Analysis of the Physical Properties and Molecular Modeling of Sec13: A WD Repeat Protein Involved in Vesicular Traffic
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- 作者：Kumkum Saxena ; Chrysanthe Gaitatzes ; Mary T. Walsh ; Michael Eck ; Eva J. Neer ; and Temple F. Smith
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：December 3, 1996
- 年：1996
- 卷：35
- 期：48
- 页码：15215 - 15221
- 全文大小：539K
- 年卷期：v.35,no.48(December 3, 1996)
- ISSN：1520-4995
文摘
WD repeat proteins are a family of proteins that contain a seriesof highly conserved internalrepeat motifs, usually ending with WD (Trp-Asp). TheG
subunit of heterotrimeric guaninenucleotidebinding protein is a member of this family, and its crystal structurehas been recently solved at highresolution (Wall et al. (1995) Cell 83, 1047-1058; Sondeket al. (1996) Nature 379, 369-374).Basedon the coordinates of G, we have constructed a model forthe structure of Sec13, a 33 kDa WD repeatprotein from Saccharomyces cerevesiae essentialfor vesicular traffic. The model has been testedusinga combination of biophysical and biochemical methods. Sec13 wasexpressed in Escherichia coli as ahexa-His-tagged protein (H6Sec13) and purified to homogeneity. Incontrast to some other WD repeatproteins that are unable to fold into monomeric structures whenexpressed in E. coli, H6Sec13 wassolubleand monomeric in the absence of detergent. The far-UV circulardichroism (CD) spectra of H6Sec13indicated less than 10% 
-helix consistent with the model whichpredicts primarily -sheets. H6Sec13shows a cooperative and irreversible thermal denaturation curveconsistent with a tightly packed structure.The CD spectrum shows an unusual positive ellipticity at 229 nmthat was attributed to interactions ofsurface tryptophans since the 229 nm maximum could be abolished bymodification of 6.3 ± 0.3 (n = 3)tryptophans (out of 15 total in the molecule) withN-bromosuccinimide. Our model predicts thatthreesets of tryptophans are clustered near the surface. As predictedby the model, purified H6Sec13 wascompletely resistant to trypsin digestion. The concordance of themodel of Sec13 presented in this paperwith the biochemical and biophysical studies suggests that this modelcan be useful as a guide to furtherexperiments designed to elucidate the function of Sec13 in vesiculartraffic.
subunit of heterotrimeric guaninenucleotidebinding protein is a member of this family, and its crystal structurehas been recently solved at highresolution (Wall et al. (1995) Cell 83, 1047-1058; Sondeket al. (1996) Nature 379, 369-374).Basedon the coordinates of G, we have constructed a model forthe structure of Sec13, a 33 kDa WD repeatprotein from Saccharomyces cerevesiae essentialfor vesicular traffic. The model has been testedusinga combination of biophysical and biochemical methods. Sec13 wasexpressed in Escherichia coli as ahexa-His-tagged protein (H6Sec13) and purified to homogeneity. Incontrast to some other WD repeatproteins that are unable to fold into monomeric structures whenexpressed in E. coli, H6Sec13 wassolubleand monomeric in the absence of detergent. The far-UV circulardichroism (CD) spectra of H6Sec13indicated less than 10% -helix consistent with the model whichpredicts primarily -sheets. H6Sec13shows a cooperative and irreversible thermal denaturation curveconsistent with a tightly packed structure.The CD spectrum shows an unusual positive ellipticity at 229 nmthat was attributed to interactions ofsurface tryptophans since the 229 nm maximum could be abolished bymodification of 6.3 ± 0.3 (n = 3)tryptophans (out of 15 total in the molecule) withN-bromosuccinimide. Our model predicts thatthreesets of tryptophans are clustered near the surface. As predictedby the model, purified H6Sec13 wascompletely resistant to trypsin digestion. The concordance of themodel of Sec13 presented in this paperwith the biochemical and biophysical studies suggests that this modelcan be useful as a guide to furtherexperiments designed to elucidate the function of Sec13 in vesiculartraffic.