A Designed Cavity in the Hydrophobic Core of a Four-α-Helix Bundle Improves Volatile Anesthetic Binding Affinity

Johansson, Jonas; Gibney, Brian R.; Rabanal, Francesc; Reddy, Konda S.; Dutton, P. Leslie

doi:10.1021/bi9721290

Cited by 56 publications

(110 citation statements)

References 51 publications

Supporting

Mentioning

101

Contrasting

Order By: Relevance

“…The cysteine-specific spin label (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)methanethiosulfonate (MTSSL) was purchased from Toronto Research Chemicals (Toronto, Canada). MTSSL was attached to the modified LD2 peptides as described earlier (26). In brief, 1.1 mM LD2 peptide and a 10-fold excess of MTSSL were mixed and stirred for 12 h in a 4:1 (vol/vol) solution of 130 mM NaCl, 20 mM sodium phosphate buffer (pH 7.2), and acetonitrile.…”

Section: Methodsmentioning

confidence: 99%

“…Paramagnetic relaxation caused by sitedirected spin labeling can be employed as a powerful tool for determining the solution structure of a molecule because the paramagnetic center can enhance the spin lattice relaxation of magnetic nuclei in a distance-dependent manner (3,13). Oxidized nitroxide has been commonly used as such a paramagnetic center, and previous reports showed that spin labeling can be achieved by the MTSSL method, which covalently links nitroxide to a solvent-exposed cysteine residue through methanethiosulfonate (26). Using a similar method, we performed site-directed labeling of the LD2 peptide.…”

Section: Structure Of the Fat Domainmentioning

confidence: 99%

“…These peptides were then subjected to MTSSL (26). In our initial screening, we found that three of the four spin-labeled peptides bound to the FAT domain with a binding affinity similar to that of the wild-type peptide.…”

Section: Structure Of the Fat Domainmentioning

confidence: 99%

See 2 more Smart Citations

Structural Insight into the Mechanisms of Targeting and Signaling of Focal Adhesion Kinase

Liu

Guibao

Zheng

2002

Molecular and Cellular Biology

View full text Add to dashboard Cite

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase whose focal adhesion targeting (FAT) domaininteracts with other focal adhesion molecules in integrin-mediated signaling. Localization of activated FAK to focal adhesions is indispensable for its function. Here we describe a solution structure of the FAT domain bound to a peptide derived from paxillin, a FAK-binding partner. The FAT domain is composed of four helices that form a "right-turn" elongated bundle; the globular fold is mainly maintained by hydrophobic interactions. The bound peptide further stabilizes the structure. Certain signaling events such as phosphorylation and molecule interplay may induce opening of the helix bundle. Such conformational change is proposed to precede departure of FAK from focal adhesions, which starts focal adhesion turnover.Adhesion of cells to the extracellular matrix (ECM) allows ECM proteins to interact with cell membrane-bound receptors. Such interactions generate intracellular signals that are important for cell growth, survival, and migration (11,12,35,48). Integrins are a large family of transmembrane receptors that have long been recognized for their roles in linking ECM proteins to the actin cytoskeleton and in regulating cell shape and tissue architecture (14,15,44). The binding of integrins to ECM proteins elicits signals that are transmitted into the cell and cause actin and associated cytoskeletal proteins, including paxillin, talin, vinculin, and tensin, to gather at cell substratum sites termed focal adhesions. Although integrins have no enzymatic activity per se, one of the most prominent alterations observed upon integrin clustering is the phosphorylation of tyrosine residues within a variety of proteins in focal adhesions (40).Focal adhesion kinase (FAK) is a nonreceptor kinase that can be activated by integrin signaling. Since the discovery of FAK in the early 1990s, this protein and its related signaling pathways have been studied in some detail (6,36,39,54). Analysis of Fak knockout mice has shown that the null mutation results in an embryonic lethal phenotype that is very similar to that of the fibronectin knockout mice (24). It is now clear that FAK plays an important role in relaying the signals that are generated by the attachment of cells to the ECM and are transmitted through integrins to cytoplasmic and nuclear targets. In this way, FAK regulates cellular processes such as migration, survival, and proliferation (6, 39).The central role of FAK in integrin signaling and the involvement of integrins in tumor progression and metastasis (15) suggest that FAK is a key player in the multistep progression toward a malignant phenotype. Indeed, mounting evidence shows that the expression as well as activity of FAK is upregulated in many cancer cells and that FAK may be involved in the development of invasive cancer (27). Furthermore, it has been demonstrated that plasma membrane-associated pY397FAK is a marker of cytotrophoblast invasion in vivo and in vitro (25). Thus, although FAK does not appear ...

