Bio

Honors & Awards


  • Honorable Mention "Zita Lobo Best Thesis Award of 2011", TIFR, Mumbai (2013)
  • Stanford Dean's Postdoctoral Fellowship, Stanford University (2012)
  • Student Travel Award, CSIR (2010)
  • Company of Biologists Student Award, Company of Biologists and Society for Experimental Biologists (2010)
  • Student Travel Award, CCSTDS and DST (2009)
  • Student Contingency Award, SPIE (2008)
  • Student Travel Award, Biophysical Society (2008)
  • International Student Travel Award, Biophysical Society (2008)

Professional Education


  • Doctor of Philosophy, Tata Institute of Fundamental Research (2011)
  • Master of Science, Tata Institute of Fundamental Research, Biophysics (2006)
  • Bachelor of Science, University of Calcutta, Chemistry (2004)

Stanford Advisors


Research & Scholarship

Current Research and Scholarly Interests


Abstract
Mutations in the beta-cardiac myosin (a molecular motor) cause disastrous effects by manifestation of hypertrophic and dilated cardiomyopathy, a leading cause of cardiac death. We hypothesize that the such mutations cause fundamental mechanistic changes in the motor which in turn affect the efficiency of the motor in several ways. My current research involves single molecule enzyme kinetics and force measurements to link intrinsic changes in motor function to the various clinical outcomes.

Detailed description
Over 250 different single point mutations in the beta-cardiac myosin heavy chain can cause either hypertrophic or dilated cardiomyopathy (HCM or DCM), but the underlying molecular effects on the myosin molecule remain elusive. The primary reason the effect of HCM and DCM mutations on purified human beta-cardiac myosin has not been elucidated is due to difficulties in expressing this protein in a functional and highly purified form. This limitation has now been eliminated by the expression of human beta-cardiac myosin in a mammalian cell line. The Spudich lab have pioneered both in vitro motility assays and single molecule techniques to assess the function of mechanoenzymes such as myosin. The lab has now developed a feedback controlled dual beam optical trap assay to measure the stroke size under low load and the maximum force that single molecules of myosin can produce. My project involves development and utilization of laser trap single molecule assays to measure these parameters using expressed and purified human beta-cardiac myosin. We hypothesize that HCM and DCM mutations affect the underlying force producing capability of human beta-cardiac myosin in different ways and that the eventual clinical phenotypes of HCM and DCM are a result of a particular fundamental mechanistic change in the human beta-cardiac myosin. As a working hypothesis, we propose that mutations that lead to increased force production lead to HCM while those that lead to decreased force production lead to DCM. Mechanisms that lead to decreased and increased force production by myosin can be varied. The inherent force producing capability of the motor, for example, could be increased or decreased by mutations that change the spring constant of the elastic element of the motor. On the other hand, the force-producing capability of the sarcomere could be changed in either direction by changes in the duty ratio of the myosin (the fraction of the ATPase cycle that the head is strongly bound to actin). I plan to study a selected group of 10 mutations, in terms of determining the biomechanical parameters of wild type human alpha- and beta-cardiac myosin and of HCM and DCM mutant human beta-cardiac myosins with respect to their velocities at ~zero load, their duty ratios, their stroke sizes, and the maximum force they produce upon interacting with actin. I plan to extend these studies with physiologically relevant muscle system which is composed of actin, tropomyosin and troponin complexes.

Lab Affiliations


Teaching

Graduate and Fellowship Programs


Publications

Journal Articles


  • Molecular consequences of the R453C hypertrophic cardiomyopathy mutation on human beta-cardiac myosin motor function PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sommese, R. F., Sung, J., Nag, S., Sutton, S., Deacon, J. C., Choe, E., Leinwand, L. A., Ruppel, K., Spudich, J. A. 2013; 110 (31): 12607-12612

