Structure of calmodulin refined at 2.2 Å resolution

Babu, Y.S.; Bugg, Charles E.; Cook, William J.

doi:10.1016/0022-2836(88)90608-0

Cited by 1,086 publications

(1,113 citation statements)

References 37 publications

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“…The Ile-27 residue is one that participates in the hydrophobic cleft that is exposed upon Ca2+ binding (Babu et al, 1985(Babu et al, , 1988. If chemical shift changes of Ile-27 CyH, are indicative of formation of the hydrophobic cleft, then the results shown here suggest that the binding of a single calcium ion in either site in the N-terminal domain can activate the conformational "switch" that organizes the residues involved in forming the hydrophobic cleft.…”

Section: Conformational Effectsmentioning

confidence: 82%

“…The CaHs from Thr-26 and Asp-64 are positioned directly across from each other in a region of 0-sheet that lies between sites I and I1 (Kinemage 4; Babu et al, 1985Babu et al, , 1988Seeholzer & Wand, 1989). In wild-type CaM, these peaks titrate upfield almost simultaneously in fast exchange with midpoints of the change in chemical shift observed at 3.1 (Thr-26) and 3.0 (Asp-64) equivalents of Ca2+ (Fig.…”

Section: Site I and Ii Mutantsmentioning

confidence: 96%

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A series of point mutations reveal interactions between the calcium‐binding sites of calmodulin

et al. 1992

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Calmodulin is a member of the "EF-hand" family of Ca'+-binding proteins. It consists of two homologous globular domains, each containing two helix-loop-helix Ca2+-binding sites. To examine the contribution of individual Ca2+-binding sites to the Ca2+-binding properties of CaM, a series of four site-directed mutants has been studied. In each, the glutamic acid at position 12 in one of the four Ca2+-binding loops has been changed to a glutamine. One-dimensional 'H-NMR has been used to monitor Ca2+-induced changes in the mutant proteins, and the spectral changes observed for each mutant have been compared to those for wild-type CaM. In this way, the effect of each mutation on both the mutated site and the other Ca2+-binding sites has been examined. The mutation of glutamate to glutamine at position 12 in any of the EF-hand Ca'+-binding loops greatly decreases the Ca2+-binding affinity at that site, yet differs in the overall effects on Ca2+ binding depending on which of the four sites is mutated. When the mutation is in site I, there is only a small decrease in the apparent Ca2+-binding affinity of site 11, and vice versa. Mutation in either site I11 or IV results in a large decrease in the apparent Ca2+-binding affinities of the partner C-terminal site. In both the N-and C-terminal domains, evidence for altered conformational effects in the partners of mutated sites is presented. In the C-terminus, the conformational consequences of mutating site 111 or site IV are strikingly different.

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Section: Conformational Effectsmentioning

confidence: 82%

Section: Site I and Ii Mutantsmentioning

confidence: 96%

A series of point mutations reveal interactions between the calcium‐binding sites of calmodulin

et al. 1992

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“…Succinimide formation at Asp and Asn is thus highly regulated by three-dimensional structure, due to the requirement that the peptide nitrogen atom be in a position to attack the side-chain carbonyl of the Asx residue [72]. The Ca# + -calmodulin crystal structure indicates that the peptide-bond nitrogen of Thr-79 is not in a position to attack the side-chain carbonyl carbon of Asp-78 and form a succinimide [90] ; however, for calmodulin, the crystal structure [90] and solution studies [91] indicate that Asp-78 and Thr-79 are in flexible regions, allowing succinimide formation. The location of the labile Asp-2 is the flexible N-terminal region of calmodulin [89].…”

Section: Aspartate Residuesmentioning

confidence: 99%

The denaturation and degradation of stable enzymes at high temperatures

Daniel

Dines

Petach³

1996

Biochemical Journal

266

175

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Now that enzymes are available that are stable above 100 mC it is possible to investigate conformational stability at this temperature, and also the effect of high-temperature degradative reactions in functioning enzymes and the inter-relationship between degradation and denaturation. The conformational stability of proteins depends upon stabilizing forces arising from a large number of weak interactions, which are opposed by an almost equally large destabilizing force due mostly to conformational entropy. The difference between these, the net free energy of stabilization, is relatively small, equivalent to a few interactions. The enhanced stability of very stable proteins can be achieved by an additional stabilizing force which is again equivalent to only a few stabilizing interactions. There is currently no strong evidence that any particular interaction (e.g. hydrogen bonds, hydrophobic interactions) plays a more important role in

