Tryptophan Fluorescence Quenching by Methionine and Selenomethionine Residues of Calmodulin:  Orientation of Peptide and Protein Binding

Yuan, Tao; Weljie, and Aalim M.; Vogel, Hans J.

doi:10.1021/bi9716579

Cited by 365 publications

(219 citation statements)

References 49 publications

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“…4)? Because selenomethionine can be biosynthetically incorporated into recombinant CaM (37), and because selenium can act as an efficient quencher of tryptophan fluorescence (31), we have previously taken advantage of these properties to determine the orientation of the complex between the myosin light chain kinase peptide and CaM by fluorescence spectroscopy (24). In agreement with NMR and x-ray studies (19,20), we have shown that the myosin light chain kinase peptide binds to CaM with topology A (see Fig.…”

Section: (Topology C) No Report Has Been Published That Describes a supporting

confidence: 59%

“…On the basis of these results, we propose that the effector domain of MRP is associated with CaM in an orientation that is similar to myosin light chain kinase (19,20,24) and CaM kinase I (24) (topology A in Fig. 4).…”

Section: (Topology C) No Report Has Been Published That Describes a mentioning

confidence: 85%

“…Pfu polymerase was from Stratagene, the Qiaquick DNA purification kit from Qiagen, restriction enzymes and T4 DNA ligase from New England Biolabs, HisBind resin from Novagen, and phenylsepharose from Pharmacia Biotech. The CaM mutants and analogs were produced as described (24). Briefly, CaM* is a CaM construct with all four methionine residues in the C-terminal domain mutated to Leu, SeMet-CaM is a CaM construct with all nine methionine residues substituted by selenomethionine, and SeMet-CaM* is a CaM construct with all five methionine residues in the N-terminal domain substituted by selenomethionine and all four methionine residues in the Cterminal domain mutated to Leu.…”

Section: Methodsmentioning

confidence: 99%

“…Quenching of the fluorescence of tryptophan residues in MRP by CaM constructs containing selenomethionine residues was measured essentially as described previously (24). The MRP mutants (0.75 M) were incubated at 20°C for 15 min in a buffer containing 10 mM Tris, pH 7.4, 0.2 mM CaCl 2 , and 1 mM DTT.…”

Section: Methodsmentioning

confidence: 99%

“…Several topologies have been reported with respect to the association of peptide ligands with the N-and C-terminal domains of CaM (see also Fig. 4): With myosin light chain kinase (19,20,24) and CaM kinase I (24), the N-terminal side of the peptides associates with the C-terminal domain of CaM, whereas the C-terminal side of the peptides associates with the N-terminal domain of CaM (topology A). This orientation is not observed for other CaM ligands: a peptide encompassing the binding site of CaM-dependent kinase kinase associates with CaM in the opposite orientation (22) (topology B), whereas a peptide corresponding to the binding site of the plasma membrane calcium pump binds solely to the C-terminal domain of CaM (23) …”

Section: Determining the Orientation Of The Effector Domain Of Mrp Inmentioning

confidence: 99%

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Mapping the interface between calmodulin and MARCKS-related protein by fluorescence spectroscopy

