Sri Rama Koti Ainavarapu - Academia.edu (original) (raw)

Papers by Sri Rama Koti Ainavarapu

Biophysical Journal, 2019

Physical Chemistry Chemical Physics

The native-state structure and folding pathways of a protein are encoded in its amino acid sequence.

bioRxiv, 2021

Plasmodium falciparum circumsporozoite protein (CSP) is a critically required abundant surface pr... more Plasmodium falciparum circumsporozoite protein (CSP) is a critically required abundant surface protein of sporozoites and a major vaccine candidate. However, neither the structure nor the role of CSP in sporozoite motility is well understood. Our recent in vitro data, from single-molecule pulling experiments suggested a mechanically pliable structure for P. falciparum CSP. By engineering vegetative cells of the cellular slime-mold Dictyostelium discoideum with regulatable CSP surface expression, we report evidence for direct involvement of CSP towards conferring elastic properties and motility of the cells. With an increase in the surface-CSP levels by 5–8-fold, the Youngs moduli of the cells, observed through atomic force microscopy, decreased around 2-fold, with a concomitant increase in motility by about 2-fold. Interestingly, only full length CSP expression conferred maximal flexibility and motility, as opposed to repeat region alone or the flanking domains of CSP. The enhanced ...

Metalloproteins are an important class of proteins involved in metal uptake, transport, and elect... more Metalloproteins are an important class of proteins involved in metal uptake, transport, and electron-transfer reactions. Mimicking the active sites of these proteins through miniaturization is an active area of research with applications in biotechnology and medicine. Azurin is a 128-residue copper-binding cupredoxin protein involved in electron-transfer reactions. Previous studies have reported on the copper-binding-induced spectroscopic and structural properties of peptide loops (11 and 13 residues) from azurin. These azurin peptides exhibited novel stoichiometries. However, the underlying mechanism of fluorescence quenching upon copper binding remains to be understood, whether it is due to electron transfer, energy transfer, or both. Here, we report nickel-binding-associated spectroscopic and structural properties of the azurin peptides. They develop a β-turn upon nickel binding as seen in circular dichroism and exhibit electronic transitions centered at 270 and 450 nm. Unlike co...

Protein folding/unfolding are complex phenomena and it is accepted that multidomain proteins gene... more Protein folding/unfolding are complex phenomena and it is accepted that multidomain proteins generally follow multiple pathways. Maltose binding protein is a large (a two-domain 370 amino acid residue) bacterial periplasmic protein involved in maltose uptake. Despite the large size, it has been shown to exhibit an apparent two-state equilibrium unfolding in bulk experiments. Single-molecule studies can uncover rare events that are masked by averaging in bulk studies. Here, we use single-molecule force spectroscopy to study the mechanical unfolding pathways of maltose binding protein (MBP) and its precursor protein (preMBP) in the presence and absence of ligands. Our results show that MBP exhibits kinetic partitioning on mechanical stretching and unfolds via two parallel pathways: one of them involves a mechanically stable intermediate (path I) while the other is devoid of it (path II). The apoMBP unfolds via path I in 62% of the mechanical unfolding events and the remaining 38% foll...

Biochemical Journal

The binuclear metalloenzyme Helicobacter pylori arginase is important for pathogenesis of the bac... more The binuclear metalloenzyme Helicobacter pylori arginase is important for pathogenesis of the bacterium in the human stomach. Despite conservation of the catalytic residues, this single Trp enzyme has an insertion sequence (–153ESEEKAWQKLCSL165–) that is extremely crucial to function. This sequence contains the critical residues, which are conserved in the homolog of other Helicobacter gastric pathogens. However, the underlying basis for the role of this motif in catalytic function is not completely understood. Here, we used biochemical, biophysical and molecular dynamics simulations studies to determine that Glu155 of this stretch interacts with both Lys57 and Ser152. These interactions are essential for positioning of the motif through Trp159, which is located near Glu155 (His122–Trp159–Tyr125 contact is essential to tertiary structural integrity). The individual or double mutation of Lys57 and Ser152 to Ala considerably reduces catalytic activity with Lys57 to Ala being more sign...

