Nanoparticle-Protein Interaction: The Significance and Role of Protein Corona.

The physico-chemical properties of nanoparticles, as characterized under idealized laboratory conditions, have been suggested to differ significantly when studied under complex physiological environments. A major reason for this variation has been the adsorption of biomolecules (mainly proteins) on the nanoparticle surface, constituting the so-called "biomolecular corona". The formation of biomolecular corona on the nanoparticle surface has been reported to influence various nanoparticle properties viz. cellular targeting, cellular interaction, in vivo clearance, toxicity, etc. Understanding the interaction of nanoparticles with proteins upon administration in vivo thus becomes important for the development of effective nanotechnology-based platforms for biomedical applications. In this chapter, we describe the formation of protein corona on nanoparticles and the differences arising in its composition due to variations in nanoparticle properties. Also discussed is the influence of protein corona on various nanoparticle activities.

Phylogenetic sequence analysis and functional studies reveal compensatory amino acid substitutions in loop 2 of human ribonucleotide reductase.

Eukaryotic class I ribonucleotide reductases (RRs) generate deoxyribonucleotides for DNA synthesis. Binding of dNTP effectors is coupled to the formation of active dimers and induces conformational changes in a short loop (loop 2) to regulate RR specificity among its nucleoside diphosphate substrates. Moreover, ATP and dATP bind at an additional allosteric site 40 Å away from loop 2 and thereby drive formation of activated or inactive hexamers, respectively. To better understand how dNTP binding influences specificity, activity, and oligomerization of human RR, we aligned >300 eukaryotic RR sequences to examine natural sequence variation in loop 2. We found that most amino acids in eukaryotic loop 2 were nearly invariant in this sample; however, two positions co-varied as nonconservative substitutions (N291G and P294K; human numbering). We also found that the individual N291G and P294K substitutions in human RR additively affect substrate specificity. The P294K substitution significantly impaired effector-induced oligomerization required for enzyme activity, and oligomerization was rescued in the N291G/P294K enzyme. None of the other mutants exhibited altered ATP-mediated hexamerization; however, certain combinations of loop 2 mutations and dNTP effectors perturbed ATP's role as an allosteric activator. Our results demonstrate that the observed compensatory covariation of amino acids in eukaryotic loop 2 is essential for its role in dNTP-induced dimerization. In contrast, defects in substrate specificity are not rescued in the double mutant, implying that functional sequence variation elsewhere in the protein is necessary. These findings yield insight into loop 2's roles in regulating RR specificity, allostery, and oligomerization.

Potent competitive inhibition of human ribonucleotide reductase by a nonnucleoside small molecule.

Human ribonucleotide reductase (hRR) is crucial for DNA replication and maintenance of a balanced dNTP pool, and is an established cancer target. Nucleoside analogs such as gemcitabine diphosphate and clofarabine nucleotides target the large subunit (hRRM1) of hRR. These drugs have a poor therapeutic index due to toxicity caused by additional effects, including DNA chain termination. The discovery of nonnucleoside, reversible, small-molecule inhibitors with greater specificity against hRRM1 is a key step in the development of more effective treatments for cancer. Here, we report the identification and characterization of a unique nonnucleoside small-molecule hRR inhibitor, naphthyl salicylic acyl hydrazone (NSAH), using virtual screening, binding affinity, inhibition, and cell toxicity assays. NSAH binds to hRRM1 with an apparent dissociation constant of 37 µM, and steady-state kinetics reveal a competitive mode of inhibition. A 2.66-Å resolution crystal structure of NSAH in complex with hRRM1 demonstrates that NSAH functions by binding at the catalytic site (C-site) where it makes both common and unique contacts with the enzyme compared with NDP substrates. Importantly, the IC50 for NSAH is within twofold of gemcitabine for growth inhibition of multiple cancer cell lines, while demonstrating little cytotoxicity against normal mobilized peripheral blood progenitor cells. NSAH depresses dGTP and dATP levels in the dNTP pool causing S-phase arrest, providing evidence for RR inhibition in cells. This report of a nonnucleoside reversible inhibitor binding at the catalytic site of hRRM1 provides a starting point for the design of a unique class of hRR inhibitors.

Nucleoside Analogue Triphosphates Allosterically Regulate Human Ribonucleotide Reductase and Identify Chemical Determinants That Drive Substrate Specificity.

