)

Papers by Mathivanan Chinnaraj

β2-glycoprotein I (β2GPI) is an abundant plasma protein displaying phospholipid-binding propertie... more β2-glycoprotein I (β2GPI) is an abundant plasma protein displaying phospholipid-binding properties. Because it binds phospholipids, it is a target of antiphospholipid antibodies (aPLs) in antiphospholipid syndrome (APS), a lifethreatening autoimmune thrombotic disease. Indeed, aPLs prefer membrane-bound β2GPI to that in solution. β2GPI exists in two almost equally populated redox states: oxidized, in which all the disulfide bonds are formed, and reduced, in which one or more disulfide bonds are broken. Furthermore, β2GPI can adopt multiple conformations (i.e. J-elongated, S-twisted, and Ocircular). While strong evidence indicates that the J-form is the structure bound to aPLs, which conformation exists and predominates in solution remains controversial, and so is the conformational pathway leading to the bound state. Here, we report that human recombinant β2GPI purified under native conditions is oxidized. Moreover, under physiological pH and salt concentrations, this oxidized form adopts a J-elongated, flexible conformation, not circular nor twisted, in which the N-terminal domain I (DI) and the C-terminal domain V (DV) are exposed to the solvent. Consistent with this model, binding kinetics and mutagenesis experiments revealed that in solution the J-form interacts with negatively charged liposomes and with MBB2, a monoclonal anti-DI antibody that recapitulates most of the features of pathogenic aPLs. We conclude that the preferential binding of aPLs to phospholipid-bound β2GPI arises from the ability of its pre-existing J-form to accumulate on the membranes, thereby offering an ideal environment for aPL binding. We propose that targeting the J-form of β2GPI may provide a strategy to block pathogenic aPLs in APS.

Blood Advances, 2019

Key Points • Type I and II are 2 novel subpopulations of immunoglobulin G aPS/PT with different m... more Key Points • Type I and II are 2 novel subpopulations of immunoglobulin G aPS/PT with different mechanisms of pro-thrombin recognition and function. • APS patients can be classified into 2 groups, A and B, according to the presence of type I and II aPS/PT autoantibodies. Anti-phosphatidylserine/prothrombin (aPS/PT) antibodies are often detected in patients with antiphospholipid syndrome (APS), but how aPS/PT engage prothrombin at the molecular level remains unknown. Here, the antigenic determinants of immunoglobulin G aPS/PT were investigated in 24 triple-positive APS patients at high risk of thrombosis by using prothrombin mutants biochemically trapped in closed and open conformations, and relevant fragments spanning the entire length of prothrombin. Two novel unexpected findings emerged from these studies. First, we discovered that some aPS/PT are unique among other anti-prothrombin antibodies insofar as they efficiently recognize prothrombin in solution after a conformational change requiring exposure of fragment-1 to the solvent. Second, we identified and characterized 2 previously unknown subpopulations of aPS/PT, namely type I and type II, which engage fragment-1 of prothrombin at different epitopes and with different mechanisms. Type I target a discontinuous density-dependent epitope, whereas type II engage the C-terminal portion of the Gla-domain, which remains available for binding even when prothrombin is bound to the phospholipids. Based on these findings, APS patients positive for aPS/PT were classified into 2 groups, group A and group B, according to their autoantibody profile. Group A contains mostly type I antibodies whereas group B contains both type I and type II antibodies. In conclusion, this study offers a first encouraging step toward unveiling the heterogeneity of anti-prothrombin antibodies in correlation with thrombosis, shedding new light on the mechanisms of antigen-autoantibody recognition in APS.

Coagulation factor II, or prothrombin, is a multi-domain glycoprotein that is essential for life ... more Coagulation factor II, or prothrombin, is a multi-domain glycoprotein that is essential for life and a key target of anticoagulant therapy. In plasma, prothrombin circulates in two forms at equilibrium, " closed " (∼80%) and " open " (∼20%), brokered by the flexibility of the linker regions. Its structure remained elusive until recently when our laboratory solved the first X-ray crystal structure of the zymogen locked in the predominant closed form. Because of this technical breakthrough, fascinating aspects of the biology of prothrombin have started to become apparent, and with this, novel and important questions arise. Here, we examine the significance of the " closed " / " open " equilibrium in the context of the mechanism of thrombin generation. Further, we discuss the potential translational opportunities for the development of next-generation anticoagulants that arise from this discovery. By providing a structural overview of each alternative conformation, this minireview also offers a relevant example of modern structural biology and establishes a practical workflow to elucidate the structural features of analogous clotting and complement factors.

