ABSTRACT
The molecular association of proteins with nucleic acids leading to the formation of macromolecular complexes is a crucial step in several biological processes. Stabilization of these complexes involves electrostatic interactions between ion pairs (salt bridges) of nucleic acid phosphates and protein side chains. The crenarchaeal DNA binding protein, Cren7 plays a key role in the regulation of chromosomal structure and gene expression in eukaryotic extremophiles. However, the molecular contacts that occur at the interface of protein-DNA complexes and their contribution to the electrostatic interaction have not been fully elucidated. This work presents a quantitative description of the mechanism of the electrostatic interaction between the protein and DNA. We have identified a few residues located at the Cren7-DNA interface that could potentially be responsible for the interaction. Structural studies using circular dichroism indicate mutation of these surface residues minimally affect their structure and stability. The binding affinity of these mutants for the DNA duplexes was examined from reverse titration, biolayer interferometry, and fluorescence anisotropy measurements with calf thymus DNA, polynucleotides, and small DNA oligonucleotides. The resulting kinetic parameters highlight a difference in electrostatic interactions potentials exhibited by residues positioned at different locations of the protein-DNA interface. Computational studies attribute this difference to their surrounding atmosphere and energetic stabilization parameters. The biophysical approach described here can be extended for other proteins that play a crucial role in DNA bending and compaction, to properly evaluate the role of specific residues on the mechanisms of DNA binding.
Subject(s)
DNA-Binding Proteins , DNA , Static Electricity , DNA/chemistry , DNA-Binding Proteins/metabolism , ThermodynamicsABSTRACT
Template mediated assembly of plasmonic nanomaterials is a promising approach to induce chirality. Naturally occurring macromolecules can self-assemble to form chiral superstructures, with dimensions extending from nanometer to micrometer length scales. These structures can serve as templates for host plasmonic nanomaterials on their surface through a variety of interactions. The arrangement of nanomaterials on these structures results in a transfer of symmetry from these templates to nanomaterials, which finally generates a chiral response in circular dichroism (CD) spectroscopy. For biosensing and in vitro applications of chiral plasmonics, long-term stability of these templates will be crucial for this approach of chirality induction. Here, we have demonstrated how protein amyloid fibrils can be used as templates to generate a chiroptical response with plasmonic nanomaterials. The temperature and ionic strength of the solution were carefully altered to convert the three-dimensional protein structure into amyloid fibrils. Changes in solution conditions affected the amyloid geometry, long-term stability, and interaction with AuNRs. The modified interactions influenced the orientation of the AuNRs, which affected the intensity of the CD response. The MTT assay indicated that the chiral AuNRs exhibited considerable cell viability, making them ideal for in vivo applications.
Subject(s)
Amyloid , Gold , Nanotubes , Amyloid/chemistry , Gold/chemistry , Nanotubes/chemistryABSTRACT
Archaea have histone homologues and chromatin proteins to organize their DNA into a compact form. This allows them to survive in extreme climates. Cren7 is one such chromatin protein conserved in Crenarchaeota. When Cren7 binds to model natural DNA, calf thymus DNA (CTD, 58% AT content) and polynucleotides under adverse solution conditions (high temperature, ionic strength), CD bands at 275-290 nm shift to higher wavelengths indicating structural changes in DNA. It formed a strong complex with CTD and poly(dA-dT)·poly(dA-dT), via a combination of electrostatic and non-electrostatic interactions. A low binding enthalpy indicated that the process was driven by entropy. The interaction was independent of the nature of the anions present in the solution. On studying the variation in protein affinity with salt concentration, it was estimated that the electrostatic interaction at the interface involves 3 pairs of ions at the protein-DNA interface. The affinity and binding site size decreased on changing the pH of the solution (between pH 6 and 8), but temperature did not result in such effects. Cren7 bound to 10 bp of DNA, increasing its flexibility and thermal stability by more than 30 °C. Increasing the amount of Cren7 produces cooperative structural transitions in DNAs without any similar transition in the protein. These crucial binding parameters, energetics, and structural changes decipher the mystery of Cren7 mediated DNA organization in Crenarchaeota.
