Molecular dynamics investigation of DNA fragments bound to the anti-HIV protein SAMHD1 reveals alterations in allosteric communications.

The sterile alpha motif and histidine-aspartate domain-containing protein 1 (or SAMHD1), a human dNTP-triphosphohydrolase, contributes to HIV-1 restriction in select terminally differentiated cells of the immune system. While the prevailing hypothesis is that the catalytically active form of the protein is an allosterically triggered tetramer, whose HIV-1 restriction properties are attributed to its dNTP - triphosphohydrolase activity, it is also known to bind to ssRNA and ssDNA oligomers. A complete picture of the structure-function relationship of the enzyme is still elusive and the function corresponding to its nucleic acid binding ability is debated. In this in silico study, we investigate the stability, preference and allosteric effects of DNA oligomers bound to SAMHD1. In particular, we compare the binding of DNA and RNA oligomers of the same sequence and also consider the binding of DNA fragments with phosphorothioate bonds in the backbone. The results are compared with the canonical form with the monomers connected by GTP/dATP crossbridges. The simulations indicate that SAMHD1 dimers preferably bind to DNA and RNA oligomers compared to GTP/dATP. However, allosteric communication channels are altered in the nucleic acid acid bound complexes compared to the canonical form. All results are consistent with the hypothesis that the DNA bound form of the protein correspond to an unproductive off-pathway state where the protein is sequestered and not available for dNTP hydrolysis.

Molecular dynamics simulations shows real-time lid opening in Hsp70 chaperone.

The stress-inducible mammalian heat shock protein Hsp70 and its bacterial orthologue DnaK are highly conserved molecular chaperones and a crucial part of the machinery responsible for protein folding and homeostasis. Hsp70 is a three-domain, 70 kDa protein that cycles between an ATP-bound state in which all three domains are securely coupled into one unit and an ADP-bound state in which they are loosely attached via a flexible interdomain linker. The Hsp70 presents an alluring novel therapeutic target since it is crucial for maintaining cellular proteostasis and is particularly crucial to cancer cells. We have performed molecular dynamics simulations of the SBD (substrate binding domain) along with the Lid domain in response to experimental efforts to identify small molecule inhibitors that impair the functioning of Hsp70. Our intent has been to characterize the motion of the SBD/Lid allosteric machinery and in, addition, to identify the effect of the PET16 molecule on this motion. Interestingly, we noticed the opening of the entire Lid domain in the apo-form of the dimer. The configuration of the open structure was very different from previously published structures (PDB 4JN4) of the open and docked conformation of the ATP bound form. MD simulations revealed the Lid to be capable of far greater dynamical excursions than has been anticipated by experimental structural biology. This is of value in future drug discovery efforts targeted to modulating Hsp70 activity. The PET16 molecule appears to be weakly bound and its effect on the dynamics of the complex is yet to be elucidated.

Quantifying Disorder in a Protein by Mapping its Locally Correlated Structure and Kinetics.

Proteins under physiological conditions are inherently mobile and sample a vast array of structures. Consequently, the need arises, on the one hand, at a local level to determine the independent moving parts and their associated conformations and kinetics, and on the other hand, at a global level, to quantify the disorder in the full protein molecule. We present an approach that provides these quantities in the form of local kinetic network models, which are constructed by analyzing the molecular dynamics (MD) trajectories of the protein molecule. Entropies of independent parts of the molecule are quantified. The method outlined here, using the Trp-cage miniprotein prototype, offers a new tool to understand the dynamic structural changes that ultimately govern the functioning of a protein. The method is particularly suited to problems where there are subtle changes in the structure or dynamics at local levels, for example, due to ligand binding.

Uncovering allostery and regulation in SORCIN through molecular dynamics simulations.

Soluble resistance-related calcium-binding protein or Sorcin is an allosteric, calcium-binding Penta-EF hand (PEF) family protein implicated in multi-drug resistant cancers. Sorcin is known to bind chemotherapeutic molecules such as Doxorubicin. This study uses in-silico molecular dynamics simulations to explore the dynamics and allosteric behavior of Sorcin in the context of Ca2+ uptake and Doxorubicin binding. The results show that Ca2+ binding induces large, but reversible conformational changes in the Sorcin structure which manifest as rigid body reorientations that preserve the local secondary structure. A reciprocal allosteric handshake centered around the EF5 hand is found to be key in Sorcin dimer formation and stabilization. Binding of Doxorubicin results in rearrangement of allosteric communities which disrupts long-range allosteric information transfer from the N-terminal domain to the middle lobe. However, this binding does not result in secondary structure destabilization. Sorcin does not appear to have a distinct Ca2+ activated mode of Doxorubicin binding.Communicated by Ramaswamy H. Sarma.