The clotting factor prothrombin exists in equilibrium between closed and open conformations, but ... more The clotting factor prothrombin exists in equilibrium between closed and open conformations, but the physiological role of these forms remains unclear. As for other allosteric proteins, elucidation of the linkage between molecular transitions and function is facilitated by reagents stabilized in each of the alternative conformations. The open form of prothrombin has been characterized structurally, but little is known about the architecture of the closed form that predominates in solution under physiological conditions. Using X-ray crystallography and single-molecule FRET, we characterize a prothrombin construct locked in the closed conformation through an engineered disulfide bond. The construct: (i) provides structural validation of the intramolecular collapse of kringle-1 onto the protease domain reported recently; (ii) documents the critical role of the linker connecting kringle-1 to kringle-2 in stabilizing the closed form; and (iii) reveals novel mechanisms to shift the equilibrium toward the open conformation. Together with functional studies, our findings define the role of closed and open conformations in the conversion of prothrombin to thrombin and establish a molecular framework for prothrombin activation that rationalizes existing phenotypes associated with prothrombin mutations and points to new strategies for therapeutic intervention. Trypsin-like proteases and their zymogen precursors play dominant roles in enzyme cascades such as blood coagulation, complement and fibrinolysis where they often appear in complexes assembled on biological membranes 1,2. The conversion of prothrombin (proT) to thrombin by the prothrombinase complex, composed of the enzyme factor Xa (fXa), the cofactor Va (fVa), Ca 2+ and phospholipids, provides a biologically relevant example 3,4. The reaction is essential for life and a key target of anticoagulant therapy 5,6. Yet, its mechanism remains poorly understood in structural terms. Prothrombin, or clotting factor II, is a modular protein composed of 579 amino acids organized into the Gla domain (residues 1–46), kringle-1 (residues 65–143), kringle-2 (residues 170–248), and the protease domain (residues 285–579) connected by three intervening linkers (Fig. 1a) 7,8. Activation of prothrombin by prothrom-binase involves cleavage at two distinct sites, Arg271 and Arg320, along two alternative pathways that generate the zymogen precursor prethrombin-2 and the active enzyme meizothrombin, respectively 3,8. Although these intermediates do not accumulate under conditions relevant to physiology 9 , selection of the pathway of activation is of mechanistic interest because it defines the rate of thrombin generation depending on the context 10–13. Recent studies with prothrombin derivatives devoid of segments of Lnk2, the flexible linker connecting kringle-1 to kringle-2, have provided new insights into the mechanism of prothrombin activation 7,14. A high-resolution crystal structure of the prothrombin mutant proTΔ154-167, lacking 14 residues of Lnk2, shows the domains of the zymogen vertically aligned along the main axis of the molecule. Interestingly, the mutant is activated by fXa in the presence of phospholipids at a rate >10-fold faster than wild-type (proTWT). The observation suggests that " shortening " of Lnk2 and stabilization of a conformation similar to that of proTΔ154-167 contributes to the 2,000-fold rate enhancement in prothrombin activation produced by cofactor fVa in prothrom-binase. The hypothesis becomes relevant in the context of the recent observation that prothrombin exists in equilibrium between " open " and " closed " conformations that differ ~50 Å in overall length and with the closed form being predominant in solution (80%) 15. The X-ray structure of proTΔ154-167 is a good representation of the

