Effects of fluorescent probe structure on the dynamics at cysteine-34 within bovine serum albumin: Evidence for probe-dependent modulation of the cybotactic region

Baker, Gary A.; Pandey, Siddharth; Kane, Maureen A.; Maloney, Todd D.; Hartnett, Ann M.; Bright, Frank V.

doi:10.1002/1097-0282(200112)59:7<502::aid-bip1055>3.0.co;2-i

Cited by 10 publications

(11 citation statements)

References 65 publications

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“…We then used frequency domain differential phase and polarized modulation ratio measurements to determine the timeresolved anisotropy decay of the fluorescein-labeled 12-RSS or nonspecific DNA in the presence or absence of StrRAG1 or HMG2 (Table I). Fitting of the anisotropy decay curves was performed using a double exponential equation (35,43) (44,45). Importantly, much lower global rotational reorientation times are reported for nonspecific DNA in the presence of StrRAG1, either at 50 or 100 nM protein concentrations, confirming the lower representation of protein-DNA species that form non-specifically in solution in our assay conditions (Table I).…”

Section: Resultsmentioning

confidence: 99%

RAG1-DNA Binding in V(D)J Recombination

Ciubotaru

Ptaszek

Baker

et al. 2003

Journal of Biological Chemistry

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The RAG1 and RAG2 proteins together constitute the nuclease that initiates the assembly of immunoglobulin and T cell receptor genes in a reaction known as V(D)J recombination. RAG1 plays a central role in recognition of the recombination signal sequence (RSS) by the RAG1/2 complex. To investigate the parameters governing the RAG1-RSS interaction, the murine core RAG1 protein (amino acids 377-1008) fused to a short Strep tag has been purified to homogeneity from bacteria. The Strep-RAG1 (StrRAG1) protein exists as a dimer at a wide range of protein concentrations (25-500 nM) in the absence of DNA and binds with reasonably high affinity and specificity (apparent K D ‫؍‬ 41 nM) to the RSS. Both electrophoretic mobility shift assays and polarization anisotropy experiments indicate that only a single StrRAG1-DNA species exists in solution. Anisotropy decay measured by frequency domain spectroscopy suggests that the complex contains a dimer of StrRAG1 bound to a single DNA molecule. Using measurements of protein intrinsic fluorescence and circular dichroism, we demonstrate that StrRAG1 undergoes a major conformational change upon binding the RSS. Steady-state fluorescence and acrylamide quenching studies reveal that this conformational change is associated with a repositioning of intrinsic protein fluorophores from a hydrophobic to a solvent-exposed environment. RSS-induced conformational changes of StrRAG1 may influence the interaction of RAG1 with RAG2 and synaptic complex formation.The genes encoding the variable domains of immunoglobulins or T cell receptors are generated during lymphocyte differentiation by a somatic recombination reaction known as V(D)J recombination (1). The reaction is initiated by DNA double strand breaks created at the junction between two coding segments (termed V, D, or J) and their flanking recombination signal sequences (RSSs). 1 Cleavage is followed by a complex repair process that results in imprecise joining of the two coding segments and typically precise joining of the two RSSs. The RSSs consist of two conserved sequence elements, the heptamer (consensus 5Ј-CACAGTG-3Ј) and the nonamer (consensus 5Ј-ACAAAAACC-3Ј), separated by a poorly conserved spacer sequence of either 12 or 23 base pairs. Efficient recombination occurs only between a 12-RSS and a 23-RSS, a phenomenon known as the 12/23 rule (for review, see Ref.2). Recognition of 12/23-RSSs and concerted cleavage at the RSScoding sequence border is performed by a complex of the RAG1 and RAG2 proteins (for reviews, see Ref. 3 and 4), the lymphoid-specific products of the recombination-activating genes RAG1 and RAG2 (5, 6). Binding and cleavage of DNA by the RAG1⅐RAG2 complex is facilitated by the ubiquitously expressed architectural DNA-binding proteins 8).Deletion mutagenesis has established the minimal "core" domains of murine RAG1 (residues 384 -1008 of the 1040 aa RAG1 protein; Fig. 1A) and RAG2 (residues 1-383 of the 527 aa RAG2 protein) required for recombination activity in transfected nonlymphoid cell lines (9 -12). Most bi...

