Protein-bound State Research Articles

Improved Analytical Performance of an Amphiphilic Probe upon Protein Encapsulation: Spectroscopic Investigation along with Computational Rationalization.

An easily synthesizable pyrene-based amphiphilic probe (Pybpa) has been developed, which exhibited no responses with metal ions in the pure aqueous medium despite possessing a metal ion-chelating bispicolyl unit. We believe that spontaneous aggregation of Pybpa in aqueous medium makes the ion binding unit not accessible to the metal ions. However, the sensitivity and selectivity of Pybpa toward Zn2+ ions drastically improve in the presence of serum albumin protein, HSA. The differences in the microenvironment inside the protein cavity, in terms of local polarity, and conformational rigidity might be attributing factors for that. The mechanistic investigations also suggest that there might be the involvement of polar amino acid residues that take part in coordination with Zn2+ ions. Pybpa shows no detectable spectroscopic changes with Zn2+ ions in aqueous medium in the absence of HSA. However, it can effectively recognize Zn2+ ions in the protein-bound form. Moreover, the photophysical behavior of Pybpa and its zinc complex have been investigated with DFT and docking studies. Noteworthy, such an unusual sensing aspect of Zn2+ exclusively in the protein-bound state and particularly in aqueous medium is truly rare and innovative.

SHAPE to Probe RNA Structure and RNA-Protein Interactions In Vitro.

Selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) is used to distinguish between the levels of flexibility of nucleotides regulated by base pairing or protein binding. In this method, a reagent reacts with the 2' hydroxyl group to form an adduct, which is then detected by reverse transcription reaction. The number of RNA molecules with an adduct at a specific nucleotide position indicates the SHAPE reactivity of that nucleotide. Here, we describe the method for probing the structure of an RNA in a protein-free or a protein-bound state by in vitro SHAPE.

Analysis of RNA-Protein Interaction Networks Using RNP-MaP.

RNA-protein interactions regulate a myriad of biological functions through formation of ribonucleoprotein complexes. These complexes may consist of one or more RNA-protein interaction network(s) providing additional layers of regulatory potential to the RNA. Moreover, since the protein-binding also regulates local and global structure of the RNA by structurally remodeling the latter, it is important to correlate RNA nucleotide flexibility with the site of protein-binding. We have discussed methods for chemical probing of structure of the RNA in the protein-free and protein-bound states in the preceding chapters. In this chapter, we describe a ribonucleoprotein mutational profiling (RNP-MaP) method for probing RNA-protein interaction networks.

Molecular recognition of two endogenous hormones by the human parathyroid hormone receptor-1.

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) are two endogenous hormones recognized by PTH receptor-1 (PTH1R), a member of class B G protein- coupled receptors (GPCRs). Both PTH and PTHrP analogs including teriparatide and abaloparatide are approved drugs for osteoporosis, but they exhibit distinct pharmacology. Here we report two cryo-EM structures of human PTH1R bound to PTH and PTHrP in the G protein-bound state at resolutions of 2.62 Åand 3.25 Å, respectively. Detailed analysis of these structures uncovers both common and unique features for the agonism of PTH and PTHrP. Molecular dynamics (MD) simulation together with site-directed mutagenesis studies reveal the molecular basis of endogenous hormones recognition specificity and selectivity to PTH1R. These results provide a rational template for the clinical use of PTH and PTHrP analogs as an anabolic therapy for osteoporosis and other disorders.

Combined Pulsed Electron Double Resonance EPR and Molecular Dynamics Investigations of Calmodulin Suggest Effects of Crowding Agents on Protein Structures.

Calmodulin (CaM) is a highly dynamic Ca2+-binding protein that exhibits large conformational changes upon binding Ca2+ and target proteins. Although it is accepted that CaM exists in an equilibrium of conformational states in the absence of target protein, the physiological relevance of an elongated helical linker region in the Ca2+-replete form has been highly debated. In this study, we use PELDOR (pulsed electron–electron double resonance) EPR measurements of a doubly spin-labeled CaM variant to assess the conformational states of CaM in the apo-, Ca2+-bound, and Ca2+ plus target peptide-bound states. Our findings are consistent with a three-state conformational model of CaM, showing a semi-open apo-state, a highly extended Ca2+-replete state, and a compact target protein-bound state. Molecular dynamics simulations suggest that the presence of glycerol, and potentially other molecular crowding agents, has a profound effect on the relative stability of the different conformational states. Differing experimental conditions may explain the discrepancies in the literature regarding the observed conformational state(s) of CaM, and our PELDOR measurements show good evidence for an extended conformation of Ca2+-replete CaM similar to the one observed in early X-ray crystal structures.