Subject(s)
Crenarchaeota , Chromatin , Crenarchaeota/metabolism , DNA/chemistry , Poly dA-dT , ThermodynamicsABSTRACT
Heat shock proteins (Hsps) stabilize the newly synthesized polypeptide chains preventing them from aggregation. They contribute to systemic response under stress and thus behave as signaling molecules. Hsp70 has been detected on the surface of stressed cells. It translocates to the extracellular environment through the plasma membrane without causing cell death. But the interaction of the protein with the membrane leading to the export process remains elusive. Hsp70 has a tendency to generate channels within lipid bilayers, and this has been a driving force for studying protein-lipid interactions. Transport of these proteins across the membrane paves their pathways for performing the desired function. We have attempted to characterize how the interaction of Hsp70 with negatively charged phospholipids affects the structure of lipids. This study will help in explaining the transport mechanism of proteins that are devoid of defined signaling pathways. The interaction of amino acids of Hsp70 with the head and tail group leads to the rearrangement of the hydration layer in contact with the bilayers. Critical analysis of the results obtained from small-angle X-ray scattering along with QCM-D provides valuable insights to analyze the effect of Hsp70 adsorption on an anionic POPS lipid bilayer.
Subject(s)
HSP70 Heat-Shock Proteins/metabolism , Phospholipids/metabolism , Cell Membrane/metabolism , HSP70 Heat-Shock Proteins/chemistry , Lipid Bilayers/chemistry , Phospholipids/chemistry , Protein Transport/physiology , Quartz Crystal Microbalance Techniques , Spectrum AnalysisABSTRACT
Biomolecules such as nucleic acids and proteins constitute the cells and its organelles that form the crucial components in all living organisms. They are associated with a variety of cellular processes during which they undergo conformational orientations. The structural rearrangements resulting from protein-protein, protein-DNA, and protein-drug interactions vary in spatial and temporal length scales. Force is one of the important key factors which regulate these interactions. The magnitude of the force can vary from sub-piconewtons to several thousands of piconewtons. Single-molecule force spectroscopy acts as a powerful tool which is capable of investigating mechanical stability and conformational rearrangements arising in biomolecules due to the above interactions. Real-time observation of conformational dynamics including access to rare or transient states and the estimation of mean dwell times using these tools aids in the kinetic analysis of these interactions. In this review, we highlight the capabilities of common force spectroscopy techniques such as optical tweezers, magnetic tweezers, and atomic force microscopy with case studies on emerging applications.
ABSTRACT
In this article, we report a simple method to synthesize biodegradable zein films functionalized with gold nanoparticles (AuNPs) with significantly improved mechanical properties, as an environmentally benign substitute to biologically hazardous polymers. Zein-coated AuNPs were synthesized using the zein protein as a reducing agent and characterized with IR, UV, CD, ζ-potential, and TEM measurements. The zein protein interaction with the negatively charged surface of AuNPs provides excellent strength to the zein thin film. For the first time, FT-IR spectral studies suggested the strong interaction between AuNPs and zein protein, which was further supported by the higher binding constant (K b) value. The films were characterized for mechanical properties with spectroscopic and physical experimental investigations. The surface morphology of AuNP-doped zein film was explored by AFM and SEM, which suggested that the AuNPs prevent the buckling of zein film and increase the strength as well as flexibility of the film.
ABSTRACT
Owing to the cooperativity of protein structures, it is often almost impossible to identify independent subunits, flexible regions, or hinges simply by visual inspection of static snapshots. Here, we use single-molecule force experiments and simulations to apply tension across the substrate binding domain (SBD) of heat shock protein 70 (Hsp70) to pinpoint mechanical units and flexible hinges. The SBD consists of two nanomechanical units matching 3D structural parts, called the α- and ß-subdomain. We identified a flexible region within the rigid ß-subdomain that gives way under load, thus opening up the α/ß interface. In exactly this region, structural changes occur in the ATP-induced opening of Hsp70 to allow substrate exchange. Our results show that the SBD's ability to undergo large conformational changes is already encoded by passive mechanics of the individual elements.