Molecular dynamics simulations suggest novel allosteric modes in the Hsp70 chaperone protein.

The Hsp70 chaperone protein system is an essential component of the protein folding and homeostasis machinery in E.Coli. Hsp70 is a three domain, 70 kDa protein which functions as an allosteric system cycling between an ADP-bound state where the three domains are loosely coupled via a flexible interdomain linker and an ATP-bound state where they are tightly coupled into a single entity. The structure-function model of this protein proposes an allosteric connection between the 45 kDa Nucleotide Binding Domain (NBD) and the 25 kDa Substrate Binding Domain (SBD) and Lid Domain which operates through the inter NBD-SBD linker. X-Ray crystallography and NMR spectroscopy have provided structures of the end states of the functional cycle of this protein, bound to ADP and ATP. We have used MD simulations to study the transitions between these end states and allosteric communication in this system. Our results largely validate the experimentally derived allosteric model of function, but shed additional light on the flow of allosteric information in the SBD + Lid. Specifically, we find that the Lid domain has a double-hinged structure with the potential for greater conformational flexibility than was hitherto expected.Communicated by Ramaswamy H. Sarma.

Differentiating Wild and Apiary Honey by Elemental Profiling: a Case Study from Mangroves of Indian Sundarban.

Honey is a natural substance produced by honeybees from the nectar or secretion of flowering plants. Along with the botanical and geographical origin, several environmental factors also play a major role in determining the characteristics of honey. The aim of this study is to determine and compare the elemental concentration of various macro and trace elements in apiary and wild honeys collected from different parts of Indian Sundarbans. The elemental analysis was performed in inductively coupled plasma optical emission spectroscopy preceded by microwave digestion method. The concentrations of 19 elements (Ag, Al, As, B, Ca, Cd, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Na, Ni, Pb, Se and Zn) were investigated from thirteen locations of Indian Sundarbans. This comparative study shows in wild honey samples, the concentration of K was highest followed by Ca, Mg and Na and Zn was lowest among all. In contrast, in apiary honey samples, Ca had maximum concentration followed by K, Mg and Na and Ag had minimum among all. The elemental concentration in honey from apiary was either equal or higher than their wild counterpart. The results of the factor analysis of PCA algorithm for wild and apiary honey samples were highly variable which implies that the elements are not coming from the same origin. The concentration of element was found to be highly variable across sites and across sources of honey samples.

Cloning and immunobiochemical analyses on recombinant chymopapain allergen Cari p 2 showing pollen-fruit cross-reaction.

Papaya is reported to trigger food and respiratory allergy. Here, we identified chymopapain Cari p 2 as an allergen that can sensitize atopic individuals through fruit consumption followed by respiratory hazards through pollen exposure. Recombinant Cari p 2 displayed IgE-reactivity with 78% of papaya allergic sera. rCari p 2 also displayed allergenic activity through basophil degranulation. rCari p 2 is correctly folded and showed irreversible denaturation in the melting curve. rCari p 2 displayed IgE-cross-reactivity with homologous cysteine proteases from kiwi and pineapple. Cari p 2 transcript was also detected in papaya pulps. rCari p 2 was resistant to pepsin digestion and retained IgE-reactivity after 60 minutes of pepsin digestion. In mouse model, rCari p 2 was found to elicit inflammatory responses in the lung and gastrointestinal epithelium. Hence, Cari p 2 is a newly characterized allergen with diagnostic and immunotherapeutic potential for managing allergic disorders in papaya sensitized individuals.

Allergenicity assessment of Delonix regia pollen grain and identification of allergens by immunoproteomic approach.