The Journal of Physical Chemistry B

Despite many studies on ligand-modulated protein mechanics, a comparative analysis of the role of... more Despite many studies on ligand-modulated protein mechanics, a comparative analysis of the role of ligand binding site on any specific protein fold is yet to be made. In this study, we explore the role of ligand binding site on the mechanical properties of β-grasp fold proteins, namely, ubiquitin and small ubiquitin related modifier 1 (SUMO1). The terminal segments directly connected through hydrogen bonds constitute the β-clamp geometry (or mechanical clamp), which confers high mechanical resilience to the β-grasp fold. Here, we study ubiquitin complexed with CUE2-1, a ubiquitin-binding domain (UBD) from yeast endonuclease protein Cue2, using a combination of single-molecule force spectroscopy (SMFS) and steered molecular dynamics (SMD) simulations. Our study reveals that CUE2-1 does not alter the mechanical properties of ubiquitin, despite directly interacting with its β-clamp. To explore the role of ligand binding site, we compare the mechanical properties of the ubiquitin/CUE2-1 complex with that of previously studied SUMO1/S12, another β-grasp protein complex, using SMD simulations. Simulations on the SUMO1/S12 complex corroborate previous experimentally observed enhancement in the mechanical stability of SUMO1, even though S12 binds away from the β-clamp. Differences in ligand binding-induced structural impact at the transition state of the two complexes explain the differences in ligand modulated protein mechanics. Contrary to previous reports, our study demonstrates that direct binding of ligands to the mechanical clamp does not necessarily alter the mechanical stability of β-grasp fold proteins. Rather, binding interactions away from the clamp can reinforce protein stability provided by the β-grasp fold. Our study highlights the importance of binding site and binding modes of ligands in modulating the mechanical stability of β-grasp fold proteins.

Protein folding can go wrong in vivo and in vitro, with significant consequences for the living c... more Protein folding can go wrong in vivo and in vitro, with significant consequences for the living cell and the pharmaceutical industry, respectively. Here we propose a general design principle for constructing small peptide-based protein-specific folding modifiers. We construct a ‘xenonucleus’, which is a pre-folded peptide that resembles the folding nucleus of a protein, and demonstrate its activity on the folding of ubiquitin. Using stopped-flow kinetics, NMR spectroscopy, Förster Resonance Energy transfer, single-molecule force measurements, and molecular dynamics simulations, we show that the ubiquitin xenonucleus can act as an effective decoy for the native folding nucleus. It can make the refolding faster by 33 ± 5% at 3 M GdnHCl. In principle, our approach provides a general method for constructing specific, genetically encodable, folding modifiers for any protein which has a well-defined contiguous folding nucleus.

Biophysical Journal, Feb 1, 2018

Calmodulin (CaM) trapping is a phenomenon where the active and inactive states of calmodulin depe... more Calmodulin (CaM) trapping is a phenomenon where the active and inactive states of calmodulin dependent protein kinase II (CaMKII) produce drastically different affinities for Ca 2þ saturated CaM. The states of CaMKII are paramount in the scheme of calcium ion signaling, which is an essential biological function whose underlying mechanism is largely unknown. Experimentally, a set of peptides modeled after CaMKII's binding domain (293-312) were created through systematic mutations of charged residues to mimic the two distinct affinity states for CaM and probe the mechanisms responsible for the observed change in kinetics. Although a model was successfully created, the observed interactions could not be explained through current protein interaction models or electrostatic steering effects. We investigate the dynamics of this experiment through the use of all atom simulations, choosing three of the mutant peptides with a length of 20 amino acids. We refer to these peptides by the 296-298 residues, specifically RRK (wildtype), RAK (1-residue mutation) and AAA (3-residue mutation), and validate our simulation findings through comparison with circular dichroism (CD) data. We demonstrate that the large side chains present in the first 6 residues of each peptide interact with each other, and that the mutation of charged residues produce global changes in sidechain conformations and disordered regions. The specific ensemble of secondary structure/rotamer conformations present in RRK, that are not observed in AAA, are essential for high affinity binding between CaMKII and CaM.

Archives of Biochemistry and Biophysics

The active sites of metalloproteins may be mimicked by designing peptides that bind to their resp... more The active sites of metalloproteins may be mimicked by designing peptides that bind to their respective metal ions. Studying the binding of protein ligands to metal ions along with the associated structural changes is important in understanding metal uptake, transport and electron transfer functions of proteins. Copper-binding metalloprotein azurin is a 128-residue electron transfer protein with a redox-active copper cofactor. Here, we report the copper-binding associated spectroscopic and structural properties of peptide loops (11 and 13 residues) from the copper-binding site of azurin. These peptides develop a β-turn upon copper-binding with a 1:1 Cu2+:peptide stoichiometry as seen in circular dichroism and exhibit electronic transitions centered at 340 nm and 540 nm. Further addition of copper develops a helical feature along with a shift in the absorption maxima to ∼360 nm and ∼580 nm at 2:1 Cu2+:peptide stoichiometry, indicating stoichiometric dependence of copper-binding geometry. Mass spectrometry indicates the copper-binding to cysteine, histidine and methionine in the peptide with 1:1 stoichiometry, and interestingly, dimerization through a disulfide linkage at 2:1 stoichiometry, as observed previously for denatured azurin. Fluorescence quenching studies on peptides with tryptophan further confirm the copper-binding induced changes in the two peptides are bi-phasic.