Class I ribonucleotide reductase (RR) maintains balanced pools of deoxyribonucleotide substrates for DNA replication by converting ribonucleoside diphosphates (NDPs) to 2'-deoxyribonucleoside diphosphates (dNDPs). Binding of deoxynucleoside triphosphate (dNTP) effectors (ATP/dATP, dGTP, and dTTP) modulates the specificity of class I RR for CDP, UDP, ADP, and GDP substrates. Crystal structures of bacterial and eukaryotic RRs show that dNTP effectors and NDP substrates bind on either side of a flexible nine-amino acid loop (loop 2). Interactions with the effector nucleobase alter loop 2 geometry, resulting in changes in specificity among the four NDP substrates of RR. However, the functional groups proposed to drive specificity remain untested. Here, we use deoxynucleoside analogue triphosphates to determine the nucleobase functional groups that drive human RR (hRR) specificity. The results demonstrate that the 5-methyl, O4, and N3 groups of dTTP contribute to specificity for GDP. The O6 and protonated N1 of dGTP direct specificity for ADP. In contrast, the unprotonated N1 of adenosine is the primary determinant of ATP/dATP-directed specificity for CDP. Structural models from X-ray crystallography of eukaryotic RR suggest that the side chain of D287 in loop 2 is involved in binding of dGTP and dTTP, but not dATP/ATP. This feature is consistent with experimental results showing that a D287A mutant of hRR is deficient in allosteric regulation by dGTP and dTTP, but not ATP/dATP. Together, these data define the effector functional groups that are the drivers of human RR specificity and provide constraints for evaluating models of allosteric regulation.

Inhibition of yeast ribonucleotide reductase by Sml1 depends on the allosteric state of the enzyme.

Sml1 is an intrinsically disordered protein inhibitor of Saccharomyces cerevisiae ribonucleotide reductase (ScRR1), but its inhibition mechanism is poorly understood. RR reduces ribonucleoside diphosphates to their deoxy forms, and balances the nucleotide pool. Multiple turnover kinetics show that Sml1 inhibition of dGTP/ADP- and ATP/CDP-bound ScRR follows a mixed inhibition mechanism. However, Sml1 cooperatively binds to the ES complex in the dGTP/ADP form, whereas with ATP/CDP, Sml1 binds weakly and noncooperatively. Gel filtration and mutagenesis studies indicate that Sml1 does not alter the oligomerization equilibrium and the CXXC motif is not involved in the inhibition. The data suggest that Sml1 is an allosteric inhibitor.

Identification of Non-nucleoside Human Ribonucleotide Reductase Modulators.

Ribonucleotide reductase (RR) catalyzes the rate-limiting step of dNTP synthesis and is an established cancer target. Drugs targeting RR are mainly nucleoside in nature. In this study, we sought to identify non-nucleoside small-molecule inhibitors of RR. Using virtual screening, binding affinity, inhibition, and cell toxicity, we have discovered a class of small molecules that alter the equilibrium of inactive hexamers of RR, leading to its inhibition. Several unique chemical categories, including a phthalimide derivative, show micromolar IC50s and KDs while demonstrating cytotoxicity. A crystal structure of an active phthalimide binding at the targeted interface supports the noncompetitive mode of inhibition determined by kinetic studies. Furthermore, the phthalimide shifts the equilibrium from dimer to hexamer. Together, these data identify several novel non-nucleoside inhibitors of human RR which act by stabilizing the inactive form of the enzyme.

The structural basis for the allosteric regulation of ribonucleotide reductase.

Ribonucleotide reductases (RRs) catalyze a crucial step of de novo DNA synthesis by converting ribonucleoside diphosphates to deoxyribonucleoside diphosphates. Tight control of the dNTP pool is essential for cellular homeostasis. The activity of the enzyme is tightly regulated at the S-phase by allosteric regulation. Recent structural studies by our group and others provided the molecular basis for understanding how RR recognizes substrates, how it interacts with chemotherapeutic agents, and how it is regulated by its allosteric regulators ATP and dATP. This review discusses the molecular basis of allosteric regulation and substrate recognition of RR, and particularly the discovery that subunit oligomerization is an important prerequisite step in enzyme inhibition.

Evaluating the therapeutic potential of a non-natural nucleotide that inhibits human ribonucleotide reductase.