Subject(s)
Adenosine Triphosphate/metabolism , HSP70 Heat-Shock Proteins/chemistry , HSP70 Heat-Shock Proteins/metabolism , Allosteric Regulation , Allosteric Site , Amino Acid Sequence , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , HSP70 Heat-Shock Proteins/ultrastructure , Kinetics , Microscopy, Atomic Force/methods , Molecular Dynamics Simulation , Protein Binding , Protein Conformation , Protein Domains , Substrate SpecificityABSTRACT
Spontaneous folding of a polypeptide chain into a knotted structure remains one of the most puzzling and fascinating features of protein folding. The folding of knotted proteins is on the timescale of minutes and thus hard to reproduce with atomistic simulations that have been able to reproduce features of ultrafast folding in great detail. Furthermore, it is generally not possible to control the topology of the unfolded state. Single-molecule force spectroscopy is an ideal tool for overcoming this problem: by variation of pulling directions, we controlled the knotting topology of the unfolded state of the 52-knotted protein ubiquitin C-terminal hydrolase isoenzyme L1 (UCH-L1) and have therefore been able to quantify the influence of knotting on its folding rate. Here, we provide direct evidence that a threading event associated with formation of either a 31 or 52 knot, or a step closely associated with it, significantly slows down the folding of UCH-L1. The results of the optical tweezers experiments highlight the complex nature of the folding pathway, many additional intermediate structures being detected that cannot be resolved by intrinsic fluorescence. Mechanical stretching of knotted proteins is also of importance for understanding the possible implications of knots in proteins for cellular degradation. Compared with a simple 31 knot, we measure a significantly larger size for the 52 knot in the unfolded state that can be further tightened with higher forces. Our results highlight the potential difficulties in degrading a 52 knot compared with a 31 knot.
Subject(s)
Protein Refolding , Protein Unfolding , Ubiquitin Thiolesterase/chemistry , Optical Tweezers , Single Molecule ImagingABSTRACT
The use of titania nanotubes (TiO2-NT) as the working electrode provides a substantial improvement in the electrochemical detection of proteins. A biosensor designed using this strategy provided a robust method to detect protein samples at very low concentrations (Cprotein ca 1ng/µl). Reproducible measurements on protein samples at this concentration (Ip,a of 80+1.2µA) could be achieved using a sample volume of ca 30µl. We demonstrate the feasibility of this strategy for the accurate detection of penicillin binding protein, PBP2a, a marker for methicillin resistant Staphylococcus aureus (MRSA). The selectivity and efficiency of this sensor were also validated using other diverse protein preparations such as a recombinant protein tyrosine phosphatase (PTP10D) and bovine serum albumin (BSA). This electrochemical method also presents a substantial improvement in the time taken (few minutes) when compared to conventional enzyme-linked immunosorbent assay (ELISA) protocols. It is envisaged that this sensor could substantially aid in the rapid diagnosis of bacterial infections in resource strapped environments.
Subject(s)
Biosensing Techniques/instrumentation , Carbon/chemistry , Nanotubes/chemistry , Proteins/analysis , Titanium/chemistry , Biomarkers/analysis , Biosensing Techniques/methods , Electrochemical Techniques , Electrodes , Feasibility Studies , Methicillin-Resistant Staphylococcus aureus/isolation & purification , Penicillin-Binding Proteins/analysis , Reproducibility of Results , Sensitivity and Specificity , Time FactorsABSTRACT
In addition to the chemical nature of the surface, the dimensions of the confining host exert a significant influence on confined protein structures; this results in immense biological implications, especially those concerning the enzymatic activities of the protein. This study probes the structure of hemoglobin (Hb), a model protein, confined inside silica tubes with pore diameters that vary by one order of magnitude (≈20-200 nm). The effect of confinement on the protein structure is probed by comparison with the structure of the protein in solution. Small-angle neutron scattering (SANS), which provides information on protein tertiary and quaternary structures, is employed to study the influence of the tube pore diameter on the structure and configuration of the confined protein in detail. Confinement significantly influences the structural stability of Hb and the structure depends on the Si-tube pore diameter. The high radius of gyration (Rg) and polydispersity of Hb in the 20 nm diameter Si-tube indicates that Hb undergoes a significant amount of aggregation. However, for Si-tube diameters greater or equal to 100 nm, the Rg of Hb is found to be in very close proximity to that obtained from the protein data bank (PDB) reported structure (Rg of native Hb=23.8 Å). This strongly indicates that the protein has a preference for the more native-like non-aggregated state if confined inside tubes of diameter greater or equal to 100 nm. Further insight into the Hb structure is obtained from the distance distribution function, p(r), and ab initio models calculated from the SANS patterns. These also suggest that the Si-tube size is a key parameter for protein stability and structure.