    Abstract

    Cardiovascular disorders are the leading cause of morbidity and mortality in the developed world, and hypertrophic cardiomyopathy (HCM) is among the most frequently occurring inherited cardiac disorders. HCM is caused by mutations in the genes encoding the fundamental force-generating machinery of the cardiac muscle, including β-cardiac myosin. Here, we present a biomechanical analysis of the HCM-causing mutation, R453C, in the context of human β-cardiac myosin. We found that this mutation causes a ∼30% decrease in the maximum ATPase of the human β-cardiac subfragment 1, the motor domain of myosin, and a similar percent decrease in the in vitro velocity. The major change in the R453C human β-cardiac subfragment 1 is a 50% increase in the intrinsic force of the motor compared with wild type, with no appreciable change in the stroke size, as observed with a dual-beam optical trap. These results predict that the overall force of the ensemble of myosin molecules in the muscle should be higher in the R453C mutant compared with wild type. Loaded in vitro motility assay confirms that the net force in the ensemble is indeed increased. Overall, this study suggests that the R453C mutation should result in a hypercontractile state in the heart muscle.

    View details for DOI 10.1073/pnas.1309493110

    View details for Web of Science ID 000322441500036

    View details for PubMedID 23798412

  • A folding transition underlies the emergence of membrane affinity in amyloid-beta PHYSICAL CHEMISTRY CHEMICAL PHYSICS Nag, S., Sarkar, B., Chandrakesan, M., Abhyanakar, R., Bhowmik, D., Kombrabail, M., Dandekar, S., Lerner, E., Haas, E., Maiti, S. 2013; 15 (44): 19129-19133

    Abstract

    Small amyloid-β (Aβ) oligomers have much higher membrane affinity compared to the monomers, but the structural origin of this functional change is not understood. We show that as monomers assemble into small n-mers (n < 10), Aβ acquires a tertiary fold that is consistent with the mature fibrils. This is an early and defining transition for the aggregating peptide, and possibly underpins its altered bioactivity.

    View details for DOI 10.1039/c3cp52732h

    View details for Web of Science ID 000326220000005

    View details for PubMedID 24121316

  • Nature of the Amyloid-beta Monomer and the Monomer-Oligomer Equilibrium JOURNAL OF BIOLOGICAL CHEMISTRY Nag, S., Sarkar, B., Bandyopadhyay, A., Sahoo, B., Sreenivasan, V. K., Kombrabail, M., Muralidharan, C., Maiti, S. 2011; 286 (16): 13827-13833

    Abstract

    The monomer to oligomer transition initiates the aggregation and pathogenic transformation of Alzheimer amyloid-? (A?) peptide. However, the monomeric state of this aggregation-prone peptide has remained beyond the reach of most experimental techniques, and a quantitative understanding of this transition is yet to emerge. Here, we employ single-molecule level fluorescence tools to characterize the monomeric state and the monomer-oligomer transition at physiological concentrations in buffers mimicking the cerebrospinal fluid (CSF). Our measurements show that the monomer has a hydrodynamic radius of 0.9 ± 0.1 nm, which confirms the prediction made by some of the in silico studies. Surprisingly, at equilibrium, both A?(40) and A?(42) remain predominantly monomeric up to 3 ?m, above which it forms large aggregates. This concentration is much higher than the estimated concentrations in the CSF of either normal or diseased brains. If A? oligomers are present in the CSF and are the key agents in Alzheimer pathology, as is generally believed, then these must be released in the CSF as preformed entities. Although the oligomers are thermodynamically unstable, we find that a large kinetic barrier, which is mostly entropic in origin, strongly impedes their dissociation. Thermodynamic principles therefore allow the development of a pharmacological agent that can catalytically convert metastable oligomers into nontoxic monomers.