Comparative Structural Dynamics of Isoforms of Helicobacter pylori Adhesin BabA Bound to Lewis b Hexasaccharide via Multiple Replica Molecular Dynamics Simulations.

BabA of Helicobacter pylori is the ABO blood group antigen-binding adhesin. Despite considerable diversity in the BabA sequence, it shows an extraordinary adaptation in attachment to mucosal layers. In the current study, multiple replica molecular dynamics simulations were conducted in a neutral aqueous solution to elucidate the conformational landscape of isoforms of BabA bound to Lewis b (Leb) hexasaccharide. In addition, we also investigated the underlying molecular mechanism of the BabA-glycan complexation using the MM/GBSA scheme. The conformational dynamics of Leb in the free and protein-bound states were also studied. The carbohydrate-binding site across the four isoforms was examined, and the conformational variability of several vital loops was observed. The cysteine–cysteine loops and the two diversity loops (DL1 and DL2) were identified to play an essential role in recognizing the glycan molecule. The flexible crown region of BabA was stabilized after association with Leb. The outward movement of the DL2 loop vanished upon ligand binding for the Spanish specialist strain (S381). Our study revealed that the S831 strain shows a stronger affinity to Leb than other strains due to an increased favorable intermolecular electrostatic contribution. Furthermore, we showed that the α1-2-linked fucose contributed most to the binding by forming several hydrogen bonds with key amino acids. Finally, we studied the effect of the acidic environment on the BabA-glycan complexation via constant pH MD simulations, which showed a reduction in the binding free energy in the acidic environment. Overall, our study provides a detailed understanding of the molecular mechanism of Leb recognition by four isoforms of H. pylori that may help the development of therapeutics targeted at inhibiting H. pylori adherence to the gastric mucosa.

Lipids and Phosphorylation Conjointly Modulate Complex Formation of β2-Adrenergic Receptor and β-arrestin2.

G protein-coupled receptors (GPCRs) are the largest class of human membrane proteins that bind extracellular ligands at their orthosteric binding pocket to transmit signals to the cell interior. Ligand binding evokes conformational changes in GPCRs that trigger the binding of intracellular interaction partners (G proteins, G protein kinases, and arrestins), which initiate diverse cellular responses. It has become increasingly evident that the preference of a GPCR for a certain intracellular interaction partner is modulated by a diverse range of factors, e.g., ligands or lipids embedding the transmembrane receptor. Here, by means of molecular dynamics simulations of the β2-adrenergic receptor and β-arrestin2, we study how membrane lipids and receptor phosphorylation regulate GPCR-arrestin complex conformation and dynamics. We find that phosphorylation drives the receptor’s intracellular loop 3 (ICL3) away from a native negatively charged membrane surface to interact with arrestin. If the receptor is embedded in a neutral membrane, the phosphorylated ICL3 attaches to the membrane surface, which widely opens the receptor core. This opening, which is similar to the opening in the G protein-bound state, weakens the binding of arrestin. The loss of binding specificity is manifested by shallower arrestin insertion into the receptor core and higher dynamics of the receptor-arrestin complex. Our results show that receptor phosphorylation and the local membrane composition cooperatively fine-tune GPCR-mediated signal transduction. Moreover, the results suggest that deeper understanding of complex GPCR regulation mechanisms is necessary to discover novel pathways of pharmacological intervention.

Seminal fluid and sperm diluent affect sperm metabolism in an insect: Evidence from NAD(P)H and flavin adenine dinucleotide autofluorescence lifetime imaging.