In this manuscript, we introduce the Cumulative Variance of Coordinate Fluctuations (CVCF) along ... more In this manuscript, we introduce the Cumulative Variance of Coordinate Fluctuations (CVCF) along atomistic MD trajectories, as a dynamical metric to examine protein dynamics and sampling convergence in MD simulations. Using model 1D and 2D PES, we first show that CVCF, which traces over the fluctuations of protein atoms as a function of sampling coordinate (time in MD simulations), captures both local and global equilibria to distinguish the underlying PES of proteins. For both model PES and protein trajectories, we compare the information content present in CVCF traces with that obtained using other measures proposed in literature to reveal conditions under which a consistent interpretation of data can be obtained. Importantly, we show that independent of convergence to either local or global equilibrium, the values and features of protein CVCF can provide a comparative assessment of the ruggedness and curvature of the underlying PES sampled by proteins along MD trajectories. Trend...

Proteins: Structure, Function, and Bioinformatics

Small ubiquitin‐related modifiers (SUMO1 and SUMO2) are ubiquitin family proteins, structurally s... more Small ubiquitin‐related modifiers (SUMO1 and SUMO2) are ubiquitin family proteins, structurally similar to ubiquitin, differing in terms of their amino acid sequence and functions. Therefore, they provide a great platform for investigating sequence‐structure‐stability‐function relationship. Here, we used chemical denaturation in comparing the folding‐unfolding pathways of the SUMO proteins with their structural homologue ubiquitin (UF45W‐pseudo wild‐type [WT] tryptophan variant) with structurally analogous tryptophan mutations (SUMO1 [S1F66W], SUMO2 [S2F62W]). Equilibrium denaturation studies report that ubiquitin is the most stable protein among the three. The observed denaturant‐dependent folding rates of SUMOs are much lower than ubiquitin and primarily exhibit a two‐state folding pathway unlike ubiquitin, which has a kinetic folding intermediate. We hypothesize that, as SUMO proteins start off as slow folders, they avoid stabilizing their folding intermediates and the presence of which might further slow‐down their folding rates. The denaturant‐dependent unfolding of ubiquitin is the fastest, followed by SUMO2, and slowest for SUMO1. However, the spontaneous unfolding rate constant is the lowest for ubiquitin (~40 times), and similar for SUMOs. This correlation between thermodynamic stability and kinetic stability is achieved by having different unfolding transition state positions with respect to the solvent‐accessible surface area, as quantified by the Tanford β u values: ubiquitin (0.42) > SUMO2 (0.20) > SUMO1 (0.16). The results presented here highlight the unique energy landscape features which help in optimizing the folding‐unfolding rates within a structurally homologous protein family.

RSC Advances

Triphala inhibits αSyn self-association by interacting with anchoring regions which are responsib... more Triphala inhibits αSyn self-association by interacting with anchoring regions which are responsible for αSyn oligomerization.

ChemPhysChem

High quantum yield, photoluminescence tunability, and sensitivity to the environment are a few di... more High quantum yield, photoluminescence tunability, and sensitivity to the environment are a few distinct trademarks that make carbon nanodots (CDs) interesting for fundamental research, with potential to replace the prevalent inorganic semiconductor quantum dots. Currently, application and fundamental understanding of CDs are constrained because it is difficult to make a quantitative comparison among different types of CDs simply because their photoluminescence properties are directly linked to their size distribution, the surface functionalization, the carbon core structures (graphitic or amorphous) and the number of defects. Herein, we report a facile one-step synthesis of mono-dispersed and highly fluorescent nanometre size CDs from a 'family' of glucose-based sugars. These CDs are stable in aqueous solutions with photoluminescence in the visible range. Our results show several common features in the family of CDs synthesized in that the fluorescence, in the visible region, is due to a weak absorption in the 300-400 nm from a heterogeneous population of fluorophores. Fluorescence quenching experiments suggest the existence of not only surface-exposed fluorophores but more importantly solvent inaccessible fluorophores present within the core of CDs. Interestingly, time-resolved fluorescence anisotropy experiments directly suggest that a fast exchange of excitation energy occurs that results in a homo-FRET based depolarization within 150 ps of excitation.

