1l54
From Proteopedia
(New page: 200px<br /><applet load="1l54" size="450" color="white" frame="true" align="right" spinBox="true" caption="1l54, resolution 1.9Å" /> '''THE STRUCTURAL AND TH...) |
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| - | [[Image:1l54.jpg|left|200px]]<br /><applet load="1l54" size=" | + | [[Image:1l54.jpg|left|200px]]<br /><applet load="1l54" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1l54, resolution 1.9Å" /> | caption="1l54, resolution 1.9Å" /> | ||
'''THE STRUCTURAL AND THERMODYNAMIC CONSEQUENCES OF BURYING A CHARGED RESIDUE WITHIN THE HYDROPHOBIC CORE OF T4 LYSOZYME'''<br /> | '''THE STRUCTURAL AND THERMODYNAMIC CONSEQUENCES OF BURYING A CHARGED RESIDUE WITHIN THE HYDROPHOBIC CORE OF T4 LYSOZYME'''<br /> | ||
==Overview== | ==Overview== | ||
| - | To determine the energetic and structural consequences of placing a | + | To determine the energetic and structural consequences of placing a charged group within the core of a protein, two "buried charge" mutants, Met 102----Lys (M102K) and Leu 133----Asp (L133D) were constructed in phage T4 lysozyme. Both proteins fold at neutral pH, although they are substantially less stable than wild type. The activity of M102K is about 35% that of wild type, while that of L133D is about 4%. M102K could be crystallized, and its structure was determined at high resolution. The crystal structure (at pH 6.8) of the mutant is very similar to that of wild type except for the alpha-helix that includes residues 108-113. In wild-type lysozyme, one side of this helix is exposed to solvent and the other contacts Met 102. In the M102K structure this alpha-helix becomes much more mobile, possibly allowing partial access of Lys 102 to solvent. The stability of M102K, determined by monitoring the unfolding of the protein with CD, is pH-dependent, consistent with the charged form of the substituted amino acid being more destabilizing than the uncharged form. The pKa of Lys 102 was estimated to be 6.5 both by differential titration and also by NMR analysis of isotopically labeled protein with 13C incorporated at the C epsilon position of all lysines. As the pH is lowered below pH 6.5, the overall three-dimensional structure of M102K at room temperature appears to be maintained to pH 3 or so, although there is evidence for some structural adjustment possibly allowing solvent accessibility to the protonated form of Lys 102. |
==About this Structure== | ==About this Structure== | ||
| - | 1L54 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Bacteriophage_t4 Bacteriophage t4]. Active as [http://en.wikipedia.org/wiki/Lysozyme Lysozyme], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.17 3.2.1.17] Full crystallographic information is available from [http:// | + | 1L54 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Bacteriophage_t4 Bacteriophage t4]. Active as [http://en.wikipedia.org/wiki/Lysozyme Lysozyme], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.2.1.17 3.2.1.17] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1L54 OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Daopin, S.]] | [[Category: Daopin, S.]] | ||
| - | [[Category: Matthews, B | + | [[Category: Matthews, B W.]] |
[[Category: hydrolase (o-glycosyl)]] | [[Category: hydrolase (o-glycosyl)]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:41:26 2008'' |
Revision as of 11:41, 21 February 2008
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THE STRUCTURAL AND THERMODYNAMIC CONSEQUENCES OF BURYING A CHARGED RESIDUE WITHIN THE HYDROPHOBIC CORE OF T4 LYSOZYME
Overview
To determine the energetic and structural consequences of placing a charged group within the core of a protein, two "buried charge" mutants, Met 102----Lys (M102K) and Leu 133----Asp (L133D) were constructed in phage T4 lysozyme. Both proteins fold at neutral pH, although they are substantially less stable than wild type. The activity of M102K is about 35% that of wild type, while that of L133D is about 4%. M102K could be crystallized, and its structure was determined at high resolution. The crystal structure (at pH 6.8) of the mutant is very similar to that of wild type except for the alpha-helix that includes residues 108-113. In wild-type lysozyme, one side of this helix is exposed to solvent and the other contacts Met 102. In the M102K structure this alpha-helix becomes much more mobile, possibly allowing partial access of Lys 102 to solvent. The stability of M102K, determined by monitoring the unfolding of the protein with CD, is pH-dependent, consistent with the charged form of the substituted amino acid being more destabilizing than the uncharged form. The pKa of Lys 102 was estimated to be 6.5 both by differential titration and also by NMR analysis of isotopically labeled protein with 13C incorporated at the C epsilon position of all lysines. As the pH is lowered below pH 6.5, the overall three-dimensional structure of M102K at room temperature appears to be maintained to pH 3 or so, although there is evidence for some structural adjustment possibly allowing solvent accessibility to the protonated form of Lys 102.
About this Structure
1L54 is a Single protein structure of sequence from Bacteriophage t4. Active as Lysozyme, with EC number 3.2.1.17 Full crystallographic information is available from OCA.
Reference
Structural and thermodynamic consequences of burying a charged residue within the hydrophobic core of T4 lysozyme., Dao-pin S, Anderson DE, Baase WA, Dahlquist FW, Matthews BW, Biochemistry. 1991 Dec 10;30(49):11521-9. PMID:1747370
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