Human ribonucleotide reductase (hRR) is the key enzyme involved in de novo dNTP synthesis and thus represents an important therapeutic target against hyperproliferative diseases, most notably cancer. The purpose of this study was to evaluate the ability of non-natural indolyl-2'-deoxynucleoside triphosphates to inhibit the activity of hRR. The structural similarities of these analogues with dATP predicted that they would inhibit hRR activity by binding to its allosteric sites. In silico analysis and in vitro characterization identified one particular analogue designated as 5-nitro-indolyl-2'-deoxyribose triphosphate (5-NITP) that inhibits hRR. 5-NITP binding to hRR was determined by isothermal titration calorimetry. X-ray crystal structure of 5-NITP bound to RR1 was determined. Cell-based studies showed the anti-cancer effects of the corresponding non-natural nucleoside against leukemia cells. 5-NITP binds to hRR with micromolar affinity. Binding does not induce hexamerization of hRR1 like dATP, the native allosteric inhibitor of hRR that binds with high affinity to the A-site. The X-ray crystal structure of Saccharomyces cerevisiae RR1-5-NITP (ScRR1-5-NITP) complex determined to 2.3 Å resolution shows that 5-NITP does not bind to the A-site but rather at the S-site. Regardless, 5-nitro-indolyl-2'-deoxynucleoside (5-NIdR) produces cytostatic and cytotoxic effects against human leukemia cells by altering cell-cycle progression. Our studies provide useful insights toward developing new inhibitors with improved potency and efficacy against hRR.

Role of arginine 293 and glutamine 288 in communication between catalytic and allosteric sites in yeast ribonucleotide reductase.

Ribonucleotide reductases (RRs) catalyze the rate-limiting step of de novo deoxynucleotide (dNTP) synthesis. Eukaryotic RRs consist of two proteins, RR1 (α) that contains the catalytic site and RR2 (ß) that houses a diferric-tyrosyl radical essential for ribonucleoside diphosphate reduction. Biochemical analysis has been combined with isothermal titration calorimetry (ITC), X-ray crystallography and yeast genetics to elucidate the roles of two loop 2 mutations R293A and Q288A in Saccharomyces cerevisiae RR1 (ScRR1). These mutations, R293A and Q288A, cause lethality and severe S phase defects, respectively, in cells that use ScRR1 as the sole source of RR1 activity. Compared to the wild-type enzyme activity, R293A and Q288A mutants show 4% and 15%, respectively, for ADP reduction, whereas they are 20% and 23%, respectively, for CDP reduction. ITC data showed that R293A ScRR1 is unable to bind ADP and binds CDP with 2-fold lower affinity compared to wild-type ScRR1. With the Q288A ScRR1 mutant, there is a 6-fold loss of affinity for ADP binding and a 2-fold loss of affinity for CDP compared to the wild type. X-ray structures of R293A ScRR1 complexed with dGTP and AMPPNP-CDP [AMPPNP, adenosine 5-(ß,γ-imido)triphosphate tetralithium salt] reveal that ADP is not bound at the catalytic site, and CDP binds farther from the catalytic site compared to wild type. Our in vivo functional analyses demonstrated that R293A cannot support mitotic growth, whereas Q288A can, albeit with a severe S phase defect. Taken together, our structure, activity, ITC and in vivo data reveal that the arginine 293 and glutamine 288 residues of ScRR1 are crucial in facilitating ADP and CDP substrate selection.

X-ray crystallography and isothermal titration calorimetry studies of the Salmonella zinc transporter ZntB.

The ZntB Zn(2+) efflux system is important for maintenance of Zn(2+) homeostasis in Enterobacteria. We report crystal structures of ZntB cytoplasmic domains from Salmonella enterica serovar Typhimurium (StZntB) in dimeric and physiologically relevant homopentameric forms at 2.3 Å and 3.1 Å resolutions, respectively. The funnel-like structure is similar to that of the homologous Thermotoga maritima CorA Mg(2+) channel and a Vibrio parahaemolyticus ZntB (VpZntB) soluble domain structure. However, the central α7 helix forming the inner wall of the StZntB funnel is oriented perpendicular to the membrane instead of the marked angle seen in CorA or VpZntB. Consequently, the StZntB funnel pore is cylindrical, not tapered, which may represent an "open" form of the ZntB soluble domain. Our crystal structures and isothermal titration calorimetry data indicate that there are three Zn(2+) binding sites in the full-length ZntB, two of which could be involved in Zn(2+) transport.

