Pyrene Excimer Fluorescence as a Probe of Protein Conformational Change

Lehrer, Sherwin S.

doi:10.1007/978-1-4899-1727-0_5

Cited by 34 publications

(28 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This excludes the possibility of intraprotomer stacking of pyrene labels in the filament, since formation of the excimer requires that the labeled sulfur atoms be within 18 Å of one another. 24 Cofilin binding to pyrenyl S265C F-actin enhanced greatly excimer formation, with a parallel decrease in the monomer pyrene peak (Figure 2, spectrum 2). This result can be rationalized in terms of a cofilininduced change in the flexibility or position of the hydrophobic loop and/or the C terminus on actin.…”

Section: Resultsmentioning

confidence: 95%

“…Evidence for the cofilin effect on the hydrophobic loop/C terminus interface is provided also by the enhancement of pyrene excimer formation in S265C F-actin ( Figure 2). However, a more quantitative interpretation of this result is difficult, because the excimer stacking of pyrene rings can occur at thiol separations between 3 Å and 18 Å , 24 and can be affected by the orientation of thiol groups, flexibility of attachment, and the interaction of the pyrene moiety with a local hydrophobic environment. These factors may improve the stacking of the pyrene rings even when the actual distance between the cysteine residues (265 and 374) is increased.…”

Section: The Environment Of Loop 262-274mentioning

confidence: 97%

See 1 more Smart Citation

Cofilin (ADF) Affects Lateral Contacts in F-actin

Bobkov

Mühlrád

Shvetsov

et al. 2004

Journal of Molecular Biology

View full text Add to dashboard Cite

Section: Resultsmentioning

confidence: 95%

Section: The Environment Of Loop 262-274mentioning

confidence: 97%

Cofilin (ADF) Affects Lateral Contacts in F-actin

Bobkov

Mühlrád

Shvetsov

et al. 2004

Journal of Molecular Biology

View full text Add to dashboard Cite

“…First, we asked whether MVT caused rearrangements in lateral and/or longitudinal interprotomer contacts. To do so, we measured fluorescence excimer formation by pyrene labeled cysteine pairs in mutant yeast actins 36 : lateral actin proximity is reported by pyrene labels at Cys265 and Cys374 (referred to as S265C because this residue is mutated) 33,37 ; longitudinal proximity is reported by pyrene bound to Cys41 and Cys374 (Q41C) 32 . In both cases, upon polymerization, two pyrene rings from neighboring protomers stack to form an excimer band.…”

Section: Resultsmentioning

confidence: 99%

Metavinculin Tunes the Flexibility and the Architecture of Vinculin-Induced Bundles of Actin Filaments

Durer

McGillivary

Kang

et al. 2015

Journal of Molecular Biology

View full text Add to dashboard Cite

Vinculin is an abundant protein found at cell-cell and cell-extracellular matrix junctions. In muscles, a longer splice-isoform of vinculin, metavinculin, is also expressed. The metavinculin-specific insert is part of the C-terminal tail domain, the actin-binding site of both isoforms. Mutations in the metavinculin-specific insert are linked to heart disease such as dilated cardiomyopathies. Vinculin tail domain (VT) both binds and bundles actin filaments. Metavinculin tail domain (MVT) binds actin filaments in a similar orientation but does not bundle filaments. Recently, MVT was reported to sever actin filaments. In this work, we asked how MVT influences F-actin alone or in combination with VT. Cosedimentation and limited proteolysis experiments indicated a similar actin binding affinity and mode for both VT and MVT. In real time TIRF microscopy experiments MVT’s severing activity was negligible. Instead, we found that MVT binding caused a two-fold reduction in F-actin’s bending persistence length and increased susceptibility to breakage. Perhaps MVT allows the load of muscle contraction to act as a signal to reorganize actin filaments. Using mutagenesis and site-directed labeling with fluorescence probes, we determined that MVT alters actin interprotomer contacts and dynamics, which presumably reflect the observed changes in bending persistence length. Finally, we found that MVT decreases the density and thickness of actin filament bundles generated by VT. Altogether, our data suggest that MVT alters actin filament flexibility and tunes filament organization in the presence of VT. Both of these activities are potentially important for muscle cell function.