Biophysical Journal

CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) pr... more CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins are part of the adaptive immune system of bacteria and archaea. CRISPRs are stretches of DNA with two distinct characteristics: the presence of nucleotide repeats and spacers, which serve as a memory bank, enabling bacteria to recognize the invading viruses. The Cas proteins are enzymes that cut foreign DNA guided by the information encoded in CRISPRs. In 2012, it has been demonstrated that bacterial CRISPR-Cas9 can be transformed into a simple, programmable genome-editing tool. Cas9 requires two RNA molecules: a CRISPR RNA (or ''crRNA'') and another tracrRNA (or ''trans-activating crRNA'') to make its cut on a target DNA with a 20-nucleotide stretch complementary to the crRNA. Recently, Cpf1 proteins have been discovered to show comparable genome-editing capability to Cas9. Cpf1 functions through a single crRNA without an additional tracrRNA. Cpf1 is an RNA-guided endonuclease of a class II CRISPR/Cas system, which is a smaller and simpler than Cas9, and overcomes some of the CRISPR/Cas9 system limitations. We are interested in rationally modifying the CRISPR-Cpf1 system to improve its genome editing efficiency. The target efficiency and specificity of the CRISPR technology are influenced by multiple factors. Our specific objective is to first perform large-scale molecular dynamics (MD) simulations to elucidate various conformational states involved in the Cpf1 catalysis (especially those upon the binding of crRNA and DNA) and the interconversion rates between different states. We further aim to use these MD simulations to uncover the molecular mechanisms underlying the higher genome editing efficiency of certain crRNAs. We will systematically explore how the incorporation of chemically and structurally modified nucleotides in crRNAs may affect the genome editing efficiency and off-target effects and to establish comprehensive structure-activity relationships of crRNA analogs.

Scientific reports, Jan 31, 2018

Metalloproteins carry out diverse biological functions including metal transport, electron transf... more Metalloproteins carry out diverse biological functions including metal transport, electron transfer, and catalysis. At present, the influence of metal cofactors on metalloprotein stability is not well understood. Here, we report the mechanical stability and unfolding pathway of azurin, a cupredoxin family protein with β-barrel topology and type I copper-binding centre. Single-molecule force spectroscopy (SMFS) experiments reveal 2-state and 3-state unfolding pathways for apo-azurin. The intermediate in the 3-state pathway occurs at an unfolding contour length of 7.5 nm from the native state. Steered molecular dynamics (SMD) simulations show that apo-azurin unfolds via a first transition state (TS) where β2Β-β8 and β7-β8 strand pairs rupture to form the intermediate, which subsequently unfolds by the collective rupture of remaining strands. SMFS experiments on holo-azurin exhibit an additional 4-state pathway besides the 2-state and 3-state pathways. The unfolding contour length lead...

Metalloproteins carry out diverse biological functions including metal transport, electron transf... more Metalloproteins carry out diverse biological functions including metal transport, electron transfer, and catalysis. At present, the influence of metal cofactors on metalloprotein stability is not well understood. Here, we report the mechanical stability and unfolding pathway of azurin, a cupredoxin family protein with β-barrel topology and type I copper-binding centre. Single-molecule force spectroscopy (SMFS) experiments reveal 2-state and 3-state unfolding pathways for apo-azurin. The intermediate in the 3-state pathway occurs at an unfolding contour length of 7.5 nm from the native state. Steered molecular dynamics (SMD) simulations show that apo-azurin unfolds via a first transition state (TS) where β2Β–β8 and β7–β8 strand pairs rupture to form the intermediate, which subsequently unfolds by the collective rupture of remaining strands. SMFS experiments on holo-azurin exhibit an additional 4-state pathway besides the 2-state and 3-state pathways. The unfolding contour length lead...

Journal of Biological Chemistry, 2016

The most effective vaccine candidate of malaria is based on the Plasmodium falciparum circumsporo... more The most effective vaccine candidate of malaria is based on the Plasmodium falciparum circumsporozoite protein (CSP), a major surface protein implicated in the structural strength, motility, and immune evasion properties of the infective sporozoites. It is suspected that reversible conformational changes of CSP are required for infection of the mammalian host, but the detailed structure and dynamic properties of CSP remain incompletely understood, limiting our understanding of its function in the infection. Here, we report the structural and mechanical properties of the CSP studied using single-molecule force spectroscopy on several constructs, one including the central region of CSP, which is rich in NANP amino acid repeats (CSP rep), and a second consisting of a near full-length sequence without the signal and anchor hydrophobic domains (CSP ⌬HP). Our results show that the CSP rep is heterogeneous, with 40% of molecules requiring virtually no mechanical force to unfold (<10 piconewtons (pN)), suggesting that these molecules are mechanically compliant and perhaps act as entropic springs, whereas the remaining 60% are partially structured with low mechanical resistance (ϳ70 pN). CSP ⌬HP having multiple force peaks suggests specifically folded domains, with two major populations possibly indicating the open and collapsed forms. Our findings suggest that the overall low mechanical resistance of the repeat region, exposed on the outer surface of the sporozoites, combined with the flexible full-length conformations of CSP, may provide the sporozoites not only with immune evasion properties, but also with lubricating capacity required during its navigation through the mosquito and vertebrate host tissues. We anticipate that these findings would further assist in the design and development of future malarial vaccines.

