The Role of β-Sheet Interactions in Domain Stability, Folding, and Target Recognition Reactions of Calmodulin

Jp, Browne; Strøm, Morten B.; [], Martin; Pm, Bayley

doi:10.1021/bi970460d

Cited by 55 publications

(70 citation statements)

References 48 publications

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“…The value of DE 220 nm increases from Ϫ549~apo! to & Bayley, 1986;Hennessey et al, 1987;Török et al, 1992;Browne et al, 1997!. CaM has a slightly greater average a-helical content than Acam in both the apo-and holo-forms, and the maximal increase in a-helical content on binding calcium is ;40% greater for CaM.…”

Section: Near-and Far-uv CDmentioning

confidence: 89%

“…One or two repeat rounds of phenyl-sepharose chromatography were sometimes required for complete purification. Drosophila CaM was expressed in E. coli and purified as described elsewhere~Maune et al, 1992b;Browne et al, 1997!. CaM and Acam were made calcium-free by incubating with 5-25 mM EGTA and then desalting by passage though two Pharmacia PD10~G25!…”

Section: Proteins and Peptidesmentioning

confidence: 99%

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Conformational and metal‐binding properties of androcam, a testis‐specific, calmodulin‐related protein from drosophila

Martin¹,

Lu²,

Xiao³

et al. 1999

Protein Science

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Androcam is a testis-specific protein of Drosophila melanogaster, with 67% sequence identity to calmodulin and four potential EF-hand calcium-binding sites. Spectroscopic monitoring of the thermal unfolding of recombinant calciumfree androcam shows a biphasic process characteristic of a two-domain protein, with the apo-N-domain less stable than the apo-C-domain. The two EF hands of the C-domain of androcam bind calcium cooperatively with 40-fold higher average affinity than the corresponding calmodulin sites. Magnesium competes with calcium binding @K a~M g! ;3 ϫ 10 3 M Ϫ1 #. Weak calcium binding is also detected at one or more N-domain sites. Compared to apo-calmodulin, apo-androcam has a smaller conformational response to calcium and a lower a-helical content over a range of experimental conditions. Unlike calmodulin, a tryptic cleavage site in the N-domain of apo-androcam remains trypsin sensitive in the presence of calcium, suggesting an altered calcium-dependent conformational change in this domain. The affinity of model target peptides for androcam is 10 3 -10 5 times lower than for calmodulin, and interaction of the N-domain of androcam with these peptides is significantly reduced. Thus, androcam shows calcium-induced conformational responses typical of a calcium sensor, but its properties indicate calcium sensitivity and target interactions significantly different from those of calmodulin. From the sequence differences and the altered calcium-binding properties it is likely that androcam differs from calmodulin in the conformation of residues in the second calcium-binding loop. Molecular modeling supports the deduction that there are significant conformational differences in the N-domain of androcam compared to calmodulin, and that these could affect the surface, conferring a different specificity on androcam in target interactions related to testis-specific calcium signaling functions.

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Section: Near-and Far-uv CDmentioning

confidence: 89%

Section: Proteins and Peptidesmentioning

confidence: 99%

Conformational and metal‐binding properties of androcam, a testis‐specific, calmodulin‐related protein from drosophila

Martin¹,

Lu²,

Xiao³

et al. 1999

Protein Science

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“…Although conclusive proof must await the determination of CALP/CAM structures, it is tempting to speculate that the antagonist action occurs as a result of the longer peptides extending out of the Ca 2ϩ -binding pockets in CaM. In particular, the three residues of the EF hand loops in CaM are involved in an anti-parallel ␤-sheet structure between two adjacent Ca 2ϩ -binding loops of CaM (44,45,53,57,58,63,64). An interaction of the four amino acid extension with this area of (41,71).…”