show abstract

“…When attached to a protein thiol, the dye produces two distinct emission peaks at 375 and 395 nm. When two pyrene-thiols interact within 6 -10 Å distance, they form excited state dimers (excimers) that emit at longer wavelengths than the lone excited fluorophore (24,25). The transporter labeled with the pyrene probe at the cysteine positions described above retained ϳ80% ATPase activity and 70 -80% maltose transport activity as compared with the wild type complex ( Table 2).…”

Section: Atp Controls the Outward Facing Conformation Of Thementioning

confidence: 99%

ATP Alone Triggers the Outward Facing Conformation of the Maltose ATP-binding Cassette Transporter

Bao¹,

Duong

2013

Journal of Biological Chemistry

View full text Add to dashboard Cite

Background:The conformational cycle of MalFGK 2 is stimulated by ATP, MalE, and maltose. Results: The transporter outward facing conformation depends on ATP only. Conclusion: MalE and maltose must therefore stimulate the return of the transporter to the inward facing state. Significance: Understanding the dynamics of the transporter is important to interpret the crystal structures.The maltose transporter MalFGK 2 is a study prototype for ABC importers. During catalysis, the MalFG membrane domain alternates between inward and outward facing conformations when the MalK dimer closes and hydrolyzes ATP. Because a rapid ATP hydrolysis depends on MalE and maltose, it has been proposed that closed liganded MalE facilitates the transition to the outward facing conformation. Here we find that, in contrast to the expected, ATP is sufficient for the closure of MalK and for the conversion of MalFG to the outward facing state. The outward facing transporter binds MalE with nanomolar affinity, yet neither MalE nor maltose is necessary or facilitates the transition. Thus, the rapid hydrolysis of ATP observed in the presence of MalE and maltose is not because closed liganded MalE accelerates the formation of the outward facing conformation. These findings have fundamental implications for the description of the transport reaction. ATP-binding cassette (ABC)3 transporters are found both in prokaryotes and eukaryotes and transport a wide variety of substrates in and out of cells. The understanding of their mode of operation is relevant for medically important processes such as multidrug resistance, chloride conductance, cholesterol transport, and surface-antigen presentation (1). In bacteria, the maltose transporter has been studied for several decades as the prototype of ABC importers (2-4). The transporter is comprised of a cytosolic homodimeric ABC domain MalK bound to a membrane domain heterodimer, MalFG. On its periplasmic side, the transporter associates with the substrate-binding protein MalE that acts as a receptor for maltose.Major progress in the description of ABC transporters has been achieved by x-ray crystallography (5-7). In the structure of MalFGK 2 without ligands, the sites for ATP binding on the MalK dimer are separated, and the MalFG membrane domain forms a cavity that is exposed to the cytosol (inward facing state) (8). In the structure obtained in the presence of MalE and nonhydrolyzable ATP analogs, the ATP-binding sites are bound together, whereas the MalFG cavity opens toward the periplasm (outward facing state) (6, 9). Together, these snapshots of the transporter structure have provided the molecular framework to explain how ATP binding and hydrolysis is coupled to membrane transport, the so-called alternating access mechanism. However, what triggers the transport reaction, why MalE stimulates ATP hydrolysis even in the absence of maltose, and how maltose is transferred from MalE to MalFG remain unclear.MalE consists of two lobes with the maltose-binding site located at the interface. When the sugar bind...

show abstract

Pyrene Excimer Fluorescence as a Probe of Protein Conformational Change

Cited by 34 publications

References 46 publications

Cofilin (ADF) Affects Lateral Contacts in F-actin

Cofilin (ADF) Affects Lateral Contacts in F-actin

Metavinculin Tunes the Flexibility and the Architecture of Vinculin-Induced Bundles of Actin Filaments

ATP Alone Triggers the Outward Facing Conformation of the Maltose ATP-binding Cassette Transporter

Contact Info

Product

Resources

About