BACKGROUND: Plantation of road-side avenue trees has become a major part of urbanization programme for megacity beautification and environmental management. Due to evergreen habit and vibrant flower colour, Delonix regia (Gulmohor/Flamboyant) is frequently selected as ornamental tree for plantation programme. However, D. regia pollen is related to IgE mediated allergic reactions and no allergen has been reported so far. OBJECTIVE: Measuring the prevalence of D. regia pollen sensitivity among the local atopic individuals and identifying the allergens using immunoproteomic tools. METHODS: Aerobiological study was conducted for a period of two years to record the D. regia pollen concentration in the outdoor ambient air. Clinico-immunological tests were performed on atopic individuals to check the prevalence of sensitivity against D. regia pollen. Allergens were detected in the pollen proteome, fractionated in 1D and 2D gel by IgE serology and finally identified by mass spectrometry. RESULT: In the study area D. regia pollen grains were present in ample amount in the air during May to July. About 38% of atopic individuals displayed positive Skin Prick Test (SPT) against D. regia pollen along with elevated level of specific IgE and histamine in the sera. Immunoproteomic analyses revealed the presence of 14 IgE reactive proteins in the 2D pollen proteome, of which 8 IgE reactive proteins were identified by MALDI TOF/TOF using homology driven proteomic approach. CONCLUSION: This study demonstrated pollen related allergy symptoms by D. regia and gave significant message regarding the plantation programme to avoid the unnecessary load of allergic pollen. Also, a panel of 8 allergens were identified for the first time from D. regia pollen. Detailed study of these allergens would help to design immunotherapeutic strategies for pollinosis management.

Identifying novel allergens from a common indoor mould Aspergillus ochraceus.

The increasing burden of respiratory disease is a rising concern in India. Although chronic colonisation is primarily caused by pathogenic fungi, the common environmental fungi also play an important role in developing sensitisation. This study aims to examine the allergenic potency of mycelial proteins of a common indoor fungus Aspergillus ochraceus to a selected atopic patient cohort as well as to identify the novel IgE-binding proteins through an immunoproteomic approach. 1-D and 2-D IgE specific western blot detected the IgE reactive proteins which were identified through MALDI-TOF/TOF and manual de novo peptide sequencing. The results revealed the detection of 10 cross-reactive IgE-binding proteins. Cluster analysis of 1-D immunoblot with individual patient sera identified NADP(+)-dependent glycerol dehydrogenase (GldB) homologous protein as a major allergen, which was further purified and the allergenicity was assessed. Other IgE-binding proteins showed homology with allergens like short-chain dehydrogenase, NAD-dependent mannitol dehydrogenase, and subtilisin-like serine protease. GldB purified under native conditions showed IgE reactivity amongst the selected patient cohort, which is reported for the first time in this study. The identified IgE-binding proteins can act as candidate molecules for developing hypoallergenic vaccines for designing specific immunotherapeutic techniques to fungal allergy. THE SIGNIFICANCE OF THE STUDY: Exposure to environmental fungal allergens is directly associated with promoting allergic response as well as complicating existing respiratory disease, leading to poor respiratory health. Amongst others, Aspergillus spp. contributes to the majority of the fungal derived atopic diseases. Aspergillus ochraceus is a common indoor mould in India, however, its allergenic potency was not explored till date. In this study, we establish A. ochraceus responsible to cause an allergic response to susceptible individuals and identified 10 IgE-binding proteins using an immunoproteomics approach for the first time. A. ochraceus being unsequenced, a homology-driven proteomics approach was used to identify the IgE-binding proteins which can be extended to identify proteins from other unsequenced species. The information on the IgE-binding proteins could be used as a step towards characterising them by molecular and structural methods to investigate the molecular basis of allergenicity. This will also help to enrich the existing database of allergenic proteins and pave a way towards developing therapeutic avenues.

Cloning, expression and immunological characterisation of Coc n 1, the first major allergen from Coconut pollen.

Coconut pollen has been documented to be a major contributor to the aeroallergen load in India, causing respiratory allergy in a large cohort of susceptible individuals. Here, we report the identification of the first major allergen from Coconut pollen, Coc n 1. The full-length sequence of the allergen was determined from previously identified peptides and overexpressed in E. coli. Recombinant Coc n 1 folded into a trimer and was found to possess allergenicity equivalent to its natural counterpart. Proteolytic processing of Coc n 1 led to the formation of an immunodominant ∼20 kDa C-terminal subunit and the site of cleavage was determined by amino acid microsequencing. Five linear IgE binding epitopes were predicted and mapped on the homology modelled structure of Coc n 1. Amongst three immunodominant epitopes, two were present towards the C-terminal end. Coc n 1 was found to belong to the highly diverse cupin superfamily and mimics its structure with known 7S globulin or vicilin allergens but lacks sequence similarity. Using sequence similarity networks, Coc n 1 clustered as a separate group containing unannotated cupin domain proteins and did not include known vicilin allergens except Gly m Bd 28 kDa, a Soybean major allergen. 7S globulins are major storage proteins and food allergens, but presence of such protein in pollen grains is reported for the first time. Further study on Coc n 1 may provide insights into its function in pollen grains and also in the development of immunotherapy to Coconut pollen allergy.