Biophysical Journal, 2019

Physical Chemistry Chemical Physics

The native-state structure and folding pathways of a protein are encoded in its amino acid sequence.

bioRxiv, 2021

Plasmodium falciparum circumsporozoite protein (CSP) is a critically required abundant surface pr... more Plasmodium falciparum circumsporozoite protein (CSP) is a critically required abundant surface protein of sporozoites and a major vaccine candidate. However, neither the structure nor the role of CSP in sporozoite motility is well understood. Our recent in vitro data, from single-molecule pulling experiments suggested a mechanically pliable structure for P. falciparum CSP. By engineering vegetative cells of the cellular slime-mold Dictyostelium discoideum with regulatable CSP surface expression, we report evidence for direct involvement of CSP towards conferring elastic properties and motility of the cells. With an increase in the surface-CSP levels by 5–8-fold, the Youngs moduli of the cells, observed through atomic force microscopy, decreased around 2-fold, with a concomitant increase in motility by about 2-fold. Interestingly, only full length CSP expression conferred maximal flexibility and motility, as opposed to repeat region alone or the flanking domains of CSP. The enhanced ...

Metalloproteins are an important class of proteins involved in metal uptake, transport, and elect... more Metalloproteins are an important class of proteins involved in metal uptake, transport, and electron-transfer reactions. Mimicking the active sites of these proteins through miniaturization is an active area of research with applications in biotechnology and medicine. Azurin is a 128-residue copper-binding cupredoxin protein involved in electron-transfer reactions. Previous studies have reported on the copper-binding-induced spectroscopic and structural properties of peptide loops (11 and 13 residues) from azurin. These azurin peptides exhibited novel stoichiometries. However, the underlying mechanism of fluorescence quenching upon copper binding remains to be understood, whether it is due to electron transfer, energy transfer, or both. Here, we report nickel-binding-associated spectroscopic and structural properties of the azurin peptides. They develop a β-turn upon nickel binding as seen in circular dichroism and exhibit electronic transitions centered at 270 and 450 nm. Unlike co...

Protein folding/unfolding are complex phenomena and it is accepted that multidomain proteins gene... more Protein folding/unfolding are complex phenomena and it is accepted that multidomain proteins generally follow multiple pathways. Maltose binding protein is a large (a two-domain 370 amino acid residue) bacterial periplasmic protein involved in maltose uptake. Despite the large size, it has been shown to exhibit an apparent two-state equilibrium unfolding in bulk experiments. Single-molecule studies can uncover rare events that are masked by averaging in bulk studies. Here, we use single-molecule force spectroscopy to study the mechanical unfolding pathways of maltose binding protein (MBP) and its precursor protein (preMBP) in the presence and absence of ligands. Our results show that MBP exhibits kinetic partitioning on mechanical stretching and unfolds via two parallel pathways: one of them involves a mechanically stable intermediate (path I) while the other is devoid of it (path II). The apoMBP unfolds via path I in 62% of the mechanical unfolding events and the remaining 38% foll...

Biochemical Journal

The binuclear metalloenzyme Helicobacter pylori arginase is important for pathogenesis of the bac... more The binuclear metalloenzyme Helicobacter pylori arginase is important for pathogenesis of the bacterium in the human stomach. Despite conservation of the catalytic residues, this single Trp enzyme has an insertion sequence (–153ESEEKAWQKLCSL165–) that is extremely crucial to function. This sequence contains the critical residues, which are conserved in the homolog of other Helicobacter gastric pathogens. However, the underlying basis for the role of this motif in catalytic function is not completely understood. Here, we used biochemical, biophysical and molecular dynamics simulations studies to determine that Glu155 of this stretch interacts with both Lys57 and Ser152. These interactions are essential for positioning of the motif through Trp159, which is located near Glu155 (His122–Trp159–Tyr125 contact is essential to tertiary structural integrity). The individual or double mutation of Lys57 and Ser152 to Ala considerably reduces catalytic activity with Lys57 to Ala being more sign...