Sperm metabolism is fundamental to sperm motility and male fertility. Its measurement is still in its infancy, and recommendations do not exist as to whether or how to standardize laboratory procedures. Here, using the sperm of an insect, the common bedbug, Cimex lectularius, we demonstrate that standardization of sperm metabolism is required with respect to the artificial sperm storage medium and a natural medium, the seminal fluid. We used fluorescence lifetime imaging microscopy (FLIM) in combination with time-correlated single-photon counting (TCSPC) to quantify sperm metabolism based on the fluorescent properties of autofluorescent coenzymes, NAD(P)H and flavin adenine dinucleotide. Autofluorescence lifetimes (decay times) differ for the free and protein-bound state of the co-enzymes, and their relative contributions to the lifetime signal serve to characterize the metabolic state of cells. We found that artificial storage medium and seminal fluid separately, and additively, affected sperm metabolism. In a medium containing sugars and amino acids (Grace's Insect medium), sperm showed increased glycolysis compared with a commonly used storage medium, phosphate-buffered saline (PBS). Adding seminal fluid to the sperm additionally increased oxidative phosphorylation, likely reflecting increased energy production of sperm during activation. Our study provides a protocol to measure sperm metabolism independently from motility, stresses that protocol standardizations for sperm measurements should be implemented and, for the first time, demonstrates that seminal fluid alters sperm metabolism. Equivalent protocol standardizations should be imposed on metabolic investigations of human sperm samples.

Profiling selective binding to promoter CpG islands by a single-DNA mechanical footprinting assay

Profiling selective binding to promoter CpG islands by a single-DNA mechanical footprinting assay

Identification of selective Lyn inhibitors from the chemical databases through integrated molecular modelling approaches

ABSTRACT In the current study, the Asinex and ChEBI databases were virtually screened for the identification of potential Lyn protein inhibitors. Therefore, a multi-steps molecular docking study was carried out using the VSW utility tool embedded in Maestro user interface of the Schrödinger suite. On initial screening, molecules having a higher XP-docking score and binding free energy compared to Staurosporin were considered for further assessment. Based on in silico pharmacokinetic analysis and a common-feature pharmacophore mapping model developed from the Staurosporin, four molecules were proposed as promising Lyn inhibitors. The binding interactions of all proposed Lyn inhibitors revealed strong ligand efficiency in terms of energy score obtained in molecular modelling analyses. Furthermore, the dynamic behaviour of each molecule in association with the Lyn protein-bound state was assessed through an all-atoms molecular dynamics (MD) simulation study. MD simulation analyses were confirmed with notable intermolecular interactions and consistent stability for the Lyn protein-ligand complexes throughout the simulation. High negative binding free energy of identified four compounds calculated through MM-PBSA approach demonstrated a strong binding affinity towards the Lyn protein. Hence, the proposed compounds might be taken forward as potential next-generation Lyn kinase inhibitors for managing numerous Lyn associated diseases or health complications after experimental validation.

Effects of amides from pungent spices on the free and protein-bound heterocyclic amine profiles of roast beef patties by UPLC–MS/MS and multivariate statistical analysis

Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), principal component analysis (PCA) and orthogonal projection to latent structure-discriminant analysis (OPLS-DA) were used to explore the effects of the amide components (capsaicin, sanshoamide, piperine) of pungent spices widely consumed in China (chili pepper, Sichuan pepper and black pepper) on the profiles of 17 heterocyclic amines (HAs) from seven categories in roast beef patties. Four groups of HAs were detected and quantified in both free and protein-bound states: imidazopyridines (DMIP, 1,5,6-TMIP), imidazoquinolines (MeIQ), imidazoquinoxalines (IQx, 4,8-MeIQx) and β-carbolines (norharman, harman). Notably, different amounts of added capsaicin, sanshoamide and piperine had significant inhibitory effects on free DMIP, MeIQx, 4,8-DiMeIQx and harman formation in roast beef patties. Additionally, all three amide components significantly inhibited the formation of protein-bound MeIQ, 4,8-DiMeIQx, harman, norharman and MeAαC. In general, amide ingredients inhibited the formation of β-carbolines and total HAs by approximately 70%. Furthermore, the addition of 0.005% and 0.010% capsaicin suppressed imidazopyridine formation by 44% and 35%, respectively, and 0.015% capsaicin inhibited imdazoquinoxaline formation by 33%. These findings shed light on the effects of amide ingredients on the formation of free and protein-bound HAs.

Simulating protein-ligand binding with neural network potentials.