Ulrich

Schmitz

Braun

et al. 2000

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

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MARCKS-related protein (MRP) isT he proteins of the myristoylated alanine-rich C kinase substrate (MARCKS) family are protein kinase C substrates that have been proposed to regulate the actin cytoskeleton (1). The family comprises two members: MARCKS itself is a ubiquitous 32-kDa protein, whereas MARCKS-related protein (MRP, also called MacMARCKS) is a 22-kDa protein expressed mainly in brain and reproductive tissues (2). MARCKS proteins share two conserved segments, namely the myristoylated N terminus and a central highly basic 24-to 25-residue-long segment, the effector domain, also called the ''phosphorylation site domain'' (1, 3). In vitro, MARCKS and MRP bind to calmodulin (CaM) with high affinity (K d Ϸ 5 nM) (4, 5). Although direct proof for an interaction between MARCKS proteins and CaM has so far not been obtained in cells, indirect evidence suggests that MARCKS proteins mediate crosstalk between the protein kinase C-and CaM-signal transduction pathways (for reviews, see refs. 6 and 7).Two segments in MARCKS proteins are of interest with respect to their interactions with CaM.(i) The Effector Domain. The structure of the effector domain of MARCKS proteins has been the subject of several reports. Fig. 1 shows the amino acid sequence of the effector domain of MRP. This segment is highly basic (12 residues of 24 are lysines or arginines), but it also contains most of the large hydrophobic residues present in the intact protein (four phenylalanines and two leucines of seven). Although sequence analysis has previously led to the conclusion that this segment forms an ␣-helix (1), circular dichroic studies with peptides (8) and proteins (5, 9) suggest an extended structure for this segment. In the absence of information on the tertiary structure of MARCKS proteins, three different topologies can be proposed for the effector domain. The effector domain could be completely buried in a hydrophobic core (model A), which is unlikely because of the high content of charged amino acid residues. The effector domain could be on the surface of MARCKS proteins and partially exposed to water (model B). This model fits the proposed amphipathic structure of the effector domain (basic͞hydrophobic residues). Finally, the effector domain could be completely exposed to water and either act as a hinge between the C-and N-terminal domains of MARCKS proteins (model C1) or form an exposed loop on the surface of the protein (model C2). The observation that MARCKS proteins are elongated molecules (5, 10) supports model C1. However, the presence of a proline at position 96 in the middle of the effector domain of MRP could potentially induce the formation of a kink or a turn (11), favoring model C2.(ii) The Myristoylated N Terminus. Covalent modification of proteins with lipids, such as myristoylation in the case of MARCKS proteins, allows the interactions of these proteins with membranes (12). Myristoylation could also mediate protein-protein interactions. However, most proteins modified with lipids interact with other proteins on the ...

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Section: (Topology C) No Report Has Been Published That Describes a supporting

confidence: 59%

Section: (Topology C) No Report Has Been Published That Describes a mentioning

confidence: 85%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Determining the Orientation Of The Effector Domain Of Mrp Inmentioning

confidence: 99%

See 3 more Smart Citations

Mapping the interface between calmodulin and MARCKS-related protein by fluorescence spectroscopy

Ulrich

Schmitz

Braun

et al. 2000

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

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Comparative modeling studies of the calmodulin-like domain of calcium-dependent protein kinase from soybean

et al. 2000

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Calmodulin-like domain protein kinases (CDPKs) represent a new class of calcium-dependent protein-phosphorylating enzymes that are not activated by calmodulin or phospholipid compounds. They have been found exclusively in plant and protozoal tissues. CDPKs are typified by four distinct domains: an N-terminal leader sequence, a protein kinase (PK) domain, a calmodulin-like domain (CLD), and a junction domain (JD) between the PK domain and CLD. Structural characterization of the CLD of CDPKalpha from soybean was undertaken based on the amino acid sequence homology of CLD to the structurally well-characterized calmodulin (CaM) family of structures. Tertiary models of apo-CLD, Ca(2+)-CLD complex, and intermolecularly bound Ca(2+)-CLD-JD complexes were obtained via automated and non-automated homology building methods. The resulting structures were compared and validated based on energy differences, phi-psi angle distribution, solvent accessibility, and hydrophobic potential. Circular dichroism, one-dimensional, and two-dimensional nuclear magnetic resonance spectroscopy studies of the CLD and peptides encompassing the JD provide experimental support to the models. The results suggest that there is a possible interaction between the CLD and JD domain similar to that of the CaM/calmodulin-dependent protein kinase II system. At low Ca(2+) levels, the JD may act as an autoinhibitory domain for kinase activity, and during calcium activation an intramolecular CLD-JD complex may form, relieving inhibition of the PK domain. Interactions between the JD and the C terminus of the CLD appear to be particularly important. The outcome of this study supports an intramolecular binding model for calcium activation of CDPK, although not exclusively.

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Calmodulin binding properties of peptide analogues and fragments of the calmodulin-binding domain of simian immunodeficiency virus transmembrane glycoprotein 41

et al. 2000

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Tryptophan Fluorescence Quenching by Methionine and Selenomethionine Residues of Calmodulin: Orientation of Peptide and Protein Binding

Cited by 365 publications

References 49 publications

Mapping the interface between calmodulin and MARCKS-related protein by fluorescence spectroscopy

Mapping the interface between calmodulin and MARCKS-related protein by fluorescence spectroscopy

Comparative modeling studies of the calmodulin-like domain of calcium-dependent protein kinase from soybean

Calmodulin binding properties of peptide analogues and fragments of the calmodulin-binding domain of simian immunodeficiency virus transmembrane glycoprotein 41

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