The Journal of Physical Chemistry B

Despite many studies on ligand-modulated protein mechanics, a comparative analysis of the role of... more Despite many studies on ligand-modulated protein mechanics, a comparative analysis of the role of ligand binding site on any specific protein fold is yet to be made. In this study, we explore the role of ligand binding site on the mechanical properties of β-grasp fold proteins, namely, ubiquitin and small ubiquitin related modifier 1 (SUMO1). The terminal segments directly connected through hydrogen bonds constitute the β-clamp geometry (or mechanical clamp), which confers high mechanical resilience to the β-grasp fold. Here, we study ubiquitin complexed with CUE2-1, a ubiquitin-binding domain (UBD) from yeast endonuclease protein Cue2, using a combination of single-molecule force spectroscopy (SMFS) and steered molecular dynamics (SMD) simulations. Our study reveals that CUE2-1 does not alter the mechanical properties of ubiquitin, despite directly interacting with its β-clamp. To explore the role of ligand binding site, we compare the mechanical properties of the ubiquitin/CUE2-1 complex with that of previously studied SUMO1/S12, another β-grasp protein complex, using SMD simulations. Simulations on the SUMO1/S12 complex corroborate previous experimentally observed enhancement in the mechanical stability of SUMO1, even though S12 binds away from the β-clamp. Differences in ligand binding-induced structural impact at the transition state of the two complexes explain the differences in ligand modulated protein mechanics. Contrary to previous reports, our study demonstrates that direct binding of ligands to the mechanical clamp does not necessarily alter the mechanical stability of β-grasp fold proteins. Rather, binding interactions away from the clamp can reinforce protein stability provided by the β-grasp fold. Our study highlights the importance of binding site and binding modes of ligands in modulating the mechanical stability of β-grasp fold proteins.

Protein folding can go wrong in vivo and in vitro, with significant consequences for the living c... more Protein folding can go wrong in vivo and in vitro, with significant consequences for the living cell and the pharmaceutical industry, respectively. Here we propose a general design principle for constructing small peptide-based protein-specific folding modifiers. We construct a ‘xenonucleus’, which is a pre-folded peptide that resembles the folding nucleus of a protein, and demonstrate its activity on the folding of ubiquitin. Using stopped-flow kinetics, NMR spectroscopy, Förster Resonance Energy transfer, single-molecule force measurements, and molecular dynamics simulations, we show that the ubiquitin xenonucleus can act as an effective decoy for the native folding nucleus. It can make the refolding faster by 33 ± 5% at 3 M GdnHCl. In principle, our approach provides a general method for constructing specific, genetically encodable, folding modifiers for any protein which has a well-defined contiguous folding nucleus.

Biophysical Journal, Feb 1, 2018

Calmodulin (CaM) trapping is a phenomenon where the active and inactive states of calmodulin depe... more Calmodulin (CaM) trapping is a phenomenon where the active and inactive states of calmodulin dependent protein kinase II (CaMKII) produce drastically different affinities for Ca 2þ saturated CaM. The states of CaMKII are paramount in the scheme of calcium ion signaling, which is an essential biological function whose underlying mechanism is largely unknown. Experimentally, a set of peptides modeled after CaMKII's binding domain (293-312) were created through systematic mutations of charged residues to mimic the two distinct affinity states for CaM and probe the mechanisms responsible for the observed change in kinetics. Although a model was successfully created, the observed interactions could not be explained through current protein interaction models or electrostatic steering effects. We investigate the dynamics of this experiment through the use of all atom simulations, choosing three of the mutant peptides with a length of 20 amino acids. We refer to these peptides by the 296-298 residues, specifically RRK (wildtype), RAK (1-residue mutation) and AAA (3-residue mutation), and validate our simulation findings through comparison with circular dichroism (CD) data. We demonstrate that the large side chains present in the first 6 residues of each peptide interact with each other, and that the mutation of charged residues produce global changes in sidechain conformations and disordered regions. The specific ensemble of secondary structure/rotamer conformations present in RRK, that are not observed in AAA, are essential for high affinity binding between CaMKII and CaM.

Archives of Biochemistry and Biophysics

The active sites of metalloproteins may be mimicked by designing peptides that bind to their resp... more The active sites of metalloproteins may be mimicked by designing peptides that bind to their respective metal ions. Studying the binding of protein ligands to metal ions along with the associated structural changes is important in understanding metal uptake, transport and electron transfer functions of proteins. Copper-binding metalloprotein azurin is a 128-residue electron transfer protein with a redox-active copper cofactor. Here, we report the copper-binding associated spectroscopic and structural properties of peptide loops (11 and 13 residues) from the copper-binding site of azurin. These peptides develop a β-turn upon copper-binding with a 1:1 Cu2+:peptide stoichiometry as seen in circular dichroism and exhibit electronic transitions centered at 340 nm and 540 nm. Further addition of copper develops a helical feature along with a shift in the absorption maxima to ∼360 nm and ∼580 nm at 2:1 Cu2+:peptide stoichiometry, indicating stoichiometric dependence of copper-binding geometry. Mass spectrometry indicates the copper-binding to cysteine, histidine and methionine in the peptide with 1:1 stoichiometry, and interestingly, dimerization through a disulfide linkage at 2:1 stoichiometry, as observed previously for denatured azurin. Fluorescence quenching studies on peptides with tryptophan further confirm the copper-binding induced changes in the two peptides are bi-phasic.