Subject(s)
Hemoglobins/chemistry , Silicon Dioxide/chemistry , Circular Dichroism , Hemoglobins/metabolism , Nanotubes/chemistry , Neutron Diffraction , Scattering, Small AngleABSTRACT
The configuration of hemoglobin in solution and confined inside silica nanotubes has been studied using synchrotron small angle X-ray scattering and electrochemical activity. Confinement inside submicron tubes of silica aid in preventing protein aggregation, which is vividly observed for unconfined protein in solution. The radius of gyration (R g) and size polydispersity (p) of confined hemoglobin was found to be lower than that in solution. This was also recently demonstrated in case of confined hemoglobin inside layered polymer capsules. The confined hemoglobin displayed a higher thermal stability with R g and p showing negligible changes in the temperature range 25-75 °C. The differences in configuration between the confined and unconfined protein were reflected in their electrochemical activity. Reversible electrochemical response (from cyclic voltammograms) obtained in case of the confined hemoglobin, in contrary to the observance of only a cathodic response for the unconfined protein, gave direct indication of the differences between the residences of the electroactive heme center in a different orientation compared to that in solution state. The confined Hb showed loss of reversibility only at higher temperatures. The electron transfer coefficient (α) and electron transfer rate constant (k s) were also different, providing additional evidence regarding structural differences between the unconfined and confined states of hemoglobin. Thus, absence of any adverse effects due to confinement of proteins inside the inorganic matrices such as silica nanotubes opens up new prospects for utilizing inorganic matrices as protein "encapsulators", as well as sensors at varying temperatures.
Subject(s)
Hemoglobins/chemistry , Nanotubes/chemistry , Scattering, Small Angle , Silicon Dioxide/chemistry , Synchrotrons , X-Ray Diffraction/instrumentation , Electrochemistry , Humans , TemperatureABSTRACT
An alternative antibody-free strategy for the rapid electrochemical detection of cardiac myoglobin has been demonstrated here using hydrothermally synthesized TiO2 nanotubes (Ti-NT). The denaturant induced unfolding of myoglobin led to easy access of the deeply buried electroactive heme center and thus the efficient reversible electron transfer from protein to electrode surface. The sensing performance of the Ti-NT modified electrodes were compared vis a vis commercially available titania and GCEs. The tubular morphology of the Ti-NT led to facile transfer of electrons to the electrode surface, which eventually provided a linear current response (obtained from cyclic voltammetry) over a wide range of Mb concentration. The sensitivity of the Ti-NT based sensor was remarkable and was equal to 18 µA mg-1 ml (detection limit = 50 nM). This coupled with the rapid analysis time of a few tens of minutes (compared to a few days for ELISA) demonstrates its potential usefulness for the early detection of acute myocardial infarction (AMI).
ABSTRACT
The effect of confinement on the structure of hemoglobin (Hb) within polymer capsules was investigated here. Hemoglobin transformed from an aggregated state in solution to a nonaggregated state when confined inside the polymer capsules. This was directly confirmed using synchrotron small-angle X-ray scattering (SAXS) studies. The radius of gyration (R(g)) and polydispersity (p) of the proteins in the confined state were smaller compared to those in solution. In fact, the R(g) value is very similar to theoretical values obtained using protein structures generated from the Protein Databank. In the temperature range (25-85 °C, Tm 59 °C), the R(g) values for the confined Hb remained constant. This observation is in contrary to the increasing R(g) values obtained for the bare Hb in solution. This suggested higher thermal stability of Hb when confined inside the polymer capsule than when in solution. Changes in protein configuration were also reflected in the protein function. Confinement resulted in a beneficial enhancement of the electroactivity of Hb. While Hb in solution showed dominance of the cathodic process (Fe(3+) â Fe(2+)), efficient reversible Fe(3+)/Fe(2+) redox response is observed in the case of the confined Hb. This has important protein functional implications. Confinement allows the electroactive heme to take up positions favorable for various biochemical activities such as sensing of analytes of various sizes from small to macromolecules and controlled delivery of drugs.