Section: Discussionmentioning

confidence: 99%

De Novo Design of Peptides Targeted to the EF Hands of Calmodulin

Villain¹,

Jackson²,

Manion³

et al. 2000

Journal of Biological Chemistry

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This report describes the use of the concept of inversion of hydropathy patterns to the de novo design of peptides targeted to a predetermined site on a protein. Eight-and 12-residue peptides were constructed with the EF hands or Ca 2؉ -coordinating sites of calmodulin as their anticipated points of interaction. These peptides, but not unrelated peptides nor those with the same amino acid composition but a scrambled sequence, interacted with the two carboxyl-terminal Ca 2؉ -binding sites of calmodulin as well as the EF hands of troponin C. The interactions resulted in a conformational change whereby the 8-mer peptide-calmodulin complex could activate phosphodiesterase in the absence of Ca 2؉ . In contrast, the 12-mer peptide-calmodulin complex did not activate phosphodiesterase but rather inhibited activation by Ca 2؉ . This inhibition could be overcome by high levels of Ca 2؉ . Thus, it would appear that the aforementioned concept can be used to make peptide agonists and antagonists that are targeted to predetermined sites on proteins such as calmodulin.Accumulating evidence suggests that a simple binary code of polar and nonpolar amino acids arranged in the appropriate order is sufficient to build helical bundle structures and artificial peptides with rudimentary function (for review see Ref. 1). Therefore, only the sequence location, not the identity, of the polar and nonpolar amino acids must be explicitly specified for the formation of a stable helical structure or biologically active peptide. Such binary coding has been successfully employed to produce biologically active analogs of corticotrophin and growth hormone-releasing hormone (2, 3), to design proteins that fold into compact ␣-helical bundles (4), and to develop computer programs that simulate or predict some aspects of protein folding (5). Considering that 20 different amino acids are encompassed by the binary code, one would expect a marked degree of sequence degeneracy for a given shape, since any one of a number of specific polar or nonpolar amino acids could occupy a given position in the sequence. Indeed, experimental evidence has confirmed that gross shape is degenerate with regard to sequence in that any number of different primary amino acid sequences with the same binary code can fold into compact ␣-helical structures (4).More recent studies have shown that, unlike simple helical structures, a five-letter amino acid alphabet is minimally required to build a well ordered, ␤-sheet containing protein architecture (6). In this particular instance, a small ␤-sheet protein, the SH3 domain, could be constructed with 95% of the residues being Ile, Lys, Glu, Ala, and Gly. Interestingly, the pattern of hydropathy of the wild type SH3 domain was largely maintained in the two sequence-simplified structures.1 This would seem once again to point to the importance of the pattern of hydropathy in building more complex structures as well as simple helical ones.If, as described above, the gross architecture of a peptide or protein is in part determined by ...

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“…Several studies have concluded that the apo N-domain is more stable than the apo C-domain both when isolated~Brzeska et al., 1983;Sorensen & Shea, 1998! and in the intact proteiñ Browne et al, 1997;Protasevich et al, 1997!. We report here the results of a systematic study of the thermodynamic stability of CaM and its constituent domains as a function of binding of Ca 2ϩ~o r Mg 2ϩ !, using thermal and chemical denaturation, and spectroscopic monitoring using far-UV CD and tyrosine fluorescence and absorption~cf. Masino et al, 1999!.…”

Section: Introductionmentioning

confidence: 99%

Ligand binding and thermodynamic stability of a multidomain protein, calmodulin

Masino¹,

Martin²,

Bayley³

2000

Protein Science

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196

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Chemical and thermal denaturation of calmodulin has been monitored spectroscopically to determine the stability for the intact protein and its two isolated domains as a function of binding of Ca 2ϩ or Mg 2ϩ . The reversible urea unfolding of either isolated apo-domain follows a two-state mechanism with relatively low DG8 20 values of ;2.7~N-domain! and ;1.9 kcal0mol~C-domain!. The apo-C-domain is significantly unfolded at normal temperatures~20-25 8C!. The greater affinity of the C-domain for Ca 2ϩ causes it to be more stable than the N-domain at @Ca 2ϩ # Ն0.3 mM. By contrast, Mg 2ϩ causes a greater stabilization of the N-rather than the C-domain, consistent with measured Mg 2ϩ affinities. For the intact protein~6Ca 2ϩ !, the bimodal denaturation profiles can be analyzed to give two DG8 20 values, which differ significantly from those of the isolated domains, with one domain being less stable and one domain more stable. The observed stability of the domains is strongly dependent on solution conditions such as ionic strength, as well as specific effects due to metal ion binding. In the intact protein, different folding intermediates are observed, depending on the ionic composition. The results illustrate that a protein of low intrinsic stability is liable to major perturbation of its unfolding properties by environmental conditions and liganding processes and, by extension, mutation. Hence, the observed stability of an isolated domain may differ significantly from the stability of the same structure in a multidomain protein. These results address questions involved in manipulating the stability of a protein or its domains by site directed mutagenesis and protein engineering.

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The Role of β-Sheet Interactions in Domain Stability, Folding, and Target Recognition Reactions of Calmodulin

Cited by 55 publications

References 48 publications

Conformational and metal‐binding properties of androcam, a testis‐specific, calmodulin‐related protein from drosophila

Conformational and metal‐binding properties of androcam, a testis‐specific, calmodulin‐related protein from drosophila

De Novo Design of Peptides Targeted to the EF Hands of Calmodulin

Ligand binding and thermodynamic stability of a multidomain protein, calmodulin

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