[21] Fluorescence quenching studies: Analysis of nonlinear Stern-Volmer data

Laws, William R.; Contino, Paul

doi:10.1016/0076-6879(92)10023-7

Cited by 70 publications

(53 citation statements)

References 9 publications

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“…Unlike plots of quencher vs. FO/F, plots of quencher vs. TO/Twere linear (Fig. 6 Upper) and affirmed a dynamic component of acrylamide quenching for a single species of 1617 and R17 (29).…”

Section: Resultsmentioning

confidence: 99%

Invariant tryptophan at a shielded site promotesfolding of the conformational unit of spectrin.

MacDonald

Musacchio

Holmgren

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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The tryptophan that is highly conserved among repeating structural units of spectrin is reported to promote the conformational stability of one such unit of chicken brain a-spectrin. Four constructs were inserted into pET vectors for overexpression in Escherichia coli of the following spectrin peptides: (i) two adjacent but separately expressed "conformationally phased" repeating units, R16 and R17, one of which (R17) contains a single tryptophan; (it) a mutant, M17, of the single tryptophan-containing unit with alanine substituted for the tryptophan; and (Wi) a conformationally unphased unit, 1617, composed of half of each of the phased units. Both the mutant unit and the unphased unit were much more readily digested by chymotrypsin and by elastase than the phased units and exhibited only 38% and 54% as much a-helical structure, respectively, as the phased units by their far UV CD spectra; 90°light scattering measurements revealed the folded peptides to be predominantly monomeric in solution, whereas the unfolded, protease-sensitive peptides consisted of dimers and/or trimers. This trend was corroborated by their dynamic light scattering. Both the blue-shifted wavelength of maximal emission and the relative inaccessibility to acrylamide of the single tryptophan in the folded unit indicate that the invariant tryptophan occupies a site that is shielded from the aqueous phase.A striking common feature of the moderately homologous structural units of a variety of a (1-4) and 1 (5-7) subunits of erythroid and nonerythroid spectrin is a single tryptophan that is conserved to a degree unmatched at any other position. Exceptions to the rule are the 10th unit of human erythroid /3-spectrin (6), certain units of Drosophila P-spectrin (5), and the 15th unit of erythroid and nonerythroid ,B3spectrin, which exhibits ankyrin-binding activity (8). Repeating units of the related a-actinin (9, 10) and dystrophin (11) also possess highly conserved tryptophans. A particular role for the invariant tryptophan is not yet apparent in models proposed thus far, which generally agree in depicting an idealized conformational unit of spectrin (1, 12, 13), a-actinin (13), and dystrophin (11, 14) as a bundle of three antiparallel helices. This structure may be stabilized by electrostatic (13) and hydrophobic (14) interactions occurring on correct juxtaposition of repeating heptads of amino acids.To determine whether the invariant tryptophan contributes to the structural stability of the conformational units of spectrin, we have cloned (i) two adjacent but separately expressed repeating units of the a subunit of chicken brain spectrin, each one "phased" (15) to insure conformational stability, (ii) a mutant of one of those units with an alanine residue substituted for the single invariant tryptophan, and (iii) a fragment of the same size but consisting of about half of each of the conformationally stable units and therefore not "phased." The expressed peptides have been examined with respect to their protease sensitivity, far UV CD s...

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“…Unlike plots of quencher vs. FO/F, plots of quencher vs. TO/Twere linear (Fig. 6 Upper) and affirmed a dynamic component of acrylamide quenching for a single species of 1617 and R17 (29).…”

Section: Resultsmentioning

confidence: 99%

Invariant tryptophan at a shielded site promotesfolding of the conformational unit of spectrin.

MacDonald

Musacchio

Holmgren

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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“…In the case the total emitted fluorescence can be separated in discrete intensity contributions, f (i), from distinct species, the overall K SV is the weighted sum of the specific quenching constant/species, ⌺f(i)K SV (i). The expression for the collisional quenching ratio is, then, given by Equation 3 (Laws and Contino, 1992),…”

Section: Methodsmentioning

confidence: 99%

“…while in the presence of a static contribution and considering the sphere of action model, the complete expression for the quenching ratio is given by Equation 4 (Laws and Contino, 1992),…”

Section: Methodsmentioning

confidence: 99%

Intrinsic Fluorescence Properties and Structural Analysis of p13 from Schizosaccharomyces pombe

Neyroz

Menna

Polverini

et al. 1996

Journal of Biological Chemistry

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p13suc1 acts in the fission yeast cell division cycle as a component of p34 cdc2 . In the present work, structural information contained in the intrinsic fluorescence of p13 suc1 has been extracted by steady-state and time-resolved fluorescence techniques. In its native form, the steady-state emission spectrum of p13 suc1 is centered at 336 nm. Upon denaturation by guanidine HCl (4.0 M), the emission spectrum is shifted to 355-360 nm and the fluorescence intensity decreases 70%. The same changes are not obtained with p13 suc1 at 56°C or after incubation at 100°C, and the protein appears to be substantially temperature-stable. The fluorescence decay of p13 suc1 is best described by three discrete lifetimes of 0.6 ns ( 1 ), 2.9 ns ( 2 ), and 6.1 ns (

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“…This is a characteristic feature for fluorophores quenched both by collisional and static quenching mechanisms. The fractional remaining fluorescence (F 0 /F) versus acrylamide concentration [Q] was fit to a second-order function (38,39),…”

Section: Methodsmentioning

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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[21] Fluorescence quenching studies: Analysis of nonlinear Stern-Volmer data

Cited by 70 publications

References 9 publications

Invariant tryptophan at a shielded site promotesfolding of the conformational unit of spectrin.

Invariant tryptophan at a shielded site promotesfolding of the conformational unit of spectrin.

Intrinsic Fluorescence Properties and Structural Analysis of p13 from Schizosaccharomyces pombe

RAG1-DNA Binding in V(D)J Recombination

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