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“…This is in sharp contrast to other calcium binding proteins such as calmodulin (25,26), troponin C (27,28), and calbindin (29) or other Gla-containing proteins (1)(2)(3). A high-resolution structure of Ca 2+ -osteocalcin will provide information on a new class of proteins.…”

mentioning

confidence: 96%

The Three-Dimensional Structure of Bovine Calcium Ion-Bound Osteocalcin Using ¹H NMR Spectroscopy

Dowd

Rosen

et al. 2003

Biochemistry

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Structural information on osteocalcin or other noncollagenous bone proteins is very limited. We have solved the three-dimensional structure of calcium bound osteocalcin using 1 H 2D NMR techniques and proposed a mechanism for mineral binding. The protons in the 49 amino acid sequence were assigned using standard two-dimensional homonuclear NMR experiments. Distance constraints, dihedral angle constraints, hydrogen bonds, and 1 H and 13 C chemical shifts were all used to calculate a family of 13 structures. The tertiary structure of the protein consisted of an unstructured N terminus and a C-terminal loop (residues 16-49) formed by long-range hydrophobic interactions. Elements of secondary structure within residues 16-49 include type III turns (residues 20-25) and two α-helical regions (residues 27-35 and 41-44). The three Gla residues project from the same face of the helical turns and are surface exposed. The genetic algorithm-molecular dynamics simulation approach was used to place three calcium atoms on the NMR-derived structure. One calcium atom was coordinated by three side chain oxygen atoms, two from Asp30, and one from Gla24. The second calcium atom was coordinated to four oxygen atoms, two from the side chain in Gla 24, and two from the side chain of Gla 21. The third calcium atom was coordinated to two oxygen atoms of the side chain of Gla17. The best correlation of the distances between the uncoordinated Gla oxygen atoms is with the intercalcium distance of 9.43 Å in hydroxyapatite. The structure may provide further insight into the function of osteocalcin.The family of vitamin K-dependent γ-carboxylated calcium binding proteins is important in a variety of tissues and cellular functions. The formation of γ-carboxyglutamic acid (Gla) 1 in the liver derived blood clotting factors, for example, results in calcium-dependent conformational changes in the proteins and functionally important interactions with acidic phospholipid surfaces (1-3). The predominant Gla protein found in bone matrix is † Support for this project was provided by NIH Grant ES-02030 to T.D., support from the Division of Environmental Sciences,Children's Hospital at Montefiore (CHAM), at the Albert Einstein College of Medicine to T.D., and NIH Grant AR-38460 to C.G. *Corresponding author. Address: Montefiore Medical Center, Moses Bldg. Rm. 401, 111 East 210th Street, Bronx, NY, 10467. Phone: 718-920-2276. Fax: 718-920-4377. dowd@aecom.yu.edu. 1 Abbreviations: Gla, γ-carboxyglutamic acid; Fmoc, 9-flourenyl-methyloxycarbonyl; CD, circular dichroism; NMR, nuclear magnetic resonance; HSQC, heteronuclear single-quantum correlation; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; rmsd, root-mean-square deviation; TOCSY, total correlation spectroscopy. HHS Public Access Author manuscriptBiochemistry. Author manuscript; available in PMC 2015 July 28. Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript osteocalcin (4, 5), a small Ca 2+ binding protein containing three Gla residues which are thought to facil...

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Structure of calmodulin refined at 2.2 Å resolution

Cited by 1,086 publications

References 37 publications

A series of point mutations reveal interactions between the calcium‐binding sites of calmodulin

A series of point mutations reveal interactions between the calcium‐binding sites of calmodulin

The denaturation and degradation of stable enzymes at high temperatures

The Three-Dimensional Structure of Bovine Calcium Ion-Bound Osteocalcin Using ¹H NMR Spectroscopy

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Structure of calmodulin refined at 2.2 Å resolution

Cited by 1,086 publications

References 37 publications

A series of point mutations reveal interactions between the calcium‐binding sites of calmodulin

A series of point mutations reveal interactions between the calcium‐binding sites of calmodulin

The denaturation and degradation of stable enzymes at high temperatures

The Three-Dimensional Structure of Bovine Calcium Ion-Bound Osteocalcin Using 1H NMR Spectroscopy

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The Three-Dimensional Structure of Bovine Calcium Ion-Bound Osteocalcin Using ¹H NMR Spectroscopy