In this manuscript, we introduce the Cumulative Variance of Coordinate Fluctuations (CVCF) along ... more In this manuscript, we introduce the Cumulative Variance of Coordinate Fluctuations (CVCF) along atomistic MD trajectories, as a dynamical metric to examine protein dynamics and sampling convergence in MD simulations. Using model 1D and 2D PES, we first show that CVCF, which traces over the fluctuations of protein atoms as a function of sampling coordinate (time in MD simulations), captures both local and global equilibria to distinguish the underlying PES of proteins. For both model PES and protein trajectories, we compare the information content present in CVCF traces with that obtained using other measures proposed in literature to reveal conditions under which a consistent interpretation of data can be obtained. Importantly, we show that independent of convergence to either local or global equilibrium, the values and features of protein CVCF can provide a comparative assessment of the ruggedness and curvature of the underlying PES sampled by proteins along MD trajectories. Trend...

Proteins: Structure, Function, and Bioinformatics

Small ubiquitin‐related modifiers (SUMO1 and SUMO2) are ubiquitin family proteins, structurally s... more Small ubiquitin‐related modifiers (SUMO1 and SUMO2) are ubiquitin family proteins, structurally similar to ubiquitin, differing in terms of their amino acid sequence and functions. Therefore, they provide a great platform for investigating sequence‐structure‐stability‐function relationship. Here, we used chemical denaturation in comparing the folding‐unfolding pathways of the SUMO proteins with their structural homologue ubiquitin (UF45W‐pseudo wild‐type [WT] tryptophan variant) with structurally analogous tryptophan mutations (SUMO1 [S1F66W], SUMO2 [S2F62W]). Equilibrium denaturation studies report that ubiquitin is the most stable protein among the three. The observed denaturant‐dependent folding rates of SUMOs are much lower than ubiquitin and primarily exhibit a two‐state folding pathway unlike ubiquitin, which has a kinetic folding intermediate. We hypothesize that, as SUMO proteins start off as slow folders, they avoid stabilizing their folding intermediates and the presence of which might further slow‐down their folding rates. The denaturant‐dependent unfolding of ubiquitin is the fastest, followed by SUMO2, and slowest for SUMO1. However, the spontaneous unfolding rate constant is the lowest for ubiquitin (~40 times), and similar for SUMOs. This correlation between thermodynamic stability and kinetic stability is achieved by having different unfolding transition state positions with respect to the solvent‐accessible surface area, as quantified by the Tanford β u values: ubiquitin (0.42) > SUMO2 (0.20) > SUMO1 (0.16). The results presented here highlight the unique energy landscape features which help in optimizing the folding‐unfolding rates within a structurally homologous protein family.

RSC Advances

Triphala inhibits αSyn self-association by interacting with anchoring regions which are responsib... more Triphala inhibits αSyn self-association by interacting with anchoring regions which are responsible for αSyn oligomerization.

ChemPhysChem

High quantum yield, photoluminescence tunability, and sensitivity to the environment are a few di... more High quantum yield, photoluminescence tunability, and sensitivity to the environment are a few distinct trademarks that make carbon nanodots (CDs) interesting for fundamental research, with potential to replace the prevalent inorganic semiconductor quantum dots. Currently, application and fundamental understanding of CDs are constrained because it is difficult to make a quantitative comparison among different types of CDs simply because their photoluminescence properties are directly linked to their size distribution, the surface functionalization, the carbon core structures (graphitic or amorphous) and the number of defects. Herein, we report a facile one-step synthesis of mono-dispersed and highly fluorescent nanometre size CDs from a 'family' of glucose-based sugars. These CDs are stable in aqueous solutions with photoluminescence in the visible range. Our results show several common features in the family of CDs synthesized in that the fluorescence, in the visible region, is due to a weak absorption in the 300-400 nm from a heterogeneous population of fluorophores. Fluorescence quenching experiments suggest the existence of not only surface-exposed fluorophores but more importantly solvent inaccessible fluorophores present within the core of CDs. Interestingly, time-resolved fluorescence anisotropy experiments directly suggest that a fast exchange of excitation energy occurs that results in a homo-FRET based depolarization within 150 ps of excitation.