The Holliday junction (HJ) is a central intermediate of various genetic processes, including homo... more The Holliday junction (HJ) is a central intermediate of various genetic processes, including homologous and site-specific DNA recombination and DNA replication. Elucidating the structure and dynamics of HJs provides the basis for understanding the molecular mechanisms of these genetic processes. Our previous single-molecule fluorescence studies led to a model according to which branch migration is a stepwise process consisting of consecutive migration and folding steps. These data led us to the conclusion that one hop can be more than 1 basepair (bp); moreover, we hypothesized that continuous runs over the entire sequence homology (5 bp) can occur. Direct measurements of the dependence of the fluorescence resonance energy transfer (FRET) value on the donor-acceptor (D-A) distance are required to justify this model and are the major goal of this article. To accomplish this goal, we performed single-molecule FRET experiments with a set of six immobile HJ molecules with varying numbers of bps between fluorescent dyes placed on opposite arms. The designs were made in such a way that the distances between the donor and acceptor were equal to the distances between the dyes formed upon 1-bp migration hops of a HJ having 10-bp homology. Using these designs, we confirmed our previous hypothesis that the migration of the junction can be measured with bp accuracy. Moreover, the FRET values determined for each acceptor-donor separation corresponded very well to the values for the steps on the FRET time trajectories, suggesting that each step corresponds to the migration of the branch at a defined depth. We used the dependence of the FRET value on the D-A distance to measure directly the size for each step on the FRET time trajectories. These data showed that one hop is not necessarily 1 bp. The junction is able to migrate over several bps, detected as one hop and confirming our model. The D-A distances extracted from the FRET properties of the immobile junctions formed the basis for modeling the HJ structures. The composite data fit a partially opened, side-by-side model with adjacent double-helical arms slightly kinked at the four-way junction and the junction as a whole adopting a global X-shaped form that mimics the coaxially stacked-X structure implicated in previous solution studies.

Biophysical Journal, 2013

made from the previous study and investigate conformational changes at atomistic details. Free en... more made from the previous study and investigate conformational changes at atomistic details. Free energy changes involved in translocation process are calculated from molecular dynamics simulations. In particular, O-helix in the finger domain and Tyr 639 residue near the active site are supposed to play important roles during each elongation cycle. Correspondingly, we examine closely the energy cost of Tyr 639 fluctuating IN and OUT of the active site, as well as that of opening and closing of O-helix. Physical insight of polymerase transcription can be gained further by combing our current free energy studies at atomistic scale with former kinetic modeling that links to experimental research. 586a

The transport of oxygen and other nonelectrolytes across lipid membranes is known to depend on bo... more The transport of oxygen and other nonelectrolytes across lipid membranes is known to depend on both diffusion and solubility in the bilayer, and to be affected by changes in the physical state and by the lipid composition, especially the content of cholesterol and unsaturated fatty acids. However, it is not known how these factors affect diffusion and solubility separately. Herein we measured the partition coefficient of oxygen in liposome membranes of dilauroyl-, dimiristoyl-and dipalmitoylphosphatidylcholine in buffer at different temperatures using the equilibrium-shift method with electrochemical detection. The apparent diffusion coefficient was measured following the fluorescence quenching of 1-pyrenedodecanoate inserted in the liposome bilayers under the same conditions. The partition coefficient varied with the temperature and the physical state of the membrane, from below 1 in the gel state to above 2.8 in the liquid-crystalline state in DMPC and DPPC membranes. The partition coefficient was directly proportional to the partial molar volume and was then associated to the increase in free-volume in the membrane as a function of temperature. The apparent diffusion coefficients were corrected by the partition coefficients and found to be nearly the same, with a null dependence on viscosity and physical state of the membrane, probably because the pyrene is disturbing the surrounding lipids and thus becoming insensitive to changes in membrane viscosity. Combining our results with those of others, it is apparent that both solubility and diffusion increase when increasing the temperature or when comparing a membrane in the gel to one in the fluid state.

The Ca 2+-induced interaction between cardiac troponin I (cTnI) and actin plays a key role in the... more The Ca 2+-induced interaction between cardiac troponin I (cTnI) and actin plays a key role in the regulation of cardiac muscle contraction and relaxation. In this report we investigated changes of this interaction in response to strong crossbridge formation between myosin S1 and actin and PKA phosphorylation of cTnI within reconstituted thin filament. The interaction was monitored by measuring Förster resonance energy transfer (FRET) between the fluorescent donor 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid (AEDANS) attached to the residues 131, 151, 160 167, 188 and 210 of cTnI and the nonfluorescent acceptor 4-dimethylaminophenylazophenyl-4′-maleimide (DABM) attached to cysteine 374 of actin. The FRET distance measurements showed that bound Ca 2+ induced large increases in the distances from actin to the cTnI sites, indicating a Ca 2+-triggered separation of actin from cTnI. Strongly bound myosin S1 induced additional increases in these distances in the presence of bound Ca 2+. These two-step changes in the observed FRET distances provide a direct link of structural changes at the interface between cTnI and actin to the three-state model of thin filament regulation of muscle contraction and relaxation. When cTnC was inactivated through mutations of key residues within the 12-residue Ca 2+-binding loop, strongly bound S1 alone induced increases in the distances in spite of the fact that the filaments no longer bound regulatory Ca 2+. These results suggest bound Ca 2+ or strongly bound S1 alone can partially activate thin filament, but full activation requires both bound Ca 2+ and strongly bound S1. The distributions of the FRET distances revealed different structural dynamics associated with different regions of cTnI in different biochemical states. The second actin-binding region appears more rigid than the inhibitory/regulatory region. In the Mg 2+ state, the regulatory region appears more flexible than the inhibitory region, and in the Ca 2+ state, the inhibitory region becomes more flexible. PKA phosphorylation of cTnI increased the FRET distance from actin to cTnI residue 131 by 2.2-5.2 Å in different biochemical states and narrowed the distributions of the distances from actin to the inhibitory and regulatory regions of cTnI. The observed phosphorylation effects are likely due to an intramolecular interaction of the phosphorylated N-terminal segment and the inhibitory region of cTnI.