Drug molecules adopt a range of conformations both in solution and in their protein-bound state. The strain and reduced flexibility of bound drugs can partially counter the intermolecular interactions that drive protein–ligand binding. To make accurate computational predictions of drug binding affinities, computational chemists have attempted to develop efficient empirical models of these interactions, although these methods are not always reliable. Machine learning has allowed the development of highly-accurate neural-network potentials (NNPs), which are capable of predicting the stability of molecular conformations with accuracy comparable to state-of-the-art quantum chemical calculations but at a billionth of the computational cost. Here, we demonstrate that these methods can be used to represent the intramolecular forces of protein-bound drugs within molecular dynamics simulations. These simulations are shown to be capable of predicting the protein–ligand binding pose and conformational component of the absolute Gibbs energy of binding for a set of drug molecules. Notably, the conformational energy for anti-cancer drug erlotinib binding to its target was found to be considerably overestimated by a molecular mechanical model, while the NNP predicts a more moderate value. Although the ANI-1ccX NNP was not trained to describe ionic molecules, reasonable binding poses are predicted for charged ligands, but this method is not suitable for modeling charged ligands in solution.

Efficient Detection of Early Events of α-Synuclein Aggregation Using a Cysteine Specific Hybrid Scaffold.

In this study, we have designed and synthesized a new hybrid ligand (SCG) that can selectively detect cysteine in the free and protein-bound states within minutes at the subnanomolar level. Photoinduced electron transfer was responsible for the visible color change as well as a large increase in steady state fluorescence. This detection was validated by using multiple model protein systems with differing cysteine environments and spatial arrangements. SCG was able to monitor the early events of the folding/aggregation kinetics of α-synuclein, a protein involved in the pathology of Parkinson's disease. The early events consisted of conformational fluctuations between different forms of the protein and oligomer formation. SCG was found to be effective in detecting early isomers of α-syn in vitro and in live cell environments.

Colorimetric indicators for specific recognition of Cu2+ and Hg2+ in physiological media: Effect of variations of signaling unit on optical response

Easy to synthesize probes are designed using bispicolyl moiety as the receptor unit with two different signaling moieties, anthraquinone and bisindolyl. Both the compounds show ‘naked-eye’ sensing of Cu2+ and Hg2+ in ∼100% aqueous medium. Not only selectivity, the compounds also show high sensitivity towards the target metal ions, as evident by their sufficiently low detection limits. The present system can detect Cu2+ as low as 0.06 ppm and Hg2+ up to 0.03 ppm from visible color change. The sensors are found equally effective in the detection of metal ions even in the protein-bound state. The impact of the signaling unit on the sensing properties of probes having same binding units has been observed and investigated. Mechanistic investigations revealed that variations in signaling unit influence the pKa of the bispicolyl nitrogens, which subsequently controls the stoichiometry of the probe-metal ion complexes as well as their luminescence property. Additionally, high specificity and good accuracy with recovery values ranging from 98.0 to 104.0% were obtained during Cu2+/Hg2+ estimation in spiked water samples. Low cost, reusable paper strips were developed for on-site detection of the metal ions which does not require the assistance of an optical instrument.

REDOR NMR Reveals Multiple Conformers for a Protein Kinase C Ligand in a Membrane Environment.

Bryostatin 1 (henceforth bryostatin) is in clinical trials for the treatment of Alzheimer’s disease and for HIV/AIDS eradication. It is also a preclinical lead for cancer immunotherapy and other therapeutic indications. Yet nothing is known about the conformation of bryostatin bound to its protein kinase C (PKC) target in a membrane microenvironment. As a result, efforts to design more efficacious, better tolerated, or more synthetically accessible ligands have been limited to structures that do not include PKC or membrane effects known to influence PKC–ligand binding. This problem extends more generally to many membrane-associated proteins in the human proteome. Here, we use rotational-echo double-resonance (REDOR) solid-state NMR to determine the conformations of PKC modulators bound to the PKCδ-C1b domain in the presence of phospholipid vesicles. The conformationally limited PKC modulator phorbol diacetate (PDAc) is used as an initial test substrate. While unanticipated partitioning of PDAc between an immobilized protein-bound state and a mobile state in the phospholipid assembly was observed, a single conformation in the bound state was identified. In striking contrast, a bryostatin analogue (bryolog) was found to exist exclusively in a protein-bound state, but adopts a distribution of conformations as defined by three independent distance measurements. The detection of multiple PKCδ-C1b-bound bryolog conformers in a functionally relevant phospholipid complex reveals the inherent dynamic nature of cellular systems that is not captured with single-conformation static structures. These results indicate that binding, selectivity, and function of PKC modulators, as well as the design of new modulators, are best addressed using a dynamic multistate model, an analysis potentially applicable to other membrane-associated proteins.