    View details for DOI 10.1074/jbc.M110.199885

    View details for Web of Science ID 000289556200005

    View details for PubMedID 21349839

  • Single Molecule In Vivo Analysis of Toll-Like Receptor 9 and CpG DNA Interaction PLOS ONE Chen, J., Nag, S., Vidi, P., Irudayaraj, J. 2011; 6 (4)

    Abstract

    Toll-like receptor 9 (TLR9) activates the innate immune system in response to oligonucleotides rich in CpG whereas DNA lacking CpG could inhibit its activation. However, the mechanism of how TLR9 interacts with nucleic acid and becomes activated in live cells is not well understood. Here, we report on the successful implementation of single molecule tools, constituting fluorescence correlation/cross-correlation spectroscopy (FCS and FCCS) and photon count histogram (PCH) with fluorescence lifetime imaging (FLIM) to study the interaction of TLR9-GFP with Cy5 labeled oligonucleotide containing CpG or lacking CpG in live HEK 293 cells. Our findings show that i) TLR9 predominantly forms homodimers (80%) before binding to a ligand and further addition of CpG or non CpG DNA does not necessarily increase the proportion of TLR9 dimers, ii) CpG DNA has a lower dissociation constant (62 nM±9 nM) compared to non CpG DNA (153 nM±26 nM) upon binding to TLR9, suggesting that a motif specific binding affinity of TLR9 could be an important factor in instituting a conformational change-dependant activation, and iii) both CpG and non CpG DNA binds to TLR9 with a 1?2 stoichiometry in vivo. Collectively, through our findings we establish an in vivo model of TLR9 binding and activation by CpG DNA using single molecule fluorescence techniques for single cell studies.

    View details for DOI 10.1371/journal.pone.0017991

    View details for Web of Science ID 000289058700007

    View details for PubMedID 21483736

  • Ultracompact alignment-free single molecule fluorescence device with a foldable light path JOURNAL OF BIOMEDICAL OPTICS Singh, N. K., Chacko, J. V., Sreenivasan, V. K., Nag, S., Maiti, S. 2011; 16 (2)

    Abstract

    Instruments with single-molecule level detection capabilities can potentially benefit a wide variety of fields, including medical diagnostics. However, the size, cost, and complexity of such devices have prevented their widespread use outside sophisticated research laboratories. Fiber-only devices have recently been suggested as smaller and simpler alternatives, but thus far, they have lacked the resolution and sensitivity of a full-fledged system, and accurate alignment remains a critical requirement. Here we show that through-space reciprocal optical coupling between a fiber and a microscope objective, combined with wavelength division multiplexing in optical fibers, allows a drastic reduction of the size and complexity of such an instrument while retaining its resolution. We demonstrate a 4 × 4 × 18 cm(3) sized fluorescence correlation spectrometer, which requires no alignment, can analyze kinetics at the single-molecule level, and has an optical resolution similar to that of much larger microscope based devices. The sensitivity can also be similar in principle, though in practice it is limited by the large background fluorescence of the commonly available optical fibers. We propose this as a portable and field deployable single molecule device with practical diagnostic applications.

    View details for DOI 10.1117/1.3548311

    View details for Web of Science ID 000288939200029

    View details for PubMedID 21361684

  • Measurement of the Attachment and Assembly of Small Amyloid-beta Oligomers on Live Cell Membranes at Physiological Concentrations Using Single-Molecule Tools BIOPHYSICAL JOURNAL Nag, S., Chen, J., Irudayaraj, J., Maiti, S. 2010; 99 (6): 1969-1975

    Abstract

    It is thought that the pathological cascade in Alzheimer's disease is initiated by the formation of amyloid-? (A?) peptide complexes on cell membranes. However, there is considerable debate about the nature of these complexes and the type of solution-phase A? aggregates that may contribute to their formation. Also, it is yet to be shown that A? attaches strongly to living cell membranes, and that this can happen at low, physiologically relevant A? concentrations. Here, we simultaneously measure the aggregate size and fluorescence lifetime of fluorescently labeled A?(1-40) on and above the membrane of cultured PC12 cells at near-physiological concentrations. We find that at 350 nM A? concentration, large (>10 nm average hydrodynamic radius) assemblies of codiffusing, membrane-attached A? molecules appear on the cell membrane together with a near-monomeric species. When the extracellular concentration is 150 nM, the membrane contains only the smaller species, but with a similar degree of attachment. At both concentrations, the extracellular solution contains only small (?2.3 nm average hydrodynamic radius) A? oligomers or monomers. We conclude that at near-physiological concentrations only the small oligomeric A? species are relevant, they are capable of attaching to the cell membrane, and they assemble in situ to form much larger complexes.