Our newly developed fluorescence resonance energy transfer (FRET) based technique, fluorescence n... more Our newly developed fluorescence resonance energy transfer (FRET) based technique, fluorescence nanotomography (FN), is used to determine the morphology and dynamics of some soft materials and bio-molecules by attaching donor (D) fluorophores and acceptors (A) to the investigated structure and using fluorescence lifetime measurements to reveal the D–A distance distribution function ρ DA (r). We report the effect of the limited sizes of the donor and acceptor, effect of porous polymer, and molecular structure and phase transition in phospholipid bilayers.

Biophysical Journal, 2011

Biophysical Journal, 2010

phosphorylation. Molecular modeling of cTnT-DK210 structure reveals changes in the electrostatic ... more phosphorylation. Molecular modeling of cTnT-DK210 structure reveals changes in the electrostatic environment of cTnT helix (residues 203-224) that lead to a more basic environment around Thr 203 , which enhances PKC-dependent phosphorylation. In addition, yeast two-hybrid assays indicate that cTnT-DK210 has enhanced binding to cTnI compared with cTnT-wt, and may impair Ca 2þ sensing/transmission leading to myofilament desensitization. Collectively, our observations suggest that cardiomyopathy-causing DK210 has far-reaching effects influencing posttranslational modifications of key sarcomeric proteins, and potentially cTnI-cTnT interaction.

Biophysical Journal, 2014

β2-glycoprotein I (β2GPI) is an abundant plasma protein displaying phospholipid-binding propertie... more β2-glycoprotein I (β2GPI) is an abundant plasma protein displaying phospholipid-binding properties. Because it binds phospholipids, it is a target of antiphospholipid antibodies (aPLs) in antiphospholipid syndrome (APS), a lifethreatening autoimmune thrombotic disease. Indeed, aPLs prefer membrane-bound β2GPI to that in solution. β2GPI exists in two almost equally populated redox states: oxidized, in which all the disulfide bonds are formed, and reduced, in which one or more disulfide bonds are broken. Furthermore, β2GPI can adopt multiple conformations (i.e. J-elongated, S-twisted, and Ocircular). While strong evidence indicates that the J-form is the structure bound to aPLs, which conformation exists and predominates in solution remains controversial, and so is the conformational pathway leading to the bound state. Here, we report that human recombinant β2GPI purified under native conditions is oxidized. Moreover, under physiological pH and salt concentrations, this oxidized form adopts a J-elongated, flexible conformation, not circular nor twisted, in which the N-terminal domain I (DI) and the C-terminal domain V (DV) are exposed to the solvent. Consistent with this model, binding kinetics and mutagenesis experiments revealed that in solution the J-form interacts with negatively charged liposomes and with MBB2, a monoclonal anti-DI antibody that recapitulates most of the features of pathogenic aPLs. We conclude that the preferential binding of aPLs to phospholipid-bound β2GPI arises from the ability of its pre-existing J-form to accumulate on the membranes, thereby offering an ideal environment for aPL binding. We propose that targeting the J-form of β2GPI may provide a strategy to block pathogenic aPLs in APS.