Biophysical Journal

CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) pr... more CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins are part of the adaptive immune system of bacteria and archaea. CRISPRs are stretches of DNA with two distinct characteristics: the presence of nucleotide repeats and spacers, which serve as a memory bank, enabling bacteria to recognize the invading viruses. The Cas proteins are enzymes that cut foreign DNA guided by the information encoded in CRISPRs. In 2012, it has been demonstrated that bacterial CRISPR-Cas9 can be transformed into a simple, programmable genome-editing tool. Cas9 requires two RNA molecules: a CRISPR RNA (or ''crRNA'') and another tracrRNA (or ''trans-activating crRNA'') to make its cut on a target DNA with a 20-nucleotide stretch complementary to the crRNA. Recently, Cpf1 proteins have been discovered to show comparable genome-editing capability to Cas9. Cpf1 functions through a single crRNA without an additional tracrRNA. Cpf1 is an RNA-guided endonuclease of a class II CRISPR/Cas system, which is a smaller and simpler than Cas9, and overcomes some of the CRISPR/Cas9 system limitations. We are interested in rationally modifying the CRISPR-Cpf1 system to improve its genome editing efficiency. The target efficiency and specificity of the CRISPR technology are influenced by multiple factors. Our specific objective is to first perform large-scale molecular dynamics (MD) simulations to elucidate various conformational states involved in the Cpf1 catalysis (especially those upon the binding of crRNA and DNA) and the interconversion rates between different states. We further aim to use these MD simulations to uncover the molecular mechanisms underlying the higher genome editing efficiency of certain crRNAs. We will systematically explore how the incorporation of chemically and structurally modified nucleotides in crRNAs may affect the genome editing efficiency and off-target effects and to establish comprehensive structure-activity relationships of crRNA analogs.

Scientific reports, Jan 31, 2018

Metalloproteins carry out diverse biological functions including metal transport, electron transf... more Metalloproteins carry out diverse biological functions including metal transport, electron transfer, and catalysis. At present, the influence of metal cofactors on metalloprotein stability is not well understood. Here, we report the mechanical stability and unfolding pathway of azurin, a cupredoxin family protein with β-barrel topology and type I copper-binding centre. Single-molecule force spectroscopy (SMFS) experiments reveal 2-state and 3-state unfolding pathways for apo-azurin. The intermediate in the 3-state pathway occurs at an unfolding contour length of 7.5 nm from the native state. Steered molecular dynamics (SMD) simulations show that apo-azurin unfolds via a first transition state (TS) where β2Β-β8 and β7-β8 strand pairs rupture to form the intermediate, which subsequently unfolds by the collective rupture of remaining strands. SMFS experiments on holo-azurin exhibit an additional 4-state pathway besides the 2-state and 3-state pathways. The unfolding contour length lead...

Metalloproteins carry out diverse biological functions including metal transport, electron transf... more Metalloproteins carry out diverse biological functions including metal transport, electron transfer, and catalysis. At present, the influence of metal cofactors on metalloprotein stability is not well understood. Here, we report the mechanical stability and unfolding pathway of azurin, a cupredoxin family protein with β-barrel topology and type I copper-binding centre. Single-molecule force spectroscopy (SMFS) experiments reveal 2-state and 3-state unfolding pathways for apo-azurin. The intermediate in the 3-state pathway occurs at an unfolding contour length of 7.5 nm from the native state. Steered molecular dynamics (SMD) simulations show that apo-azurin unfolds via a first transition state (TS) where β2Β–β8 and β7–β8 strand pairs rupture to form the intermediate, which subsequently unfolds by the collective rupture of remaining strands. SMFS experiments on holo-azurin exhibit an additional 4-state pathway besides the 2-state and 3-state pathways. The unfolding contour length lead...

Journal of Biological Chemistry, 2016

The most effective vaccine candidate of malaria is based on the Plasmodium falciparum circumsporo... more The most effective vaccine candidate of malaria is based on the Plasmodium falciparum circumsporozoite protein (CSP), a major surface protein implicated in the structural strength, motility, and immune evasion properties of the infective sporozoites. It is suspected that reversible conformational changes of CSP are required for infection of the mammalian host, but the detailed structure and dynamic properties of CSP remain incompletely understood, limiting our understanding of its function in the infection. Here, we report the structural and mechanical properties of the CSP studied using single-molecule force spectroscopy on several constructs, one including the central region of CSP, which is rich in NANP amino acid repeats (CSP rep), and a second consisting of a near full-length sequence without the signal and anchor hydrophobic domains (CSP ⌬HP). Our results show that the CSP rep is heterogeneous, with 40% of molecules requiring virtually no mechanical force to unfold (<10 piconewtons (pN)), suggesting that these molecules are mechanically compliant and perhaps act as entropic springs, whereas the remaining 60% are partially structured with low mechanical resistance (ϳ70 pN). CSP ⌬HP having multiple force peaks suggests specifically folded domains, with two major populations possibly indicating the open and collapsed forms. Our findings suggest that the overall low mechanical resistance of the repeat region, exposed on the outer surface of the sporozoites, combined with the flexible full-length conformations of CSP, may provide the sporozoites not only with immune evasion properties, but also with lubricating capacity required during its navigation through the mosquito and vertebrate host tissues. We anticipate that these findings would further assist in the design and development of future malarial vaccines.