Subject(s)
Capsules/chemistry , Hemoglobins/chemistry , Polymers/chemistry , Capsules/chemical synthesis , Electrochemistry , Hemoglobins/metabolism , Humans , Microscopy, Electron, Transmission , Polymers/chemical synthesis , Scattering, Small Angle , SynchrotronsABSTRACT
The ethanol extract of Coccos nucifera (EECN) was tested for possible pharmacological effects on experimental animals. EECN significantly potentiated the sleeping time of mice induced by standard hypnotics viz. pentobarbital sodium, diazepam, and meprobamate in a dose dependent manner. EECN showed significant analgesic properties as evidenced by the significant reduction in the number of writhes and stretches induced in mice by 1.2% acetic acid solution. It also potentiated analgesia induced by morphine and pethidine in mice. Pretreatment with EECN caused significant protection against pentylenetetrazole-induced convulsions. The behavioral studies on mice indicate CNS depressant activity of the ethanol extract of C. nucifera.
Subject(s)
Behavior, Animal/drug effects , Central Nervous System Depressants/pharmacology , Cocos , Plant Extracts/pharmacology , Acetic Acid , Analgesics/pharmacology , Animals , Anticonvulsants/pharmacology , Central Nervous System Depressants/toxicity , Disease Models, Animal , Dose-Response Relationship, Drug , Ethanol/chemistry , Female , Hypnotics and Sedatives/pharmacology , Lethal Dose 50 , Male , Mice , Pain/chemically induced , Pain/prevention & control , Pentylenetetrazole , Plant Extracts/toxicity , Plant Roots , Seizures/chemically induced , Seizures/prevention & control , Sleep/drug effects , Solvents/chemistryABSTRACT
Sensing and photocatalysis of textile industry effluents such as dyes using mesoporous anatase titania nanowires are discussed here. Spectroscopic investigations show that the titania nanowires preferentially sense cationic (e.g. Methylene Blue, Rhodamine B) over anionic (e.g. Orange G, Remazol Brilliant Blue R) dyes. The adsorbed dye concentration on titania nanowires increased with increase in nanowire dimensions and dye solution pH. Electrochemical sensing directly corroborated spectroscopic findings. Electrochemical detection sensitivity for Methylene Blue increased by more than two times in magnitude with tripling of nanowire average length. Photodegradation of Methylene Blue using titania nanowires is also more efficient than the commercial P25-TiO(2) nanopowders. Keeping illumination protocol and observation times constant, the Methylene Blue concentration in solution decreased by only 50% in case of P25-TiO(2) nanoparticles compared to a 100% decrease for titania nanowires. Photodegradation was also found to be function of exposure times and dye solution pH. Excellent sensing ability and photocatalytic activity of the titania nanowires is attributed to increased effective reaction area of the controlled nanostructured morphology.
Subject(s)
Nanowires/chemistry , Photochemical Processes , Textile Industry , Titanium/chemistry , Water Pollutants, Chemical/analysis , Water Pollutants, Chemical/chemistry , Adsorption , Coloring Agents , Hydrogen-Ion Concentration , Industrial Waste/analysis , Photolysis , Water Purification/methodsABSTRACT
Investigations on the structure and function of hemoglobin (Hb) confined inside sol-gel template synthesized silica nanotubes (SNTs) have been discussed here. Immobilization of hemoglobin inside SNTs resulted in the enhancement of direct electron transfer during an electrochemical reaction. Extent of influence of nanoconfinement on protein activity is further probed via ligand binding and thermal stability studies. Electrochemical investigations show reversible binding of n-donor liquid ligands, such as pyridine and its derivatives, and predictive variation in their redox potentials suggests an absence of any adverse effect on the structure and function of Hb confined inside nanometer-sized channels of SNTs. Immobilization also resulted in enhanced thermal stability of Hb. The melting or denaturation temperature of Hb immobilized inside SNTs increase by approximately 4 degrees C as compared with that of free Hb in solution.