Blood Advances, 2019

Key Points • Type I and II are 2 novel subpopulations of immunoglobulin G aPS/PT with different m... more Key Points • Type I and II are 2 novel subpopulations of immunoglobulin G aPS/PT with different mechanisms of pro-thrombin recognition and function. • APS patients can be classified into 2 groups, A and B, according to the presence of type I and II aPS/PT autoantibodies. Anti-phosphatidylserine/prothrombin (aPS/PT) antibodies are often detected in patients with antiphospholipid syndrome (APS), but how aPS/PT engage prothrombin at the molecular level remains unknown. Here, the antigenic determinants of immunoglobulin G aPS/PT were investigated in 24 triple-positive APS patients at high risk of thrombosis by using prothrombin mutants biochemically trapped in closed and open conformations, and relevant fragments spanning the entire length of prothrombin. Two novel unexpected findings emerged from these studies. First, we discovered that some aPS/PT are unique among other anti-prothrombin antibodies insofar as they efficiently recognize prothrombin in solution after a conformational change requiring exposure of fragment-1 to the solvent. Second, we identified and characterized 2 previously unknown subpopulations of aPS/PT, namely type I and type II, which engage fragment-1 of prothrombin at different epitopes and with different mechanisms. Type I target a discontinuous density-dependent epitope, whereas type II engage the C-terminal portion of the Gla-domain, which remains available for binding even when prothrombin is bound to the phospholipids. Based on these findings, APS patients positive for aPS/PT were classified into 2 groups, group A and group B, according to their autoantibody profile. Group A contains mostly type I antibodies whereas group B contains both type I and type II antibodies. In conclusion, this study offers a first encouraging step toward unveiling the heterogeneity of anti-prothrombin antibodies in correlation with thrombosis, shedding new light on the mechanisms of antigen-autoantibody recognition in APS.

Coagulation factor II, or prothrombin, is a multi-domain glycoprotein that is essential for life ... more Coagulation factor II, or prothrombin, is a multi-domain glycoprotein that is essential for life and a key target of anticoagulant therapy. In plasma, prothrombin circulates in two forms at equilibrium, " closed " (∼80%) and " open " (∼20%), brokered by the flexibility of the linker regions. Its structure remained elusive until recently when our laboratory solved the first X-ray crystal structure of the zymogen locked in the predominant closed form. Because of this technical breakthrough, fascinating aspects of the biology of prothrombin have started to become apparent, and with this, novel and important questions arise. Here, we examine the significance of the " closed " / " open " equilibrium in the context of the mechanism of thrombin generation. Further, we discuss the potential translational opportunities for the development of next-generation anticoagulants that arise from this discovery. By providing a structural overview of each alternative conformation, this minireview also offers a relevant example of modern structural biology and establishes a practical workflow to elucidate the structural features of analogous clotting and complement factors.

The clotting factor prothrombin exists in equilibrium between closed and open conformations, but ... more The clotting factor prothrombin exists in equilibrium between closed and open conformations, but the physiological role of these forms remains unclear. As for other allosteric proteins, elucidation of the linkage between molecular transitions and function is facilitated by reagents stabilized in each of the alternative conformations. The open form of prothrombin has been characterized structurally, but little is known about the architecture of the closed form that predominates in solution under physiological conditions. Using X-ray crystallography and single-molecule FRET, we characterize a prothrombin construct locked in the closed conformation through an engineered disulfide bond. The construct: (i) provides structural validation of the intramolecular collapse of kringle-1 onto the protease domain reported recently; (ii) documents the critical role of the linker connecting kringle-1 to kringle-2 in stabilizing the closed form; and (iii) reveals novel mechanisms to shift the equilibrium toward the open conformation. Together with functional studies, our findings define the role of closed and open conformations in the conversion of prothrombin to thrombin and establish a molecular framework for prothrombin activation that rationalizes existing phenotypes associated with prothrombin mutations and points to new strategies for therapeutic intervention. Trypsin-like proteases and their zymogen precursors play dominant roles in enzyme cascades such as blood coagulation, complement and fibrinolysis where they often appear in complexes assembled on biological membranes 1,2. The conversion of prothrombin (proT) to thrombin by the prothrombinase complex, composed of the enzyme factor Xa (fXa), the cofactor Va (fVa), Ca 2+ and phospholipids, provides a biologically relevant example 3,4. The reaction is essential for life and a key target of anticoagulant therapy 5,6. Yet, its mechanism remains poorly understood in structural terms. Prothrombin, or clotting factor II, is a modular protein composed of 579 amino acids organized into the Gla domain (residues 1–46), kringle-1 (residues 65–143), kringle-2 (residues 170–248), and the protease domain (residues 285–579) connected by three intervening linkers (Fig. 1a) 7,8. Activation of prothrombin by prothrom-binase involves cleavage at two distinct sites, Arg271 and Arg320, along two alternative pathways that generate the zymogen precursor prethrombin-2 and the active enzyme meizothrombin, respectively 3,8. Although these intermediates do not accumulate under conditions relevant to physiology 9 , selection of the pathway of activation is of mechanistic interest because it defines the rate of thrombin generation depending on the context 10–13. Recent studies with prothrombin derivatives devoid of segments of Lnk2, the flexible linker connecting kringle-1 to kringle-2, have provided new insights into the mechanism of prothrombin activation 7,14. A high-resolution crystal structure of the prothrombin mutant proTΔ154-167, lacking 14 residues of Lnk2, shows the domains of the zymogen vertically aligned along the main axis of the molecule. Interestingly, the mutant is activated by fXa in the presence of phospholipids at a rate >10-fold faster than wild-type (proTWT). The observation suggests that " shortening " of Lnk2 and stabilization of a conformation similar to that of proTΔ154-167 contributes to the 2,000-fold rate enhancement in prothrombin activation produced by cofactor fVa in prothrom-binase. The hypothesis becomes relevant in the context of the recent observation that prothrombin exists in equilibrium between " open " and " closed " conformations that differ ~50 Å in overall length and with the closed form being predominant in solution (80%) 15. The X-ray structure of proTΔ154-167 is a good representation of the