Purification and biochemical characterization of Hel a 6, a cross-reactive pectate lyase allergen from Sunflower (Helianthus annuus L.) pollen.

Sunflower pollen was reported to contain respiratory allergens responsible for occupational allergy and pollinosis. The present study describes the comprehensive characterization of a major sunflower allergen Hel a 6. Natural Hel a 6 was purified from sunflower pollen by anion exchange and gel filtration chromatography. Hel a 6 reacted with IgE-antibodies from 57% of 39 sunflower-sensitized patient sera suggesting it to be a major allergen. The patients were of Indian origin and suffering from pollinosis and allergic rhinitis. Hel a 6 exhibited allergenic activity by stimulating mediator release from basophils. Monomeric Hel a 6 displayed pectate lyase activity. The effect of various physicochemical parameters such as temperature, pH, and calcium ion on the functional activity of Hel a 6 revealed a stable nature of the protein. Hel a 6 was folded, and its melting curve showed reversible denaturation in which it refolded back to its native conformation from a denatured state. Hel a 6 displayed a high degree of sequence conservation with the pectate lyase allergens from related taxonomic families such as Amb a 1 (67%) and Art v 6 (57%). The IgE-cross reactivity was observed between Hel a 6 and its ragweed and mugwort homologs. The cross-reactivity was further substantiated by the mediator release when Hel a 6-sensitized effector cells were cross-stimulated with Art v 6 and Amb a 1. Several putative B cell epitopes were predicted and mapped on these 3 allergens. Two antigenic regions were found to be commonly shared by these 3 allergens, which could be crucial for cross-reactivity. In conclusion, Hel a 6 serves as a candidate molecule for diagnosis and immunotherapy for weed allergy.

Dimeric Hold States of Anti-HIV Protein SAMHD1 are Redox Tunable.

The sterile α motif and histidine-aspartate domain-containing protein 1 (or SAMHD1) is a human protein that restricts HIV-1 in select terminally differentiated cells of the immune system by acting as a triphosphohydrolase, lowering dNTP pools. The functionally active form of the protein has been reported to be a tetramer where adjacent monomers are linked by GTP-Mg+2-dNTP cross-bridges, although some studies have also suggested the existence of a dimeric form of this protein. In this in silico study, we have investigated the stability of SAMHD1 dimeric "hold states" as well as the role of intrachain disulfide bonds. We have found that dimeric-GTP bound SAMHD1 can exist as a viable meso-stable hold state with extensive motion in the C-terminal domain, which is quenched upon tetramer assembly. The redox switch comprised of residues C341, C350, and C522 was found to be linked to changes in the allosteric site, suggesting a mechanism for initiating tetramer disassembly. The disulfide state of the protein dimer (C341-S-S-C350 vs C341-S-S-C522) also plays a role in driving affinities for the allosteric dATP molecules. In sum, our results suggest a model wherein dimeric SAMHD1 exists as a "hold state" in the cytosol, ready to be activated by dATP concentrations, where the "tunability" of this activation is further regulated by the redox state of the enzyme.

Identification, cloning, and immunological studies on a major eggplant (Solanum melongena L.) allergen Sola m 1: A new member of profilin allergen family.

Eggplant or brinjal (Solanum melongena L.) is widely consumed worldwide and thought to trigger allergic reactions in sensitive individuals. So far, no molecular information is available on the allergy-eliciting components of eggplant. In this study, a 17 kDa profilin, Sola m 1, was identified from eggplant by employing an immunoproteomic approach. Based on MALDI-TOF/TOF derived sequences, the full-length cDNA of Sola m 1 was PCR amplified and then cloned. Recombinant (r) Sola m 1 was expressed in E. coli and then purified by metal affinity and gel filtration. rSola m 1 reacted with IgE-antibodies in the sera from all eggplant allergic patients. rSola m 1 also displayed allergenic activity by stimulating histamine release. rSola m 1 was monomeric, and the CD spectra revealed it to be folded with a mixture of α-helices and ß-strands. In the melting curve, rSola m 1 exhibited an irreversible denaturation where no refolding took place. Sola m 1 was found to share >80 % sequence identity with Bet v 2, which was further validated by confirming the presence of significant cross-reactivity with Bet v 2 in IgE-inhibition assay. IgE-cross reactivity was also observed between rSola m 1 and profilins from six other foods. In SGF assay, no rSola m 1-derived fragments exhibited IgE-reactivity after prolonged digestion suggesting the association of rSola m 1 with the oral allergy syndromes. Immunofluorescence localization revealed a high abundance of Sola m 1 allergen in eggplant seeds as compared to other edible parts. Taken together, Sola m 1 is the first major eggplant allergen reported in this study, which has the potential of being used as a candidate antigen in component-resolved diagnosis and immunotherapy.