A molecular dynamics-based algorithm for evaluating the glycosaminoglycan mimicking potential of synthetic, homogenous, sulfated small molecules.

Glycosaminoglycans (GAGs) are key natural biopolymers that exhibit a range of biological functions including growth and differentiation. Despite this multiplicity of function, natural GAG sequences have not yielded drugs because of problems of heterogeneity and synthesis. Recently, several homogenous non-saccharide glycosaminoglycan mimetics (NSGMs) have been reported as agents displaying major therapeutic promise. Yet, it remains unclear whether sulfated NSGMs structurally mimic sulfated GAGs. To address this, we developed a three-step molecular dynamics (MD)-based algorithm to compare sulfated NSGMs with GAGs. In the first step of this algorithm, parameters related to the range of conformations sampled by the two highly sulfated molecules as free entities in water were compared. The second step compared identity of binding site geometries and the final step evaluated comparable dynamics and interactions in the protein-bound state. Using a test case of interactions with fibroblast growth factor-related proteins, we show that this three-step algorithm effectively predicts the GAG structure mimicking property of NSGMs. Specifically, we show that two unique dimeric NSGMs mimic hexameric GAG sequences in the protein-bound state. In contrast, closely related monomeric and trimeric NSGMs do not mimic GAG in either the free or bound states. These results correspond well with the functional properties of NSGMs. The results show for the first time that appropriately designed sulfated NSGMs can be good structural mimetics of GAGs and the incorporation of a MD-based strategy at the NSGM library screening stage can identify promising mimetics of targeted GAG sequences.

Organic Cyanide Decorated SERS Active Nanopipettes for Quantitative Detection of Hemeproteins and Fe3+ in Single Cells.

It is challenging to develop a robust nanoprobe for real-time operational and accurate detection of heavy metals in single cells. Fe-CN coordination chemistry has been well studied to determine the structural characteristics of hemeproteins by different techniques. However, the frequently used cyanide ligands are inorganic molecules that release cyanide anion under particular conditions and cause cyanide poisoning. In the present study, organic cyanide (4-mercaptobenzonitrile, MBN) was utilized for the first time in developing a facile nanoprobe based on surface-enhanced Raman scattering (SERS) for quantitative detection of hemeproteins (oxy-Hb) and trivalent iron (Fe3+) ions. The nanoprobe prepared by coating the glass capillary tip (100 nm) with a thin gold film, which enables highly localized study in living cell system. The cyanide stretching vibration in MBN was highly sensitive and selective to Fe3+ and oxy-Hb with excellent binding affinity (Kd 0.4 pM and 0.1 nM, respectively). The high sensitivity of the nanoprobe to analyte (Fe3+) was attributed to the two adsorption conformations (-SH and -CN) of MBN to the gold surface. Therefore, MBN showed an exceptional dual-peak (2126 and 2225 cm-1) behavior. Furthermore, the special Raman peaks of cyanide in 2100-2300 cm-1 (silent region of SERS spectra) are distinguishable from other biomolecules characteristic peaks. The selective detection of Fe3+ in both free and protein-bound states in aqueous solution is achieved with 0.1 pM and 0.08 μM levels of detection limits, respectively. Furthermore, practical applicability of fabricated nanoprobe was validated by detection of free Fe3+ in pretreated living HeLa cells by direct insertion of a SERS active nanoprobe. Regarding the appropriate precision, good reproducibility (relative standard deviation, RSD 7.2-7.6%), and recyclability (retain good Raman intensity even after three renewing cycles) of the method, the developed sensing strategy on a nanopipette has potential benefits for label-free, qualitative and quantitative recognition of heavy metal ions within nanoliter volumes.

