Panchali Goswami - Academia.edu (original) (raw)

Papers by Panchali Goswami

Methods in molecular biology, 2018

High-resolution image acquisition and structure determination by cryo-electron microscopy is beco... more High-resolution image acquisition and structure determination by cryo-electron microscopy is becoming increasingly streamlined. Preparing electron-microscopy grids of suitable quality remains, however, a critical bottleneck. Strategies to achieve particle monodispersity, optimal sample concentration and suitable ice thickness can vary from specimen to specimen. In this book chapter we describe our protocols for negative-stain grid and cryo-grid preparation, which we apply to studying protein-nucleic acid complexes.

International Journal of Molecular Sciences, Mar 24, 2023

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

International Journal of Molecular Sciences

The presentation of viral antigens on nanoparticles in multivalent arrays has emerged as a valuab... more The presentation of viral antigens on nanoparticles in multivalent arrays has emerged as a valuable technology for vaccines. On the nanoparticle surface, highly ordered, repetitive arrays of antigens can mimic their geometric arrangement on virion surfaces and elicit stronger humoral responses than soluble viral antigens. More recently, bacterial antigens have been presented on self-assembling protein nanoparticles and have elicited protective antibody and effective T-helper responses, further supporting the nanoparticle platform as a universal approach for stimulating potent immunogenicity. Here, we present the rational design, structural analysis, and immunogenicity of self-assembling ferritin nanoparticles displaying eight copies of the Neisseria meningitidis trimeric adhesin NadA. We engineered constructs consisting of two different NadA fragments, head only and head with stalk, that we fused to ferritin and expressed in Escherichia coli. Both fusion constructs self-assembled in...

Proceedings of the National Academy of Sciences, 2019

Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and Leishmania... more Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and Leishmania infantum , is one of the major parasitic diseases worldwide. There is an urgent need for new drugs to treat VL, because current therapies are unfit for purpose in a resource-poor setting. Here, we describe the development of a preclinical drug candidate, GSK3494245/DDD01305143/compound 8, with potential to treat this neglected tropical disease. The compound series was discovered by repurposing hits from a screen against the related parasite Trypanosoma cruzi . Subsequent optimization of the chemical series resulted in the development of a potent cidal compound with activity against a range of clinically relevant L. donovani and L. infantum isolates. Compound 8 demonstrates promising pharmacokinetic properties and impressive in vivo efficacy in our mouse model of infection comparable with those of the current oral antileishmanial miltefosine. Detailed mode of action studies confirm that ...

Methods in molecular biology (Clifton, N.J.), 2018

Proceedings of the National Academy of Sciences of the United States of America, Apr 18, 2017

The replisome unwinds and synthesizes DNA for genome duplication. In eukaryotes, the Cdc45-MCM-GI... more The replisome unwinds and synthesizes DNA for genome duplication. In eukaryotes, the Cdc45-MCM-GINS (CMG) helicase and the leading-strand polymerase, Pol epsilon, form a stable assembly. The mechanism for coupling DNA unwinding with synthesis is starting to be elucidated, however the architecture and dynamics of the replication fork remain only partially understood, preventing a molecular understanding of chromosome replication. To address this issue, we conducted a systematic single-particle EM study on multiple permutations of the reconstituted CMG-Pol epsilon assembly. Pol epsilon contains two flexibly tethered lobes. The noncatalytic lobe is anchored to the motor of the helicase, whereas the polymerization domain extends toward the side of the helicase. We observe two alternate configurations of the DNA synthesis domain in the CMG-bound Pol epsilon. We propose that this conformational switch might control DNA template engagement and release, modulating replisome progression.

PROTEOMICS, 2015

The quantification of plasma proteins using the high resolution and accurate mass (HR/AM)‐based p... more The quantification of plasma proteins using the high resolution and accurate mass (HR/AM)‐based parallel reaction monitoring (PRM) method provides an immediate benefit over the conventional SRM‐based method in terms of selectivity. In this study, multiplexed PRM assays were developed to analyze isotypes of serum amyloid A (SAA) proteins in human plasma with a focus on SAA1 and SAA2. Elevated plasma levels of these proteins in patients diagnosed with lung cancer have been reported in previous studies. Since SAA1 and SAA2 are highly homologous, the available immunoassays tend to overestimate their concentrations due to cross‐reactivity. On the other hand, when mass spectrometry (MS)‐based assays are used, the presence of the several allelic variants may result in a problem of underestimation. In the present study, eight peptides that represent the target proteins at three different levels: isotype‐specific (SAA1α, SAA 1β, SAA1γ, SAA2α, SAA2β), protein‐specific (SAA1 or SAA2), and ...