    View details for DOI 10.1016/j.bpj.2010.07.020

    View details for Web of Science ID 000282197500035

    View details for PubMedID 20858443

  • On the Stability of the Soluble Amyloid Aggregates BIOPHYSICAL JOURNAL Sahoo, B., Nag, S., Sengupta, P., Maiti, S. 2009; 97 (5): 1454-1460

    Abstract

    Many amyloid proteins form metastable soluble aggregates (or protofibrils, or protein nanoparticles, with characteristic sizes from approximately 10 to a few hundred nm). These can coexist with protein monomers and amyloid precipitates. These soluble aggregates are key determinants of the toxicity of these proteins. It is therefore imperative to understand the physical basis underlying their stability. Simple nucleation theory, typically applied to explain the kinetics of amyloid precipitation, fails to predict such intermediate stable states. We examine stable nanoparticles formed by the Alzheimer's amyloid-beta peptide (40 and 42 residues), and by the protein barstar. These molecules have different hydrophobicities, and therefore have different short-range attractive interactions between the molecules. We also vary the pH and the ionic strength of the solution to tune the long-range electrostatic repulsion between them. In all the cases, we find that increased long-range repulsion results in smaller stable nanoparticles, whereas increased hydrophobicity produces the opposite result. Our results agree with a charged-colloid type of model for these particles, which asserts that growth-arrested colloid particles can result from a competition between short-range attraction and long-range repulsion. The nanoparticle size varies superlinearly with the ionic strength, possibly indicating a transition from an isotropic to a linear mode of growth. Our results provide a framework for understanding the stability and growth of toxic amyloid nanoparticles, and provide cues for designing effective destabilizing agents.

    View details for DOI 10.1016/j.bpj.2009.05.055

    View details for Web of Science ID 000269429400025

    View details for PubMedID 19720034

  • Spatial pH Jump Measures Chemical Kinetics in a Steady-State System JOURNAL OF PHYSICAL CHEMISTRY A Nag, S., Bandyopadhyay, A., Maiti, S. 2009; 113 (18): 5269-5272

    Abstract

    We measure chemical kinetics in a steady-state solution where we create a microscopic open region with conditions different from the bulk. Individual reactant molecules spontaneously diffuse through this "reaction volume". We measure the changes which take place within their short residence time in the volume. The advantage of this approach is that the time resolution is limited only by the residence time tau(D) of the molecules in the reaction volume (which can easily be <50 mus), while the time taken to average the data can be arbitrarily long. In addition, if the chemical changes are reversible, the system is always in a steady state, and no replenishment of the reactants is necessary. Also, the total specimen volume required can be very small (<20 muL). We demonstrate the scheme by measuring the protonation induced changes of the fluorescence properties of fluorescein. We first show that a pH jump of >1 unit can be achieved by multiphoton excitation of ortho-nitro benzaldehyde (o-NBA). We then perform fluorescence correlation spectroscopy (FCS) to show that the residence time tau(D) of fluorescein in this low-pH region is approximately 30 mus. Subsequently, we use time correlated single photon counting (a widely used probing technique with an inherently long averaging time), and show that the data can be averaged for an arbitrarily long time, yet it captures the fluorescence lifetime of the low-pH species which exists only for the short time tau(D). Finally, we show that the time resolution can be tuned by over 3 orders of magnitude, by changing the focal volume and by changing the viscosity of the solution. The latter experiment also shows that small chemically induced changes in the fluorescence lifetime can be resolved by our technique.