Histidine hydrogen-deuterium exchange mass spectrometry for probing the microenvironment of histidine residues in dihydrofolate reductase.

BACKGROUND: Histidine Hydrogen-Deuterium Exchange Mass Spectrometry (His-HDX-MS) determines the HDX rates at the imidazole C(2)-hydrogen of histidine residues. This method provides not only the HDX rates but also the pK(a) values of histidine imidazole rings. His-HDX-MS was used to probe the microenvironment of histidine residues of E. coli dihydrofolate reductase (DHFR), an enzyme proposed to undergo multiple conformational changes during catalysis. METHODOLOGY/PRINCIPAL FINDINGS: Using His-HDX-MS, the pK(a) values and the half-lives (t(1/2)) of HDX reactions of five histidine residues of apo-DHFR, DHFR in complex with methotrexate (DHFR-MTX), DHFR in complex with MTX and NADPH (DHFR-MTX-NADPH), and DHFR in complex with folate and NADP+ (DHFR-folate-NADP+) were determined. The results showed that the two parameters (pK(a) and t(1/2)) are sensitive to the changes of the microenvironment around the histidine residues. Although four of the five histidine residues are located far from the active site, ligand binding affected their pK(a), t(1/2) or both. This is consistent with previous observations of ligand binding-induced distal conformational changes on DHFR. Most of the observed pK(a) and t(1/2) changes could be rationalized using the X-ray structures of apo-DHFR, DHFR-MTX-NADPH, and DHFR-folate-NADP+. The availability of the neutron diffraction structure of DHFR-MTX enabled us to compare the protonation states of histidine imidazole rings. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate the usefulness of His-HDX-MS in probing the microenvironments of histidine residues within proteins.

Structural basis for allosteric regulation of human ribonucleotide reductase by nucleotide-induced oligomerization.

Ribonucleotide reductase (RR) is an α(n)ß(n) (RR1-RR2) complex that maintains balanced dNTP pools by reducing NDPs to dNDPs. RR1 is the catalytic subunit, and RR2 houses the free radical required for catalysis. RR is allosterically regulated by its activator ATP and its inhibitor dATP, which regulate RR activity by inducing oligomerization of RR1. Here, we report the first X-ray structures of human RR1 bound to TTP alone, dATP alone, TTP-GDP, TTP-ATP, and TTP-dATP. These structures provide insights into regulation of RR by ATP or dATP. At physiological dATP concentrations, RR1 forms inactive hexamers. We determined the first X-ray structure of the RR1-dATP hexamer and used single-particle electron microscopy to visualize the α(6)-ßß'-dATP holocomplex. Site-directed mutagenesis and functional assays confirm that hexamerization is a prerequisite for inhibition by dATP. Our data indicate a mechanism for regulating RR activity by dATP-induced oligomerization.

Targeting the Large Subunit of Human Ribonucleotide Reductase for Cancer Chemotherapy.

Ribonucleotide reductase (RR) is a crucial enzyme in de novo DNA synthesis, where it catalyses the rate determining step of dNTP synthesis. RRs consist of a large subunit called RR1 (α), that contains two allosteric sites and one catalytic site, and a small subunit called RR2 (ß), which houses a tyrosyl free radical essential for initiating catalysis. The active form of mammalian RR is an α(n)ß(m) hetero oligomer. RR inhibitors are cytotoxic to proliferating cancer cells. In this brief review we will discuss the three classes of RR, the catalytic mechanism of RR, the regulation of the dNTP pool, the substrate selection, the allosteric activation, inactivation by ATP and dATP, and the nucleoside drugs that target RR. We will also discuss possible strategies for developing a new class of drugs that disrupts the RR assembly.

X-ray structure of the ternary MTX.NADPH complex of the anthrax dihydrofolate reductase: a pharmacophore for dual-site inhibitor design.

