Kinetics and mechanism of base hydrolysis of tris(3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine)iron(II) in aqueous and micellar media

The kinetics of base hydrolysis of tris(3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine)iron(II), \( {\text{Fe(PDT)}}_{ 3}^{2 + } \) has been studied in aqueous, cetyltrimethyl ammonium bromide (CTAB) and sodium dodecyl sulphate (SDS) media at 25, 35 and 45 °C under pseudo-first-order conditions, i.e. \( [ {\text{OH}}^{ - } ]\gg [{\text{Fe(PDT)}}_{ 3}^{2 + } ] \). The reactions are first order in both of substrate \( {\text{Fe(PDT)}}_{ 3}^{2 + } \) and hydroxide ion. The rates decrease with increasing ionic strength in aqueous and CTAB media, whereas SDS medium shows little ionic strength effect. The rate also increases with CTAB concentration but decreases with SDS. The specific rate constant, k and thermodynamic parameters (E _a, ΔH ^#, ΔS ^# and ΔG ^#) have also been evaluated. The near equal values of ΔG ^# obtained in aqueous and CTAB media suggest that these reactions occur essentially by the same mechanism such that \( {\text{Fe(PDT)}}_{ 3}^{2 + } \) reacts with OH^? in the rate-determining step. The ionic strength effect in SDS medium suggests that the rate-determining step involves an ion and a neutral species. The results in this study are compared with those obtained for other iron(II)-bipyridine complexes.     

Metal Affinity-Based Purification of a Red Fluorescent Protein

A red fluorescent protein, DsRed, which emits fluorescence in the red region of the spectrum has become a popular alternative to green fluorescent protein as a label in biochemical and bioanalytical applications. In this study, we have developed a simple purification method for DsRed variants utilizing their inherent copper binding property. A purification procedure was developed and optimized using immobilized copper ions yielding a single strong band corresponding to purified DsRed proteins on the SDS-PAGE gel. A purification efficiency of higher than 95% was achieved. A spectral analysis and copper binding study was performed to verify activity of the purified proteins. The development of this method allows DsRed to play a dual role as a fluorescent reporter protein and as a purification affinity tag for a target protein. This simpler approach of purification should expand the utility of DsRed.     

Luminescent proteins from Aequorea victoria: applications in drug discovery and in high throughput analysis

Recent progress in generating a vast number of drug targets through genomics and large compound libraries through combinatorial chemistry have stimulated advancements in drug discovery through the development of new high throughput screening (HTS) methods. Automation and HTS techniques are also highly desired in fields such as clinical diagnostics. Luminescence-based assays have emerged as an alternative to radiolabel-based assays in HTS as they approach the sensitivity of radioactive detection along with ease of operation, which makes them amenable to miniaturization. Luminescent proteins provide the advantage of reduced reagent and operating costs because they can be produced in unlimited amounts through the use of genetic engineering tools. In that regard, the use of two naturally occurring and recombinantly produced luminescent proteins from the jellyfish Aequorea victoria, namely, aequorin and the green fluorescent protein (GFP), has attracted attention in a number of analytical applications in diverse research areas. Aequorin is naturally bioluminescent and has therefore, virtually no associated background signal, which allows its detection down to attomole levels. GFP has become the reporter of choice in a variety of applications given that it is an autofluorescent protein that does not require addition of any co-factors for fluorescence emission. Furthermore, the generation of various mutants of GFP with differing luminescent and spectral properties has spurred additional interest in this protein. In this review, we focus on the use of aequorin and GFP in the development of highly sensitive assays that find applications in drug discovery and in high throughput analysis.     

