1efq
From Proteopedia
(New page: 200px<br /><applet load="1efq" size="450" color="white" frame="true" align="right" spinBox="true" caption="1efq, resolution 1.60Å" /> '''Q38D mutant of LEN''...) |
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| - | [[Image:1efq.jpg|left|200px]]<br /><applet load="1efq" size=" | + | [[Image:1efq.jpg|left|200px]]<br /><applet load="1efq" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1efq, resolution 1.60Å" /> | caption="1efq, resolution 1.60Å" /> | ||
'''Q38D mutant of LEN'''<br /> | '''Q38D mutant of LEN'''<br /> | ||
==Overview== | ==Overview== | ||
| - | Asp residues are significantly under represented in beta-sheet regions of | + | Asp residues are significantly under represented in beta-sheet regions of proteins, especially in the middle of beta-strands, as found by a number of studies using statistical, modeling, or experimental methods. To further understand the reasons for this under representation of Asp, we prepared and analyzed mutants of a beta-domain. Two Gln residues of the immunoglobulin light-chain variable domain (V(L)) of protein Len were replaced with Asp, and then the effects of these changes on protein stability and protein structure were studied. The replacement of Q38D, located at the end of a beta-strand, and that of Q89D, located in the middle of a beta-strand, reduced the stability of the parent immunoglobulin V(L) domain by 2.0 kcal/mol and 5.3 kcal/mol, respectively. Because the Q89D mutant of the wild-type V(L)-Len domain was too unstable to be expressed as a soluble protein, we prepared the Q89D mutant in a triple mutant background, V(L)-Len M4L/Y27dD/T94H, which was 4.2 kcal/mol more stable than the wild-type V(L)-Len domain. The structures of mutants V(L)-Len Q38D and V(L)-Len Q89D/M4L/Y27dD/T94H were determined by X-ray diffraction at 1.6 A resolution. We found no major perturbances in the structures of these Q-->D mutant proteins relative to structures of the parent proteins. The observed stability changes have to be accounted for by cumulative effects of the following several factors: (1) by changes in main-chain dihedral angles and in side-chain rotomers, (2) by close contacts between some atoms, and, most significantly, (3) by the unfavorable electrostatic interactions between the Asp side chain and the carbonyls of the main chain. We show that the Asn side chain, which is of similar size but neutral, is less destabilizing. The detrimental effect of Asp within a beta-sheet of an immunoglobulin-type domain can have very serious consequences. A somatic mutation of a beta-strand residue to Asp could prevent the expression of the domain both in vitro and in vivo, or it could contribute to the pathogenic potential of the protein in vivo. |
==About this Structure== | ==About this Structure== | ||
| - | 1EFQ is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens] with IUM and ZN as [http://en.wikipedia.org/wiki/ligands ligands]. Full crystallographic information is available from [http:// | + | 1EFQ is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens] with <scene name='pdbligand=IUM:'>IUM</scene> and <scene name='pdbligand=ZN:'>ZN</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1EFQ OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Cai, X.]] | [[Category: Cai, X.]] | ||
[[Category: Gu, M.]] | [[Category: Gu, M.]] | ||
| - | [[Category: Pokkuluri, P | + | [[Category: Pokkuluri, P R.]] |
[[Category: Schiffer, M.]] | [[Category: Schiffer, M.]] | ||
| - | [[Category: Stevens, F | + | [[Category: Stevens, F J.]] |
[[Category: IUM]] | [[Category: IUM]] | ||
[[Category: ZN]] | [[Category: ZN]] | ||
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[[Category: uranyl ion in crystal contact]] | [[Category: uranyl ion in crystal contact]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:27:16 2008'' |
Revision as of 10:27, 21 February 2008
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Q38D mutant of LEN
Overview
Asp residues are significantly under represented in beta-sheet regions of proteins, especially in the middle of beta-strands, as found by a number of studies using statistical, modeling, or experimental methods. To further understand the reasons for this under representation of Asp, we prepared and analyzed mutants of a beta-domain. Two Gln residues of the immunoglobulin light-chain variable domain (V(L)) of protein Len were replaced with Asp, and then the effects of these changes on protein stability and protein structure were studied. The replacement of Q38D, located at the end of a beta-strand, and that of Q89D, located in the middle of a beta-strand, reduced the stability of the parent immunoglobulin V(L) domain by 2.0 kcal/mol and 5.3 kcal/mol, respectively. Because the Q89D mutant of the wild-type V(L)-Len domain was too unstable to be expressed as a soluble protein, we prepared the Q89D mutant in a triple mutant background, V(L)-Len M4L/Y27dD/T94H, which was 4.2 kcal/mol more stable than the wild-type V(L)-Len domain. The structures of mutants V(L)-Len Q38D and V(L)-Len Q89D/M4L/Y27dD/T94H were determined by X-ray diffraction at 1.6 A resolution. We found no major perturbances in the structures of these Q-->D mutant proteins relative to structures of the parent proteins. The observed stability changes have to be accounted for by cumulative effects of the following several factors: (1) by changes in main-chain dihedral angles and in side-chain rotomers, (2) by close contacts between some atoms, and, most significantly, (3) by the unfavorable electrostatic interactions between the Asp side chain and the carbonyls of the main chain. We show that the Asn side chain, which is of similar size but neutral, is less destabilizing. The detrimental effect of Asp within a beta-sheet of an immunoglobulin-type domain can have very serious consequences. A somatic mutation of a beta-strand residue to Asp could prevent the expression of the domain both in vitro and in vivo, or it could contribute to the pathogenic potential of the protein in vivo.
About this Structure
1EFQ is a Single protein structure of sequence from Homo sapiens with and as ligands. Full crystallographic information is available from OCA.
Reference
Factors contributing to decreased protein stability when aspartic acid residues are in beta-sheet regions., Pokkuluri PR, Gu M, Cai X, Raffen R, Stevens FJ, Schiffer M, Protein Sci. 2002 Jul;11(7):1687-94. PMID:12070321
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