Identification and biochemical characterization of Asp t 36, a new fungal allergen from Aspergillus terreus.

Aspergillus terreus is an allergenic fungus, in addition to causing infections in both humans and plants. However, the allergens in this fungus are still unknown, limiting the development of diagnostic and therapeutic strategies. We used a proteomic approach to search for allergens, identifying 16 allergens based on two-dimensional immunoblotting with A. terreus susceptible patient sera. We further characterized triose-phosphate isomerase (Asp t 36), one of the dominant IgE (IgE)-reactive proteins. The gene was cloned and expressed in Escherichia coli. Phylogenetic analysis showed Asp t 36 to be highly conserved with close similarity to the triose-phosphate isomerase protein sequence from Dermatophagoides farinae, an allergenic dust mite. We identified four immunodominant epitopes using synthetic peptides, and mapped them on a homology-based model of the tertiary structure of Asp t 36. Among these, two were found to create a continuous surface patch on the 3D structure, rendering it an IgE-binding hotspot. Biophysical analysis indicated that Asp t 36 shows similar secondary structure content and temperature sensitivity with other reported triose-phosphate isomerase allergens. In vivo studies using a murine model displayed that the recombinant Asp t 36 was able to stimulate airway inflammation, as demonstrated by an influx of eosinophils, goblet cell hyperplasia, elevated serum Igs, and induction of Th2 cytokines. Collectively, our results reveal the immunogenic property of Asp t 36, a major allergen from A. terreus, and define a new fungal allergen more broadly. This allergen could serve as a potent candidate for investigating component resolved diagnosis and immunotherapy.

Molecular characterization of a fungal cyclophilin allergen Rhi o 2 and elucidation of antigenic determinants responsible for IgE-cross-reactivity.

Cyclophilins are structurally conserved pan-allergens showing extensive cross-reactivity. So far, no precise information on cross-reactive IgE-epitopes of cyclophilins is available. Here, an 18-kDa IgE-reactive cyclophilin (Rhi o 2) was purified from Rhizopus oryzae, an indoor mold causing allergic sensitization. Based on LC-MS/MS-derived sequences of natural Rhi o 2, the full-length cDNA was cloned, and expressed as recombinant (r) allergen. Purified rRhi o 2 displayed IgE-reactivity and basophil degranulation with sera from all cyclophilin-positive patients. The melting curve of properly folded rRhi o 2 showed partial refolding after heat denaturation. The allergen displayed monomeric functional peptidyl-prolyl cis-trans isomerase (PPIase) activity. In IgE-inhibition assays, rRhi o 2 exhibited extensive cross-reactivity with various other cyclophilins reported as allergens from diverse sources including its homologous human autoantigen. By generating a series of deletion mutants, a conserved 69-residue (Asn81-Asn149) fragment at C terminus of Rhi o 2 was identified as crucial for IgE-recognition and cross-reactivity. Grafting of the Asn81-Asn149 fragment within the primary structure of yeast cyclophilin CPR1 by replacing its homologous sequence resulted in a hybrid molecule with structural folds similar to Rhi o 2. The IgE-reactivity and allergenic activity of the hybrid cyclophilin were greater than that of CPR1. Therefore, the Asn81-Asn149 fragment can be considered as the site of IgE recognition of Rhi o 2. Hence, Rhi o 2 serves as a candidate antigen for the molecular diagnosis of mold allergy, and determination of a major cross-reactive IgE-epitope has clinical potential for the design of next-generation immunotherapeutics against cyclophilin-induced allergies.

Allergenicity assessment of fungal species using immunoclinical and proteomic techniques: a study on Fusarium lateritium.