Energetics and protomer communication in the dynamical structure of S100A13 in free and protein-bound states

S100A13 is S100 family of EF-hand-containing calcium-binding protein involved in the secretion of some growth factors and pro-inflammatory cytokines lacking signal peptides. The involvement of S100A13 in cancer progression and inflammatory diseases has been reported. In this study, structures generated during atomistic molecular dynamics simulation were studied. Dynamical network analysis data revealed that native inter-protomer communication network driven principally by vdW interaction (~550 kj/mol) is altered (Receptor for advanced glycation end products (RAGE) C2- and Fibroblast growth factor (FGF)-1-bound S100A13) or completely abolished (interleukin-1 (IL1)-α- and C2A-p40Syt1-bound S100A13) in protein-bound S100A13 homodimer. Bulk water density (weighted atomic density) around exposed S100A13 homodimer surface explored tends to follow the dynamical network lead as S100A13 homodimer appeared densely solvated in C2A-p40Syt1- and IL1)-α-bound states but not in RAGE C2- and FGF-1-bound biosystems. Furthermore, projection of radius of gyration and root mean square deviation (from native structure) variables of the generated structures along the 3D-free energy surface showed anti-parallel β-sheet proximal to Ca2+-binding loops-I/II in most metastable complexes retrieved from energy minima state with strong indications for β-sheet network formation during protein binding. Interaction between S100A13 homodimer and ligand–proteins may be dictated by the strength of vdW and electrostatic interaction with possible involvement of bulk water desolvation in some complexes. All these results strongly suggest that disruption of multiprotein receptor complex can be achieved by designing specific compounds targeting a specific aspect of S100A13/protein interaction; such drugs may have clinical usefulness in blocking angiogenesis, reversing cell proliferation and attenuating inflammatory processes.

Structure analysis of free and bound states of an RNA aptamer against ribosomal protein S8 from Bacillus anthracis.

Several protein-targeted RNA aptamers have been identified for a variety of applications and although the affinities of numerous protein-aptamer complexes have been determined, the structural details of these complexes have not been widely explored. We examined the structural accommodation of an RNA aptamer that binds bacterial r-protein S8. The core of the primary binding site for S8 on helix 21 of 16S rRNA contains a pair of conserved base triples that mold the sugar-phosphate backbone to S8. The aptamer, which does not contain the conserved sequence motif, is specific for the rRNA binding site of S8. The protein-free RNA aptamer adopts a helical structure with multiple non-canonical base pairs. Surprisingly, binding of S8 leads to a dramatic change in the RNA conformation that restores the signature S8 recognition fold through a novel combination of nucleobase interactions. Nucleotides within the non-canonical core rearrange to create a G-(G-C) triple and a U-(A-U)-U quartet. Although native-like S8-RNA interactions are present in the aptamer-S8 complex, the topology of the aptamer RNA differs from that of the helix 21-S8 complex. This is the first example of an RNA aptamer that adopts substantially different secondary structures in the free and protein-bound states and highlights the remarkable plasticity of RNA secondary structure.

Role of structure and dynamics of DNA with cisplatin and oxaliplatin adducts in various sequence contexts on binding of HMGB1a

Anti-cancer drugs, such as cisplatin and oxaliplatin, covalently bind to adjacent guanine bases in DNA to form intra-strand adducts. Differential recognition of drug–DNA adducts by the protein HMGB1a has been related to the differences in efficacy of these drugs in tumours. Additionally, the bases flanking the adduct (the sequence context) also have a marked effect on HMGB1a binding affinity. We perform atomistic molecular dynamics simulations of DNA with cisplatin and oxaliplatin adducts in four sequence contexts (AGGC, CGGA, TGGA and TGGT) in the absence and presence of HMGB1a. The structure of HMGB1a-bound drug–DNA molecules is independent of sequence and drug identity, confirming that differential recognition cannot be explained by the protein-bound structure. The differences in the static and conformational dynamics of the drug–DNA structures in the absence of the protein explain some but not all trends in differential binding affinity of HMGB1a. Since the minor groove width and helical bend of all drug–DNA molecules in the unbound state are lower than the protein-bound state, HMGB1a must actively deform the DNA during binding. The thermodynamic pathway between the unbound and protein-bound states could be an additional factor in the binding affinity of HMGB1a for drug–DNA adducts in various sequence contexts.