The Holliday junction (HJ) is a central intermediate of various genetic processes, including homo... more The Holliday junction (HJ) is a central intermediate of various genetic processes, including homologous and site-specific DNA recombination and DNA replication. Elucidating the structure and dynamics of HJs provides the basis for understanding the molecular mechanisms of these genetic processes. Our previous single-molecule fluorescence studies led to a model according to which branch migration is a stepwise process consisting of consecutive migration and folding steps. These data led us to the conclusion that one hop can be more than 1 basepair (bp); moreover, we hypothesized that continuous runs over the entire sequence homology (5 bp) can occur. Direct measurements of the dependence of the fluorescence resonance energy transfer (FRET) value on the donor-acceptor (D-A) distance are required to justify this model and are the major goal of this article. To accomplish this goal, we performed single-molecule FRET experiments with a set of six immobile HJ molecules with varying numbers of bps between fluorescent dyes placed on opposite arms. The designs were made in such a way that the distances between the donor and acceptor were equal to the distances between the dyes formed upon 1-bp migration hops of a HJ having 10-bp homology. Using these designs, we confirmed our previous hypothesis that the migration of the junction can be measured with bp accuracy. Moreover, the FRET values determined for each acceptor-donor separation corresponded very well to the values for the steps on the FRET time trajectories, suggesting that each step corresponds to the migration of the branch at a defined depth. We used the dependence of the FRET value on the D-A distance to measure directly the size for each step on the FRET time trajectories. These data showed that one hop is not necessarily 1 bp. The junction is able to migrate over several bps, detected as one hop and confirming our model. The D-A distances extracted from the FRET properties of the immobile junctions formed the basis for modeling the HJ structures. The composite data fit a partially opened, side-by-side model with adjacent double-helical arms slightly kinked at the four-way junction and the junction as a whole adopting a global X-shaped form that mimics the coaxially stacked-X structure implicated in previous solution studies.

Biophysical Journal, 2013

made from the previous study and investigate conformational changes at atomistic details. Free en... more made from the previous study and investigate conformational changes at atomistic details. Free energy changes involved in translocation process are calculated from molecular dynamics simulations. In particular, O-helix in the finger domain and Tyr 639 residue near the active site are supposed to play important roles during each elongation cycle. Correspondingly, we examine closely the energy cost of Tyr 639 fluctuating IN and OUT of the active site, as well as that of opening and closing of O-helix. Physical insight of polymerase transcription can be gained further by combing our current free energy studies at atomistic scale with former kinetic modeling that links to experimental research. 586a