Airborne fungal spores are extensively reported as the elicitors of respiratory allergies in human. Fusarium lateritium is one such fungal species reported for eliciting significant skin prick results from India. The present study aims to analyze the allergenic potential of F. lateritium followed by the identification of allergens. The total protein of F. lateritium was subjected to 1dimensional (1D) and 2D gel electrophoresis followed by corresponding IgE-specific immunoblots. We found 8 immunoreactive bands/zones in (1D) immunoblot using 11 F. lateritium-sensitised patient sera. In 1D immunoblot, a 34 kDa band was detected in >80% of the patients and hence considered as a potential allergen of F. lateritium. Corresponding 34 kDa spot in 2D-immunoblot was analyzed by mass spectrometric analysis and identified as Glyceraldehyde 3-phosphate dehydrogenase. The identified F. lateritium allergen holds the potential to instigate vaccine development for immunotherapy of F. lateritium sensitized patients.

Pilot-Scale Study Of Human Plasma Proteomics Identifies ApoE And IL33 As Markers In Atopic Asthma.

BACKGROUND: The pathobiology of atopic asthma is complex and the symptoms similar to other respiratory diseases. As such, identification of biomarkers of atopic asthma is of prime importance for better diagnosis and control of the disease. OBJECTIVES: We sought to study the changes in plasma proteome and cytokine-expression profile across healthy and atopic asthmatics for identifying biomarkers and exploring aberrant pathways for atopic asthma. METHODS: A pilot-scale study in humans was performed to identify differentially expressed proteins in blood plasma of healthy controls (n=5) and treatment-naïve atopic asthma patients (n=5) using quantitative label-free liquid chromatography-tandem mass spectrometry proteomics and ELISA. RESULTS: Mass spectrometry-based proteomic analysis revealed ApoE to be significantly downregulated in atopic asthmatics compared to healthy volunteers. Decreased expression of ApoE in atopic asthmatics was validated by immunoblotting (50.74% decrease). Comparison with atopic asthmatics and COPD patients showed that ApoE was decreased (36.33%) in atopic asthma compared to COPD. IL33 was significantly upregulated in atopic asthmatics compared to healthy subjects (3.84-fold). CONCLUSION: ApoE was downregulated and IL33 upregulated in atopic asthma patients compared to healthy volunteers. These two proteins' profiles were distinct in atopic asthma from healthy and COPD plasma samples. Differential expression of these proteins could serve as a probable candidate for a two-protein classifier-based prognostic biomarker of atopic asthma.

Molecular dynamics investigation of a redox switch in the anti-HIV protein SAMHD1.

HIV-1 is restricted in macrophages and certain quiescent myeloid cells due to a "Scorched Earth" dNTP starvation strategy attributed to the sterile alpha motif and HD domain protein-SAMHD1. Active SAMHD1 tetramers are assembled by GTP-Mg+2-dNTP cross bridges and cleave the triphosphate groups of dNTPs at a K m of ~10 µM, which is consistent with dNTP concentrations in cycling cells, but far higher than the equivalent concentration in quiescent cells. Given the substantial disparity between the dNTP concentrations required to activate SAMHD1 tetramers (~10 µM) and the dNTP concentrations in noncycling cells (~10 nM), the possibility of alternate enzymatically active forms of SAMHD1, including monomers remains open. In particular, the possibility of redox regulation of such monomers is also an open question. There have been experimental studies on the regulation of SAMHD1 by Glutathione driven redox reactions recently. Therefore, in this work, we have performed all-atom molecular dynamics simulations to study the dynamics of monomeric SAMHD1 constructs in the context of the three redox-susceptible Cysteine residues and compared them to monomers assembled within a tetramer. Our results indicate that assembly into a tetramer causes ordering of the catalytic core and increased solvent accessibility of the Catalytic Site. We have also found that glutathionylation of surface exposed C522 causes long range allosteric disruptions extending into the protein core. Finally, we see evidence suggesting a transient interaction between C522 and C341. Such a disulfide linkage has been hypothesized by experimental models, but has never been observed in crystal structures before.

Uncertainty quantification for Markov state models of biomolecules constructed using rare event acceleration techniques.

Markov state models (MSMs) of biomolecular systems are often constructed using the molecular dynamics (MD) technique. Despite having very long MD trajectories, some states and pathways can be missing in the MD data, which may make the MSMs incomplete. Consequently, uncertainty quantification for the resulting MSM becomes important. Using deca-alanine as a prototype system, we demonstrate that rare-event acceleration techniques can be employed to greatly lower the MSM uncertainty with a high computational efficiency with the assumption that the rare-event acceleration technique is able to determine most pathways that are relevant to the dynamics. In particular, we explore applications of steered MD to construct MSMs. Upper and lower bounds for uncertainty in the resulting MSM are derived. Safeguards are built into our approach to handle scenarios where the rare-event acceleration technique is unable to discover some important pathways.