    View details for DOI 10.1021/jp901296w

    View details for Web of Science ID 000265631000001

    View details for PubMedID 19402714

  • Protein aggregation probed by two-photon fluorescence correlation spectroscopy of native tryptophan JOURNAL OF CHEMICAL PHYSICS Sahoo, B., Balaji, J., Nag, S., Kumar, S. K., Maitia, S. 2008; 129 (7)

    Abstract

    Fluorescence correlation spectroscopy (FCS) has proven to be a powerful tool for the study of a range of biophysical problems including protein aggregation. However, the requirement of fluorescent labeling has been a major drawback of this approach. Here we show that the intrinsic tryptophan fluorescence, excited via a two-photon mechanism, can be effectively used to study the aggregation of tryptophan containing proteins by FCS. This method can also yield the tryptophan fluorescence lifetime in parallel, which provides a complementary parameter to understand the aggregation process. We demonstrate that the formation of soluble aggregates of barstar at pH 3.5 shows clear signatures both in the two-photon tryptophan FCS data and in the tryptophan lifetime analysis. The ability to probe the soluble aggregates of unmodified proteins is significant, given the major role played by this species in amyloid toxicity.

    View details for DOI 10.1063/1.2969110

    View details for Web of Science ID 000258643300053

    View details for PubMedID 19044804

  • Intermolecular association provides specific optical and NMR signatures for serotonin at intravesicular concentrations BIOPHYSICAL JOURNAL Nag, S., Balaji, J., Madhu, P. K., Maiti, S. 2008; 94 (10): 4145-4153

    Abstract

    Neurotransmitter vesicles contain biomolecules at extraordinarily high concentrations (hundreds of millimoles/liter). Such concentrations can drive intermolecular associations, which may affect vesicular osmolarity and neuronal signaling. Here we investigate whether aqueous serotonin (a monoamine neurotransmitter) forms oligomers at intravesicular concentrations and whether these oligomers have specific spectroscopic signatures that can potentially be used for monitoring neuronal storage and release. We report that, as serotonin concentration is increased from 60 microM to 600 mM, the normalized fluorescence spectrum of serotonin displays a growing long-wavelength tail, with an isoemissive point at 376 nm. The fluorescence decay is monoexponential with a lifetime of 4 ns at low concentrations but is multiexponential with an average lifetime of 0.41 ns at 600 mM. A 600 mM serotonin solution has 30% less osmolarity than expected for monomeric serotonin, indicating oligomer formation. The proton NMR chemical shifts move upfield by as much as 0.3 ppm at 600 mM compared to those at 10 mM, indicating a stacking of the serotonin indole moieties. However, no intermolecular crosspeak is evident in the two-dimensional NMR rotating frame Overhauser effect spectroscopy spectrum even at 600 mM, suggesting that oligomeric structures are possibly weakly coupled. The appearance of a single peak for each proton suggests that the rate of interconversion between the monomeric and the oligomeric structures is faster than 240 Hz. A stopped-flow kinetic experiment also confirms that the rate of dissociation is faster than 100 ms. We conclude that serotonin forms oligomers at intravesicular concentrations but becomes monomeric quickly on dilution. NMR signatures of the oligomers provide potential contrast agents for monitoring the activity of serotonergic neurons in vivo.

    View details for DOI 10.1529/biophysj.107.121384

    View details for Web of Science ID 000255309700041

    View details for PubMedID 18234835

  • A high-resolution large area serotonin map of a live rat brain section NEUROREPORT Kaushalya, S. K., Nag, S., Ghosh, H., Arumugam, S., Maiti, S. 2008; 19 (7): 717-721

    Abstract

    We employ three-photon microscopy to produce a high-resolution map of serotonin autofluorescence in a rat midbrain section (covering more than half of the brain), to quantitatively characterize serotonin distribution and release in different areas of a live brain slice. The map consists of a tiling of approximately 160 contiguous optical images (covering an area of approximately 27 mm with sub-mum resolution in 20 min), and is recorded before and after inducing depolarization. We observe that the total serotonin exocytosed from the somata in the raphe is quantitatively comparable with regions containing a high density of serotonergic processes. Our results demonstrate that high-resolution, wide-area, dynamic neurotransmitter mapping is now possible.

    View details for Web of Science ID 000255530600003

    View details for PubMedID 18418245

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