Nature Communications, 2018

Eukaryotic origin firing depends on assembly of the Cdc45-MCM-GINS (CMG) helicase. A key step is ... more Eukaryotic origin firing depends on assembly of the Cdc45-MCM-GINS (CMG) helicase. A key step is the recruitment of GINS that requires the leading-strand polymerase Pol epsilon, composed of Pol2, Dpb2, Dpb3, Dpb4. While a truncation of the catalytic N-terminal Pol2 supports cell division, Dpb2 and C-terminal Pol2 (C-Pol2) are essential for viability. Dpb2 and C-Pol2 are non-catalytic modules, shown or predicted to be related to an exonuclease and DNA polymerase, respectively. Here, we present the cryo-EM structure of the isolated C-Pol2/Dpb2 heterodimer, revealing that C-Pol2 contains a DNA polymerase fold. We also present the structure of CMG/C-Pol2/Dpb2 on a DNA fork, and find that polymerase binding changes both the helicase structure and fork-junction engagement. Inter-subunit contacts that keep the helicase-polymerase complex together explain several cellular phenotypes. At least some of these contacts are preserved during Pol epsilon-dependent CMG assembly on path to origin fi...

Na+/H+ antiporters are ubiquitous membrane proteins involved in ion homeostasis and pH sensing. T... more Na+/H+ antiporters are ubiquitous membrane proteins involved in ion homeostasis and pH sensing. The amino acid sequence of one such antiporter, MjNhaP1, from Methanococcus jannaschii, shows a significant homology to eukaryotic sodium proton exchangers like NHE1 from Homo sapiens and SOS1 of Arabidopsis thaliana than to the well-characterized Escherichia coli NhaA or NhaB. MjNhaP1 shows activity at acidic pH unlike NhaA, which is active at basic pH. 13 transmembrane helices have been predicted to be present in NhaP1. A projection map, calculated by Cryo-EM of 2D crystals of MjNhaP1 grown at pH 4, showed it to be a dimer containing elongated densities in the centre of the dimer and a cluster of density peaks on either side of the dimer core (Vinothkumar et al., 2005). Incubation of 2D crystals at pH 8 on the EM grid resulted in well-defined conformational changes, clearly evident in a difference map as a major change in density distribution within the helix bundle (Vinothkumar et al.,...

Molecular Cell, 2016

Highlights d Ctf4 is a replisomal interaction hub that recruits CIP box client proteins d Chl1 he... more Highlights d Ctf4 is a replisomal interaction hub that recruits CIP box client proteins d Chl1 helicase is the Ctf4 client that links replication and cohesion establishment d Ctf4 interaction, but not helicase activity, conveys Chl1 cohesion function d Chl1 physically engages with cohesin during cohesion establishment

Annals of The Rheumatic Diseases, 2006

Background: The contribution of the environment to rheumatoid arthritis (RA) remains uncertain. I... more Background: The contribution of the environment to rheumatoid arthritis (RA) remains uncertain. Intrauterine and early postnatal life may be important. Rheumatoid factor (RF) found in around 10% of the normal population confers a risk of developing RA and may be present years before onset of clinical disease. The immune pathology leading to RA and RF may have similar genetic and

The EMBO Journal, 2011

We have determined the structure of the archaeal sodium/ proton antiporter NhaP1 at 7 Å resolutio... more We have determined the structure of the archaeal sodium/ proton antiporter NhaP1 at 7 Å resolution by electron crystallography of 2D crystals. NhaP1 is a dimer in the membrane, with 13 membrane-spanning a-helices per protomer, whereas the distantly related bacterial NhaA has 12. Dimer contacts in the two antiporters are very different, but the structure of a six-helix bundle at the tip of the protomer is conserved. The six-helix bundle of NhaA contains two partially unwound a-helices thought to harbour the ion-translocation site, which is thus similar in NhaP1. A model of NhaP1 based on detailed sequence comparison and the NhaA structure was fitted to the 7 Å map. The additional N-terminal helix 1 of NhaP1, which appears to be an uncleaved signal sequence, is located near the dimer interface. Similar sequences are present in many eukaryotic homologues of NhaP1, including NHE1. Although fully folded and able to dimerize, NhaP1 constructs without helix 1 are inactive. Possible reasons are investigated and discussed.

Lupus, 2006

There has been limited success defining environmental factors important to the development of con... more There has been limited success defining environmental factors important to the development of connective tissue diseases such as systemic lupus erythematosus (SLE). Recent work has suggested that the perinatal environment may be important. To investigate this we measured antinuclear antibodies (ANA) in a general population with well-defined early lives to see whether fetal and infant growth and infections were associated with ANA positivity in adult life. Included in our investigation were 1334 individuals (668 men, 666 women) from the Hertfordshire cohort study. ANA was measured using an ANA ELISA and confirmed using immunofluorescence. We investigated associations between the presence of ANA and early growth and infectious exposure in infancy in men and women combined, but with adjustment for gender throughout. A positive ANA was present in 73 (10.9%) of men and 81 (12.2%) women. Of these, 26 women and 14 men were positive using IF on HEP2 cells. Sharing a bedroom during childhood was associated with a higher risk of being ANA positive (odds ratio (OR), 1.42, 95% confidence interval (CI) 1.00-2.01, P = 0.05). A record of diarrhoeal illness (OR 2.12 95% CI 1.07, 4.23, P = 0.03) and rubella or mumps during the first year of life (OR 16.12, 95% CI 2.92, 88.94, P = 0.001) was also significantly associated with ANA in adult life. Higher ANA titres by Inova ELISA were associated with infections in the first year of life from mumps (2.74-fold higher, 95% CI 0.98, 7.64, P = 0.05) and rubella (3.90-fold higher, 95% CI 1.89, 8.04, P &amp;lt; 0.001). In addition, higher ANA titres were also associated with mumps (1.26-fold higher, 95% CI 1.02, 1.56, P = 0.03) between one and five years of age. Our results suggest that a developing immune system exposed to increased infection is more likely to produce ANA in adult life and perhaps begin the pathological process that leads to SLE.

