Quenching-resolved emission anisotropy studies with single and multitryptophan-containing proteins

Eftink, Maurice R.

doi:10.1016/s0006-3495(83)84356-2

Cited by 74 publications

(48 citation statements)

References 51 publications

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“…The limiting anisotropy for a completely immobilized Trp is 0.31 (28). Because of the size of F 1 , the depolarization caused by rotational diffusion of the protein (estimated rotational correlation time Ϸ200 ns) during the fluorescence lifetime of Trp (Ͻ10 ns; see ref.…”

Section: Subunitmentioning

confidence: 99%

Identification of the β _TP site in the x-ray structure of F ₁ -ATPase as the high-affinity catalytic site

Mao

Weber²

2007

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

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ATP synthase uses a unique rotary mechanism to couple ATP synthesis and hydrolysis to transmembrane proton translocation. The F1 subcomplex has three catalytic nucleotide binding sites, one on each ␤ subunit, with widely differing affinities for MgATP or MgADP. During rotational catalysis, the sites switch their affinities. The affinity of each site is determined by the position of the central ␥ subunit. The site with the highest nucleotide binding affinity is catalytically active. From the available x-ray structures, it is not possible to discern the high-affinity site. Using fluorescence resonance energy transfer between tryptophan residues engineered into ␥ and trinitrophenyl nucleotide analogs on the catalytic sites, we were able to determine that the high-affinity site is close to the C-terminal helix of ␥, but at considerable distance from its N terminus. Thus, the ␤TP site in the ATP synthase ͉ catalytic mechanism ͉ rotation F 1 F o -ATP synthase is responsible for the bulk of ATP synthesis from ADP and P i in most organisms. F 1 F o -ATP synthase consists of the membrane embedded F o subcomplex with, in Escherichia coli, a subunit composition of ab 2 c 10 , and the peripheral F 1 subcomplex, with a subunit composition of ␣ 3 ␤ 3 ␥␦ . The energy necessary for ATP synthesis is derived from an electrochemical transmembrane proton (or, in some organisms, sodium ion) gradient. Proton flow, down the gradient, through F o is coupled to ATP synthesis on F 1 by a unique rotary mechanism. The protons flow through channels at the interface of a and c subunits, which drives rotation of the ring of c subunits. The c 10 ring, together with F 1 subunits ␥ and , forms the rotor. Rotation of ␥ leads to conformational changes in the catalytic nucleotide binding sites on the ␤ subunits, where ADP and P i are bound. The conformational changes result in formation and release of ATP. Thus, ATP synthase converts electrochemical energy, the proton gradient, into mechanical energy in form of subunit rotation, and back into chemical energy as ATP. In bacteria, under certain physiological conditions, the process runs in reverse. ATP is hydrolyzed to generate a transmembrane proton gradient which the bacterium requires for such functions as nutrient import and locomotion (for reviews, see refs.

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Section: Subunitmentioning

confidence: 99%

Identification of the β _TP site in the x-ray structure of F ₁ -ATPase as the high-affinity catalytic site

Mao

Weber²

2007

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

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“…Fluorescence anisotropy was analyzed by the Perrin equation (3) where A is the anisotropy, A 0 is the limiting anisotropy in the absence of molecular motion, τ is the lifetime, and θ is the rotational correlation time (34). The limiting anisotropy A 0 is obtained by plotting 1/A versus F/F 0 since the relative change in the lifetime is as follows τ ≡ τ rel = τ/τ 0 = F/F 0 .…”

Section: Fluorescence and Absorption Spectroscopymentioning

confidence: 99%

Influence of the N-Terminal Domain and Divalent Cations on Self-Association and DNA Binding by the Saccharomyces cerevisiae TATA Binding Protein

Khrapunov

Brenowitz

2007

Biochemistry

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The localization of a single tryptophan to the N-terminal domain and six tyrosines to the C-terminal domain of TBP allows intrinsic fluorescence to separately report on the structures and dynamics of the full-length TATA binding protein (TBP) of Saccharomyces cerevisiae and its C-terminal DNA binding domain (TBPc) as a function of self-association and DNA binding. TBPc is more compact than the C-terminal domain within the full-length protein. Quenching of the intrinsic fluorescence by DNA and external dynamic quenchers shows that the observed tyrosine fluorescence is due to the four residues surrounding the "DNA binding saddle" of the C-terminal domain. TBP's N-terminal domain unfolds and changes its position relative to the C-terminal domain upon DNA binding. It partially shields the DNA binding saddle in octameric TBP, shifting upon dissociation to monomers to expose the saddle to DNA. Structure-energetic correlations were obtained by comparing the contribution that electrostatic interactions make to DNA binding by TBP and TBPc; DNA binding by TBPc is more hydrophobic than that by TBP, suggesting that the N-terminal domain either interacts with bound DNA directly or screens a part of the C-terminal domain, diminishing its electronegativity. The competition between divalent cations, K + , and DNA is not straightforward. Divalent cations strengthen binding of TBP to DNA and do so more strongly for TBPc. We suggest that divalent cations affect the structure of the bound DNA perhaps by stabilizing its distorted conformation in complexes with TBPc and TBP and that the N-terminal domain mimics the effects of divalent cations. These data support an autoinhibitory mechanism in which competition between the N-terminal domain and DNA for the saddle diminishes the DNA binding affinity of the fulllength protein.The initiation of gene transcription in eukaryotes requires assembly of a multiprotein preinitiation complex (PIC)1 that recruits RNA polymerase to a promoter. The binding of the TATA binding protein (TBP) to a TATA box sequence is a key step in PIC assembly at many promoters (1). Crystallographic and solution studies have established the formation of a 1:1 complex of TBP and TATA box (e.g., refs 2-5). TBP consists of a 180-residue C-terminal domain (TBPc) that is ~80% identical in sequence among all eukaryotes and a N-terminal domain that varies in both length and sequence (2). The shortest N-terminal domain consists of four residues in Pyrococcus woesei TBP (6) with the 158 residues of human TBP being the longest (7). The crystal structures of yeast, human, Arabidopsis, and archael TBP molecules that have been determined either lack or have rudimentary N-terminal domains (6,(8)(9)(10).