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Section: Resultsmentioning

confidence: 99%

RAG1-DNA Binding in V(D)J Recombination

Ciubotaru

Ptaszek

Baker

et al. 2003

Journal of Biological Chemistry

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“…All chemical shifts regarding 11 B and 19 F were referenced using the absolute referencing procedure standard on digital spectrometers. For 11 B chemical shifts the 0 ppm position corresponds to the chemical shift of BF 3 •Et 2 O (15% in CDCl 3 ) whereas for 19 F the reference compound is CCl 3 F. Coupling constants (J) are given in Hertz (Hz). Mass spectra were obtained using TOF and LCQ Duo ion trap instruments operating in ESI +/− or APCI mode, as indicated.…”

Section: Methodsmentioning

confidence: 99%

“…The 1 H NMR spectrum (Supporting Information, Figure S3a) of the mixture contained two sets (4:1 ratio) of vinylic protons between 6.00 and 5.20 ppm. Although the 11 B NMR triplet diagnostic was too broad to reveal detailed information beyond confirming the presence of F-BODIPYs, the 19 F NMR spectrum clearly displayed two overlapping quartets, thereby also suggesting the presence of two F-BODIPYs in Alongside two-dimensional NMR analysis, mass spectrometric analysis of the mixture enabled the identification of these two products as the bromovinyl-and enol-containing F-BODIPYs 5 and 6, respectively, with the bromovinyl-derivative as the major constituent (Scheme 2). Both materials presumably formed as a result of addition across the triple bond, with 6 as a result of exposure to water in the work-up.…”

Section: Bond Length (å)mentioning

confidence: 99%

“…[15][16][17] However, the emission wavelength of the BODIPY core (generally <600 nm) is outside of the 650-1000 nm biological window, where auto-fluorescence and light scattering are minimized. 18 Furthermore, conjugation of BODIPYs to large biomolecules is rather underdeveloped 19,20 and relies on synthetic methodology involving extended reaction times and harsh conditions. A bathochromic shift of the emission wavelength has been achieved by extending the π-conjugation of the dipyrrinato ligand of BODIPYs (Figure 1).…”

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confidence: 99%

“…The 4,4′-difluoro-4-bora-(3a,4a)-diaza-s-indacenes ( F -BODIPYs) are popular courtesy of their robustness in chemical and physiological environments, tunable nanosecond fluorescence lifetime, negligible triple-state population, and high quantum yield. − Indeed, the electronic properties of BODIPYs have recently been exploited in chromogenic and pH probes in biomolecular environments, , drug delivery agents, , fluorescent switches, electroluminescent films, , and photosensitizers in both solar cells and photodynamic therapy. − However, the emission wavelength of the BODIPY core (generally < 600 nm) is outside of the 650–1000 nm biological window, where autofluorescence and light scattering are minimized . Furthermore, conjugation of BODIPYs to large biomolecules is rather underdeveloped , and relies on synthetic methodology involving extended reaction times and harsh conditions. A bathochromic shift of the emission wavelength has been achieved by extending the π-conjugation of the dipyrrinato ligand of BODIPYs (Figure ).…”

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confidence: 99%

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Asymmetric Dipyrrin and F-BODIPYs Conjugated to Terminal Alkynes and Alkenes

et al. 2017

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An asymmetric meso-H dipyrrin featuring a conjugated terminal alkyne substituent was converted to its corresponding difluoro boron complex, and the extent of π-conjugation was extended using Sonogashira cross-coupling. Treatment of the alkyne-substituted dipyrrin with BF·OEt and NEt revealed the reactivity of the conjugated terminal alkyne toward Lewis-activated electrophilic substitution and led to the isolation of F-BODIPYs bearing terminal bromovinyl and enol substituents.

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BODIPY on Board of Sugars: A Short Enlightened Journey up to the Cells

et al. 2020

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BODIPY dyes have unique properties including: good stability in many solvents, high extinction coefficients, stability to light, sharp fluorescence emissions in the visible region with high fluorescence quantum yields. Moreover, through small chemical modifications of the skeleton, their luminescence properties can be finely tuned. As a consequence, during the last decades, this family of chromophores found extensive application in a wide range of fields. Carbohydrates play a relevant role in living organisms: their tasks range from energy storage to structural constituents of cells and tissues, they are involved in a variety of molecular recognition phenomena of physiological and biological relevance. The cyclic structure of natural D‐monosaccharides, also offers the conditions to use carbohydrates as building‐blocks in the construction of more complex, supramolecular structures. With these assumptions, in the last years our research in this field has focused on the study of synthesis, the photophysical features, and the application in the biomedical field of molecules that bear the synergic presence of these two tools. Pointing on the rationalization of this research, this article will provide a synthetic review of our last‐year's results in the field of BODIPY/glycosyl species, that can be classified in two principal sections: a) the rational synthesis and the photophysical studies of BODIPY dyads and antennas containing different kind of sugar units as scaffolds, ranging from mono‐ to disaccharide patterns, and b) the synthetic functionalization and modification of suitable BODIPY structures, for their punctual application in the field of bio‐imaging.

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Effects of fluorescent probe structure on the dynamics at cysteine-34 within bovine serum albumin: Evidence for probe-dependent modulation of the cybotactic region

Cited by 10 publications

References 65 publications

RAG1-DNA Binding in V(D)J Recombination

RAG1-DNA Binding in V(D)J Recombination

Asymmetric Dipyrrin and F-BODIPYs Conjugated to Terminal Alkynes and Alkenes

BODIPY on Board of Sugars: A Short Enlightened Journey up to the Cells

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