For reasons of bioterrorism and drug resistance, it is imperative to identify and develop new molecular points of intervention against anthrax. Dihydrofolate reductase (DHFR) is a highly conserved enzyme and an established target in a number of species for a variety of chemotherapeutic programs. Recently, the crystal structure of Bacillus anthracis DHFR (baDHFR) in complex with methotrexate (MTX) was determined and, based on the structure, proposals were made for drug design strategies directed against the substrate-binding site. However, little is gleaned about the binding site for NADPH, the cofactor responsible for hydride transfer in the catalytic mechanism. In the present study, X-ray crystallography at 100 K was used to determine the structure of baDHFR in complex with MTX and NADPH. Although the NADPH binding mode is nearly identical to that seen in other DHFR ternary complex structures, the adenine moiety adopts an off-plane tilt of nearly 90 degrees and this orientation is stabilized by hydrogen bonds to functionally conserved Arg residues. A comparison of the binding site, focusing on this region, between baDHFR and the human enzyme is discussed, with an aim at designing species-selective therapeutics. Indeed, the ternary model, refined to 2.3 A resolution, provides an accurate template for testing the feasibility of identifying dual-site inhibitors, compounds that target both the substrate and cofactor-binding site. With the ternary model in hand, using in silico methods, several compounds were identified which could potentially form key bonding contacts in the substrate and cofactor-binding sites. Ultimately, two structurally distinct compounds were verified that inhibit baDHFR at low microM concentrations. The apparent Kd for one of these, (2-(3-(2-(hydroxyimino)-2-(pyridine-4-yl)-6,7-dimethylquinoxalin-2-yl)-1-(pyridine-4-yl)ethanone oxime), was measured by fluorescence spectroscopy to be 5.3 microM.

Selective Cu2+ binding, redox silencing, and cytoprotective effects of the small heat shock proteins alphaA- and alphaB-crystallin.

Oxidative stress and Cu(2+) have been implicated in several neurodegenerative diseases and in cataract. Oxidative stress, as well as Cu(2+), is also known to induce the expression of the small heat shock proteins alpha-crystallins. However, the role of alpha-crystallins in oxidative stress and in Cu(2+)-mediated processes is not clearly understood. We demonstrate using fluorescence and isothermal titration calorimetry that alpha-crystallins (alphaA- and alphaB-crystallin and its phosphorylation mimic, 3DalphaB-crystallin) bind Cu(2+) with close to picomolar range affinity. The presence of other tested divalent cations such as Zn(2+), Mg(2+), and Ca(2+) does not affect Cu(2+) binding, indicating selectivity of the Cu(2+)-binding site(s) in alpha-crystallins. Cu(2+) binding induces structural changes and increase in the hydrodynamic radii of alpha-crystallins. Cu(2+) binding increases the stability of alpha-crystallins towards guanidinium chloride-induced unfolding. Chaperone activity of alphaA-crystallin increases significantly upon Cu(2+) binding. Alpha-crystallins rescue amyloid beta peptide, Abeta(1-40), from Cu(2+)-induced aggregation in vitro. Alpha-crystallins inhibit Cu(2+)-induced oxidation of ascorbate and, hence, prevent the generation of reactive oxygen species. Interestingly, alpha-synuclein, a Cu(2+)-binding protein, does not inhibit this oxidation process significantly. We find that the Cu(2+)-sequestering (or redox-silencing) property of alpha-crystallins confers cytoprotection. To the best of our knowledge, this is the first study to reveal high affinity (close to picomolar) for Cu(2+) binding and redox silencing of Cu(2+) by any heat shock protein. Thus, our study ascribes a novel functional role to alpha-crystallins in Cu(2+) homeostasis and helps in understanding their protective role in neurodegenerative diseases and cataract.

Effect of phosphorylation on alpha B-crystallin: differences in stability, subunit exchange and chaperone activity of homo and mixed oligomers of alpha B-crystallin and its phosphorylation-mimicking mutant.