Journal of Molecular Biology, 2006

Using single-molecule force spectroscopy we probed molecular interactions within native bovine rh... more Using single-molecule force spectroscopy we probed molecular interactions within native bovine rhodopsin and discovered structural segments of welldefined mechanical stability. Highly conserved residues among G proteincoupled receptors were located at the interior of individual structural segments, suggesting a dual role for these segments in rhodopsin. Firstly, structural segments stabilize secondary structure elements of the native protein, and secondly, they position and hold the highly conserved residues at functionally important environments. Two main classes of force curves were observed. One class corresponded to the unfolding of rhodopsin with the highly conserved Cys110-Cys187 disulfide bond remaining intact and the other class corresponded to the unfolding of the entire rhodopsin polypeptide chain. In the absence of the Cys110-Cys187 bond, the nature of certain molecular interactions within folded rhodopsin was altered. These changes highlight the structural importance of this disulfide bond and may form the basis of dysfunctions associated with its absence.

Journal of Allergy and Clinical Immunology, 2008

Background-Acute wheezing illnesses in preschoolers need better management strategies to reduce m... more Background-Acute wheezing illnesses in preschoolers need better management strategies to reduce morbidity.

Indian Journal of Radiology and Imaging, 2006

Indian Journal of Radiology and Imaging, 2005

Heart, 2007

Subjects with rheumatoid arthritis have an increased prevalence of ischaemic heart disease (IHD).... more Subjects with rheumatoid arthritis have an increased prevalence of ischaemic heart disease (IHD). This is most likely in those people with the autoantibody rheumatoid factor (RF). RF is strongly associated with rheumatoid arthritis (RA) but is also present in up to 15% of all adults. To determine whether RF might identify people in a general population who also share an increased likelihood of developing IHD. Subjects from the Hertfordshire Cohort Study were investigated for the presence of RF. Subjects completed a questionnaire and attended a clinic where a history of IHD was recorded (ECG, coronary artery bypass grafting, Rose chest pain). Associations between the presence of RF, antinuclear antibodies (ANA), anticardiolipin antibodies (ACA) and IHD in 567 men and 589 women were investigated and compared with traditional risk factors for IHD. RF was associated with an increased likelihood of IHD in men (odds ratio (OR) = 3.1, 95% CI 1.7 to 5.4, p&amp;amp;amp;lt;0.001). This increased risk could not be explained by traditional risk factors for IHD (mutually adjusted OR for RF 2.9 (95% CI 1.6 to 5.3), p&amp;amp;amp;lt;0.001). There was no significant association between RF in women or between ANA or ACA with IHD in men or women. This work suggests that RF is an independent risk factor for IHD in the general population. It lends support to the importance of inflammation in atherosclerosis and suggests that autoimmune processes may be involved. In addition, it raises the intriguing possibility that RF may have a direct role in the pathogenesis of IHD in some subjects.

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2009

In this study, structural, functional, and mechanistic properties of the Na + /H + antiporter MjN... more In this study, structural, functional, and mechanistic properties of the Na + /H + antiporter MjNhaP1 from Methanococcus jannaschii were analyzed by infrared spectroscopic techniques. Na + /H + antiporters are generally responsible for the regulation of cytoplasmic pH and Na + concentration. MjNhaP1 is active in the pH range between pH 6 and pH 6.5; below and above it is inactive. The secondary structure analysis on the basis of ATR-IR spectra provides the first insights into the structural changes between inactive (pH 8) and active (pH 6) state of MjNhaP1. It results in decreased ordered structural elements with increasing the pHvalue i.e. with inactivation of the protein. Analysis of temperature-dependent FTIR spectra indicates that MjNhaP1 in the active state exhibits a much higher unfolding temperature in the spectral region assigned to α-helical segments. In contrast, the temperature-induced structural changes for β-sheet structure are similar for inactive and active state. Consequently, this structure element is not the part of the activation region of the protein. The surface accessibility of the protein was analyzed by following the extent of H/D exchange. Due to higher content of unordered structural elements a higher accessibility for amide protons is observed for the inactive as compared to the active state of MjNhaP1. Altogether, the results present the active state of MjNhaP1 as the state with ordered structural elements which exhibit high thermal stability and increased hydrophobicity.