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Structure Of the Fat Domainmentioning

confidence: 99%

See 1 more Smart Citation

Structural Insight into the Mechanisms of Targeting and Signaling of Focal Adhesion Kinase

Liu

Guibao

Zheng

2002

Molecular and Cellular Biology

View full text Add to dashboard Cite

show abstract

“…All fits showed an R 2 value Ͼ 0.99. core in these GPCRs. Furthermore, with the use of designed four-␣-helical bundles (Johansson et al, 1998(Johansson et al, , 2000, and other soluble proteins with hydrophobic cavities Eriksson et al, 1992), it has been shown that preformed accessible cavities are important features to facilitate anesthetic binding. Although inhaled anesthetics have divergent chemical structures, the molecules are relatively small (90 -160 Å 3 ) and have few interactive atoms or groups to provide much selectivity, compared with other molecules that interact with GPCRs, such as biogenic amines and retinal.…”

Section: Table 1 Effects Of Halothane On Retinal Binding Kineticsmentioning

confidence: 99%

G Protein-Coupled Receptors as Direct Targets of Inhaled Anesthetics

et al. 2002

View full text Add to dashboard Cite

The molecular pharmacology of inhalational anesthetics remains poorly understood. Despite accumulating evidence suggesting that neuronal membrane proteins are potential targets of inhaled anesthetics, most currently favored membrane protein targets lack any direct evidence for anesthetic binding. We report herein the location of the binding site for the inhaled anesthetic halothane at the amino acid residue level of resolution in the ligand binding cavity in a prototypical G proteincoupled receptor, bovine rhodopsin. Tryptophan fluorescence quenching and direct photoaffinity labeling with [ 14 C]halothane suggested an interhelical location of halothane with a stoichiometry of 1 (halothane/rhodopsin molar ratio). Radiosequence analysis of [14 C]halothane-labeled rhodopsin revealed that halothane contacts an amino acid residue (Trp265) lining the ligand binding cavity in the transmembrane core of the receptor. The predicted functional consequence, competition between halothane and the ligand retinal, was shown here by spectroscopy and is known to exist in vivo. These data suggest that competition with endogenous ligands may be a general mechanism of the action of halothane at this large family of signaling proteins.

show abstract

“…Such a designed fourhelix bundle protein, (␣ 2 ) 2 , has recently been described and used as a template for anesthetic binding studies (8).…”

mentioning

confidence: 99%

Folding of a de Novo Designed Native-like Four-helix Bundle Protein

Chapeaurouge¹,

Johansson²,

Ferreira³

2002

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The folding of a model native-like dimeric four-helix bundle protein, (␣ 2 ) 2 , was investigated using guanidine hydrochloride, hydrostatic pressure, and low temperature. Unfolding by guanidine hydrochloride followed by circular dichroism and intrinsic fluorescence spectroscopy revealed a highly cooperative transition between the native-like and unfolded states, with free energy of unfolding determined from CD data, ⌬G unf ‫؍‬ 14.3 ؎ 0.8 kcal/mol. However, CD and intrinsic fluorescence data were not superimposable, indicating the presence of an intermediate state during the folding transition. To stabilize the folding intermediate, we used hydrostatic pressure and low temperature. In both cases, dissociation of the dimeric native-like (␣ 2 ) 2 into folded monomers (␣ 2 ) was observed. van't Hoff analysis of the low temperature experiments, assuming a two-state dimer 171-monomer transition, yielded a free energy of dissociation of (␣ 2 ) 2 of ⌬G diss ‫؍‬ 11.4 ؎ 0.4 kcal/mol, in good agreement with the free energy determined from pressure dissociation experiments (⌬G diss ‫؍‬ 10.5 ؎ 0.1 kcal/ mol). Binding of the hydrophobic fluorescent probe 4,4-dianilino-1,1-binaphthyl-5,5-disulfonic acid (bis-ANS) to the pressure-and cold-dissociated states of (␣ 2 ) 2 indicated the existence of molten-globule monomers. In conclusion, we demonstrate that the folding pathway of (␣ 2 ) 2 can be described by a three-state transition including a monomeric molten globule-like state.

show abstract

A Designed Cavity in the Hydrophobic Core of a Four-α-Helix Bundle Improves Volatile Anesthetic Binding Affinity

Cited by 56 publications

References 51 publications

Structural Insight into the Mechanisms of Targeting and Signaling of Focal Adhesion Kinase

Structural Insight into the Mechanisms of Targeting and Signaling of Focal Adhesion Kinase

G Protein-Coupled Receptors as Direct Targets of Inhaled Anesthetics

Folding of a de Novo Designed Native-like Four-helix Bundle Protein

Contact Info

Product

Resources

About