The transport of oxygen and other nonelectrolytes across lipid membranes is known to depend on bo... more The transport of oxygen and other nonelectrolytes across lipid membranes is known to depend on both diffusion and solubility in the bilayer, and to be affected by changes in the physical state and by the lipid composition, especially the content of cholesterol and unsaturated fatty acids. However, it is not known how these factors affect diffusion and solubility separately. Herein we measured the partition coefficient of oxygen in liposome membranes of dilauroyl-, dimiristoyl-and dipalmitoylphosphatidylcholine in buffer at different temperatures using the equilibrium-shift method with electrochemical detection. The apparent diffusion coefficient was measured following the fluorescence quenching of 1-pyrenedodecanoate inserted in the liposome bilayers under the same conditions. The partition coefficient varied with the temperature and the physical state of the membrane, from below 1 in the gel state to above 2.8 in the liquid-crystalline state in DMPC and DPPC membranes. The partition coefficient was directly proportional to the partial molar volume and was then associated to the increase in free-volume in the membrane as a function of temperature. The apparent diffusion coefficients were corrected by the partition coefficients and found to be nearly the same, with a null dependence on viscosity and physical state of the membrane, probably because the pyrene is disturbing the surrounding lipids and thus becoming insensitive to changes in membrane viscosity. Combining our results with those of others, it is apparent that both solubility and diffusion increase when increasing the temperature or when comparing a membrane in the gel to one in the fluid state.

The Ca 2+-induced interaction between cardiac troponin I (cTnI) and actin plays a key role in the... more The Ca 2+-induced interaction between cardiac troponin I (cTnI) and actin plays a key role in the regulation of cardiac muscle contraction and relaxation. In this report we investigated changes of this interaction in response to strong crossbridge formation between myosin S1 and actin and PKA phosphorylation of cTnI within reconstituted thin filament. The interaction was monitored by measuring Förster resonance energy transfer (FRET) between the fluorescent donor 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid (AEDANS) attached to the residues 131, 151, 160 167, 188 and 210 of cTnI and the nonfluorescent acceptor 4-dimethylaminophenylazophenyl-4′-maleimide (DABM) attached to cysteine 374 of actin. The FRET distance measurements showed that bound Ca 2+ induced large increases in the distances from actin to the cTnI sites, indicating a Ca 2+-triggered separation of actin from cTnI. Strongly bound myosin S1 induced additional increases in these distances in the presence of bound Ca 2+. These two-step changes in the observed FRET distances provide a direct link of structural changes at the interface between cTnI and actin to the three-state model of thin filament regulation of muscle contraction and relaxation. When cTnC was inactivated through mutations of key residues within the 12-residue Ca 2+-binding loop, strongly bound S1 alone induced increases in the distances in spite of the fact that the filaments no longer bound regulatory Ca 2+. These results suggest bound Ca 2+ or strongly bound S1 alone can partially activate thin filament, but full activation requires both bound Ca 2+ and strongly bound S1. The distributions of the FRET distances revealed different structural dynamics associated with different regions of cTnI in different biochemical states. The second actin-binding region appears more rigid than the inhibitory/regulatory region. In the Mg 2+ state, the regulatory region appears more flexible than the inhibitory region, and in the Ca 2+ state, the inhibitory region becomes more flexible. PKA phosphorylation of cTnI increased the FRET distance from actin to cTnI residue 131 by 2.2-5.2 Å in different biochemical states and narrowed the distributions of the distances from actin to the inhibitory and regulatory regions of cTnI. The observed phosphorylation effects are likely due to an intramolecular interaction of the phosphorylated N-terminal segment and the inhibitory region of cTnI.

Our newly developed fluorescence resonance energy transfer (FRET) based technique, fluorescence n... more Our newly developed fluorescence resonance energy transfer (FRET) based technique, fluorescence nanotomography (FN), is used to determine the morphology and dynamics of some soft materials and bio-molecules by attaching donor (D) fluorophores and acceptors (A) to the investigated structure and using fluorescence lifetime measurements to reveal the D–A distance distribution function ρ DA (r). We report the effect of the limited sizes of the donor and acceptor, effect of porous polymer, and molecular structure and phase transition in phospholipid bilayers.

Biophysical Journal, 2011

Biophysical Journal, 2010

phosphorylation. Molecular modeling of cTnT-DK210 structure reveals changes in the electrostatic ... more phosphorylation. Molecular modeling of cTnT-DK210 structure reveals changes in the electrostatic environment of cTnT helix (residues 203-224) that lead to a more basic environment around Thr 203 , which enhances PKC-dependent phosphorylation. In addition, yeast two-hybrid assays indicate that cTnT-DK210 has enhanced binding to cTnI compared with cTnT-wt, and may impair Ca 2þ sensing/transmission leading to myofilament desensitization. Collectively, our observations suggest that cardiomyopathy-causing DK210 has far-reaching effects influencing posttranslational modifications of key sarcomeric proteins, and potentially cTnI-cTnT interaction.

Biophysical Journal, 2014