Molecular biosensing system based on intrinsically disordered proteins

Intrinsically disordered proteins (IDPs) that undergo structural transition upon binding their target molecules are becoming increasingly known. IDPs, because of their binding specificity and induced folding properties, can serve as biological recognition elements for sensing applications. In this paper, BRCA1, an IDP, was utilized as the biological recognition element to detect tumor suppressor protein p53 through the BRCA1/p53 binding interaction to serve as a proof-of-concept for the use of IDPs as recognition elements. The binding resulted in a disordered-to-ordered BRCA1 conformational change, as seen in our circular dichroism (CD) measurements. This conformational change in BRCA1 (residues 219-498) was utilized in the detection of p53 (residues 311-393) via both intrinsic and extrinsic fluorescent probes. Intrinsic tryptophan residues within the BRCA1 sequence detected p53 (311-393) with a detection limit of 0.559 nM (0.112 pmol). Two environmentally sensitive fluorophores, tetramethylrhodamine-5-maleimide (TMR) and 6-((5-dimethylaminonaphthalene-1-sulfonyl)amino)hexanoic acid, succinimidyl ester (dansyl-X, SE) were conjugated to BRCA1 (219-498). Dansyl-X, SE-conjugated BRCA1 (219-498) detected p53 (311-393) with a detection limit of 1.50 nM (0.300 pmol). The sensitivities for TMR and dansyl-X, SE-conjugated BRCA1 for the detection of p53 were nearly threefold and twofold higher, respectively, than the sensitivity reported using intrinsic BRCA1 tryptophan fluorescence. CD measurements did not reveal a disruption of p53/dye-conjugated BRCA1 binding, thus validating the applicability of environmentally sensitive fluorophores as transduction moieties to detect molecules which bind to IDPs and induce a structural change.     

Glucose Responsive Hydrogel Networks Based on Protein Recognition

Stimuli‐responsive materials capable of manifesting physical changes in response to environmental signals are valuable tools for use in a variety of biomedical applications. Herein we describe one such smart glucose‐responsive hydrogel material prepared by immobilizing the glucose/galactose binding protein within an acrylamide hydrogel network. This hydrogel demonstrates a quantitative “accordion”‐like dynamic response in the presence of glucose. We further show the feasibility of employing this responsive smart material as a gating agent for controlled drug delivery, thus, demonstrating that these hydrogels may eventually lead to the development of implantable drug delivery systems for diabetes management applications.

     

Anthozoa red fluorescent protein in biosensing

The identification and cloning of a red fluorescent protein (DsRed) obtained from Anthozoa corals has provided an alternative to commonly used green fluorescent proteins (GFPs) in bioanalytical and biomedical research. DsRed in tandem with GFPs has enhanced the feasibility of multicolor labeling studies. Properties of DsRed, for example high photostability, red-shifted fluorescence emission, and stability to pH changes have proven valuable in its use as a fluorescent tag in cell-biology applications. DsRed has some limitations, however. Its slow folding and tendency to form tetramers have been a hurdle. Several different mutational studies have been performed on DsRed to overcome these problems. In this paper, applications of DsRed in biosensing, specifically in FRET/BRET assays, whole-cell assays, and in biosensors, is discussed. In the future, construction of DsRed mutants with unique characteristics will further expand its applications in bioanalysis.     

Structural and thermodynamic properties of a linearly perturbed matrix model for RNA folding

The structural and thermodynamic properties of a matrix model of homo-RNA folding with linear external interaction are studied. The interaction distinguishes paired bases of the homo-RNA chain from the unpaired bases hence dividing the possible RNA structures given by the linear model into two structural regimes. The genus distribution functions show that the total number of structures for any given length of the chain are reduced for the simple linear interaction considered. The partition function of the model exhibits a scaling relation with the matrix model in which the base pairing strength parameter is re-scaled (G. Vernizzi, H. Orland, A. Zee, Phys. Rev. Lett. 94, 168103 (2005)). The thermodynamics of the model are computed for i) largely secondary structures, (with tertiary structures suppressed by a factor 10^-4) and ii) secondary plus tertiary structures. A structural change for large even lengths is observed in the free energy and specific heat. This change with largely secondary structures appears much before (with respect to length of the chain) than when all the structures (secondary and pseudoknots) are considered. The appearance of different structures which dominate the ensemble with varying temperatures is also found as a function of the interaction parameter for different types of structures (given by different numbers of pairings).