Phosphorylation appears to be one of the modulators of chaperone functions of small heat shock proteins. However, the role of phosphorylation is not completely understood. We have investigated the structural and functional consequences of a phosphorylation-mimicking mutation in alpha B-crystallin, a small heat shock protein with chaperone activity. We have used a phosphorylation-mimicking mutant, 3D alpha B-crystallin, in which all the three phosphorylatable serine residues are replaced with aspartic acid. 3D alpha B-Crystallin showed enhanced chaperone-like activity towards DTT-induced aggregation of insulin, heat-induced aggregation of citrate synthase and SDS-induced amyloid fibril formation of alpha-synuclein. Fluorescence and circular dichroism spectroscopic studies showed that 3D alpha B-crystallin exhibits lower stability towards urea-induced denaturation compared to alpha B-crystallin. Subunit exchange studies using fluorescence resonance energy transfer showed that 3D alpha B-crystallin exhibits an observable increase in subunit exchange compared to alpha B-crystallin. Since only part of alpha B-crystallin is phosphorylated in vivo, our subunit exchange studies indicate that formation of mixed oligomers between the unphosphorylated and phosphorylated subunits are likely to play a role in vivo. Our study shows that mixed-oligomer formation modulates the chaperone-like activity. We propose that the degree of phosphorylation of the alpha B-crystallin oligomers and temperature are key modulators to achieve a wide range of chaperone capabilities of the small heat shock protein, alpha -crystallin.

Fibrillogenic and non-fibrillogenic ensembles of SDS-bound human alpha-synuclein.

Fibril formation of alpha-synuclein is associated with several neurodegenerative diseases, including Parkinson's disease in humans. The anionic detergent sodium dodecyl sulfate (SDS) can accelerate the fibril formation in vitro. However, the molecular basis of this acceleration is not clear. Our study shows that native alpha-synuclein exhibits relatively less fibril growth despite providing fibril seeds for nucleation. The presence of SDS promotes the seeded fibril growth in a concentration-dependent manner, with an optimal concentration of 0.5-0.75 mM. We used isothermal calorimetry, hydrophobic dye binding and circular dichroism spectroscopy to characterize the protein-detergent interactions as a function of the concentration of SDS. Interaction of SDS with alpha-synuclein when studied by isothermal titration calorimetry and hydrophobic dye-binding reveals a similar characteristic optimal behavior between 0.5 mM and 0.75 mM SDS. The study shows two types of ensembles of alpha-synuclein and SDS: the fibrillogenic ensembles formed with optimal concentration of SDS around 0.5-0.75 mM are characterized by enhanced accessible hydrophobic surfaces and extended to partially helical conformation, while the less or non-fibrillogenic ensembles formed above 2 mM SDS are characterized by less accessible hydrophobic surfaces and maximal helical content. Little or no fibrillogenicity of the ensembles observed above 2 mM SDS could be partly because of the observed intrinsic instability of the fibrils under the condition.

The acid-induced state of glucose oxidase exists as a compact folded intermediate.

A systematic investigation of the acid-induced unfolding of glucose oxidase (beta-D-glucose: oxygen 1-oxidoreductase) (GOD) from Aspergillus niger was made using steady-state tryptophan fluorescence, circular dichroism (CD), and ANS (1-anilino 8-naphthalene sulfonic acid) binding. Intrinsic tryptophan fluorescence studies showed a maximally unfolded state at pH 2.6 and the presence of a non-native intermediate in the vicinity of pH 1.4. Flavin adenine dinucleotide (FAD) fluorescence measurements indicate that the bound cofactors are released at low pH. In the pH range studied, near- and far-UV CD spectra show maximal loss of tertiary as well as secondary structure (40%) at pH 2.6 although glucose oxidase at this pH is relatively less denatured as compared to the conformation in 6M GdnHCl. Interestingly, in the vicinity of pH 1.4, glucose oxidase shows a refolded conformation (A-state) with approximately 90% of native secondary structure and native-like near-UV CD spectral features. ANS fluorescence studies, however, show maximal binding of the dye to the protein at pH 1.4, indicating a "molten-globule"-like conformation with enhanced exposure of hydrophobic surface area. Acrylamide quenching data exhibit reduced accessibility of quencher to tryptophan, suggesting a more compact conformation at low pH. Thermal stability of this state was assessed by ellipticity changes at 222 nm relative to native protein. While native glucose oxidase showed a completely reversible thermal denaturation profile, the state at pH 1.4 showed approximately 50% structural loss and the denatured state appeared to be in a different conformation exhibiting prominent beta-sheet structure (around 85 degrees C) that was not reversible. To summarize; the A-state of GOD exists as a compact folded intermediate with "molten-globule"-like characteristics, viz., native-like secondary structure but with non-native cofactor environment, enhanced hydrophobic surface area and non-cooperative thermal unfolding. That the A-state also possesses significant tertiary structure is an interesting observation made in this study.