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“…Proteins are complex systems often exhibiting both soft regions and rigid and compact domains [9,10]. Using the fluorescence approach, many authors have reported movements of Tyr and Trp residues in various proteins [11–14]. Other studies were carried out on a time‐dependent dipolar re‐orientation of the environment surrounding the Trp residue [15].…”

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confidence: 99%

Heterogeneous motions within human apohemoglobin

Haouz

Mohsni

Zentz

et al. 1999

European Journal of Biochemistry

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Conservation of the secondary and tertiary protein organization of human apohemoglobin was observed at temperatures ranging from 7 to 25 8C using CD spectra in the far-UV (200±250 nm) and near-UV (250±300 nm) regions. The dynamics of apohemoglobin were probed using fluorescence quenching experiments on the Trp residues and an extrinsic dye (ANS or bis-ANS) located in the heme cavities. The long decay time of the dye emission (. 10 ns) reveals the dynamics of the protein matrix averaged over the whole molecule. The short decay time of the Trp residue emission (ù3 ns) probes the dynamics of their close vicinities. When the temperature rises from 10 to 20 8C, the average intraproteic motions throughout the whole apohemoglobin matrix are greatly accelerated, whereas the hydrophobic protein regions around the a14, b15 and b37 Trp residues appear much less animated. These dynamic differences between the behavior of the softer matrix and the packed rigid regions containing the tryptophans could be one of the requisites for apohemoglobin stability. We suspect that the highly rigid tryptophan domains in human apohemoglobin are likely to be knots.Keywords: apohemoglobin; dynamics; protein domains; Trp fluorescence.The ability of a protein to undergo conformational change is a direct consequence of its flexibility, the origin of which is to be found in fluctuations in the amino acid residues. Small changes in local configuration allow the great stereo-organization of macromolecules. Both immediate and indirect evidence show that all proteins are within a fluctuating system [1±3], with interconversion rates between their protein conformational substates [4,5]. Linderstro Èm-Lang and Schellman [6] were the first to suggest that a protein does not possess a unique conformation but exists as a group of structures not too different in free energy, but frequently differing in enthalpy and entropy. Klotz [7] and Weber [8] have also suggested that proteins are not rigid, but rather that a protein is dynamic and exists in many conformational states. Proteins are complex systems often exhibiting both soft regions and rigid and compact domains [9,10]. Using the fluorescence approach, many authors have reported movements of Tyr and Trp residues in various proteins [11±14]. Other studies were carried out on a time-dependent dipolar re-orientation of the environment surrounding the Trp residue [15]. The majority of these fluorescence studies focused on the dynamics of particular areas within the protein [16,17], leaving other areas within the macromolecule uninvestigated. We therefore investigated the dynamic differences of intraproteic motions as opposed to those of any one particular area.Indeed protein matrix mobility is an average of the many contributions of local protein fluctuations; the various regions cannot have the same dynamic behavior and should be more or less sensitive to thermic energy. Therefore, the present study introduces a new approach, which gives an insight into an integrated picture of these various dynamics. To ...

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Quenching-resolved emission anisotropy studies with single and multitryptophan-containing proteins

Cited by 74 publications

References 51 publications

Identification of the β _TP site in the x-ray structure of F ₁ -ATPase as the high-affinity catalytic site

Identification of the β _TP site in the x-ray structure of F ₁ -ATPase as the high-affinity catalytic site

Influence of the N-Terminal Domain and Divalent Cations on Self-Association and DNA Binding by the Saccharomyces cerevisiae TATA Binding Protein

Heterogeneous motions within human apohemoglobin

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Quenching-resolved emission anisotropy studies with single and multitryptophan-containing proteins

Cited by 74 publications

References 51 publications

Identification of the β TP site in the x-ray structure of F 1 -ATPase as the high-affinity catalytic site

Identification of the β TP site in the x-ray structure of F 1 -ATPase as the high-affinity catalytic site

Influence of the N-Terminal Domain and Divalent Cations on Self-Association and DNA Binding by the Saccharomyces cerevisiae TATA Binding Protein

Heterogeneous motions within human apohemoglobin

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Identification of the β _TP site in the x-ray structure of F ₁ -ATPase as the high-affinity catalytic site

Identification of the β _TP site in the x-ray structure of F ₁ -ATPase as the high-affinity catalytic site