1t2h: Difference between revisions

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Y81W mutant of RNase Sa from Streptomyces aureofaciens

OverviewOverview

Ribonuclease Sa (RNase Sa) contains no tryptophan (Trp) residues. We have added single Trp residues to RNase Sa at sites where Trp is found in four other microbial ribonucleases, yielding the following variants of RNase Sa: Y52W, Y55W, T76W, and Y81W. We have determined crystal structures of T76W and Y81W at 1.1 and 1.0 A resolution, respectively. We have studied the fluorescence properties and stabilities of the four variants and compared them to wild-type RNase Sa and the other ribonucleases on which they were based. Our results should help others in selecting sites for adding Trp residues to proteins. The most interesting findings are: 1), Y52W is 2.9 kcal/mol less stable than RNase Sa and the fluorescence intensity emission maximum is blue-shifted to 309 nm. Only a Trp in azurin is blue-shifted to a greater extent (308 nm). This blue shift is considerably greater than observed for Trp71 in barnase, the Trp on which Y52W is based. 2), Y55W is 2.1 kcal/mol less stable than RNase Sa and the tryptophan fluorescence is almost completely quenched. In contrast, Trp59 in RNase T1, on which Y55W is based, has a 10-fold greater fluorescence emission intensity. 3), T76W is 0.7 kcal/mol more stable than RNase Sa, indicating that the Trp side chain has more favorable interactions with the protein than the threonine side chain. The fluorescence properties of folded Y76W are similar to those of the unfolded protein, showing that the tryptophan side chain in the folded protein is largely exposed to solvent. This is confirmed by the crystal structure of the T76W which shows that the side chain of the Trp is only approximately 7% buried. 4), Y81W is 0.4 kcal/mol less stable than RNase Sa. Based on the crystal structure of Y81W, the side chain of the Trp is 87% buried. Although all of the Trp side chains in the variants contribute to the unusual positive circular dichroism band observed near 235 nm for RNase Sa, the contribution is greatest for Y81W.

About this StructureAbout this Structure

1T2H is a Single protein structure of sequence from Streptomyces aureofaciens with as ligand. Active as Ribonuclease T(1), with EC number 3.1.27.3 Full crystallographic information is available from OCA.

ReferenceReference

Contribution of single tryptophan residues to the fluorescence and stability of ribonuclease Sa., Alston RW, Urbanikova L, Sevcik J, Lasagna M, Reinhart GD, Scholtz JM, Pace CN, Biophys J. 2004 Dec;87(6):4036-47. Epub 2004 Sep 17. PMID:15377518

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-[[Image:1t2h.gif|left|200px]]<br /><applet load="1t2h" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1t2h.gif|left|200px]]<br /><applet load="1t2h" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1t2h, resolution 1.00&Aring;" />
 '''Y81W mutant of RNase Sa from Streptomyces aureofaciens'''<br />
 ==Overview==
-Ribonuclease Sa (RNase Sa) contains no tryptophan (Trp) residues. We have, added single Trp residues to RNase Sa at sites where Trp is found in four, other microbial ribonucleases, yielding the following variants of RNase, Sa: Y52W, Y55W, T76W, and Y81W. We have determined crystal structures of, T76W and Y81W at 1.1 and 1.0 A resolution, respectively. We have studied, the fluorescence properties and stabilities of the four variants and, compared them to wild-type RNase Sa and the other ribonucleases on which, they were based. Our results should help others in selecting sites for, adding Trp residues to proteins. The most interesting findings are: 1), Y52W is 2.9 kcal/mol less stable than RNase Sa and the fluorescence, intensity emission maximum is blue-shifted to 309 nm. Only a Trp in azurin, is blue-shifted to a greater extent (308 nm). This blue shift is, considerably greater than observed for Trp71 in barnase, the Trp on which, Y52W is based. 2), Y55W is 2.1 kcal/mol less stable than RNase Sa and the, tryptophan fluorescence is almost completely quenched. In contrast, Trp59, in RNase T1, on which Y55W is based, has a 10-fold greater fluorescence, emission intensity. 3), T76W is 0.7 kcal/mol more stable than RNase Sa, indicating that the Trp side chain has more favorable interactions with, the protein than the threonine side chain. The fluorescence properties of, folded Y76W are similar to those of the unfolded protein, showing that the, tryptophan side chain in the folded protein is largely exposed to solvent., This is confirmed by the crystal structure of the T76W which shows that, the side chain of the Trp is only approximately 7% buried. 4), Y81W is 0.4, kcal/mol less stable than RNase Sa. Based on the crystal structure of, Y81W, the side chain of the Trp is 87% buried. Although all of the Trp, side chains in the variants contribute to the unusual positive circular, dichroism band observed near 235 nm for RNase Sa, the contribution is, greatest for Y81W.
+Ribonuclease Sa (RNase Sa) contains no tryptophan (Trp) residues. We have added single Trp residues to RNase Sa at sites where Trp is found in four other microbial ribonucleases, yielding the following variants of RNase Sa: Y52W, Y55W, T76W, and Y81W. We have determined crystal structures of T76W and Y81W at 1.1 and 1.0 A resolution, respectively. We have studied the fluorescence properties and stabilities of the four variants and compared them to wild-type RNase Sa and the other ribonucleases on which they were based. Our results should help others in selecting sites for adding Trp residues to proteins. The most interesting findings are: 1), Y52W is 2.9 kcal/mol less stable than RNase Sa and the fluorescence intensity emission maximum is blue-shifted to 309 nm. Only a Trp in azurin is blue-shifted to a greater extent (308 nm). This blue shift is considerably greater than observed for Trp71 in barnase, the Trp on which Y52W is based. 2), Y55W is 2.1 kcal/mol less stable than RNase Sa and the tryptophan fluorescence is almost completely quenched. In contrast, Trp59 in RNase T1, on which Y55W is based, has a 10-fold greater fluorescence emission intensity. 3), T76W is 0.7 kcal/mol more stable than RNase Sa, indicating that the Trp side chain has more favorable interactions with the protein than the threonine side chain. The fluorescence properties of folded Y76W are similar to those of the unfolded protein, showing that the tryptophan side chain in the folded protein is largely exposed to solvent. This is confirmed by the crystal structure of the T76W which shows that the side chain of the Trp is only approximately 7% buried. 4), Y81W is 0.4 kcal/mol less stable than RNase Sa. Based on the crystal structure of Y81W, the side chain of the Trp is 87% buried. Although all of the Trp side chains in the variants contribute to the unusual positive circular dichroism band observed near 235 nm for RNase Sa, the contribution is greatest for Y81W.
 ==About this Structure==
-T2H is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Streptomyces_aureofaciens Streptomyces aureofaciens] with SO4 as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Ribonuclease_T(1) Ribonuclease T(1)], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.27.3 3.1.27.3] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1T2H OCA].
+T2H is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Streptomyces_aureofaciens Streptomyces aureofaciens] with <scene name='pdbligand=SO4:'>SO4</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Ribonuclease_T(1) Ribonuclease T(1)], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.27.3 3.1.27.3] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1T2H OCA].
 ==Reference==
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 [[Category: ribonuclease]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Wed Nov 21 02:56:22 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 15:09:10 2008''