Abstract
Mutations in the human cellular retinaldehyde binding protein (CRALBP) gene cause retinal pathology. To understand the molecular basis of impaired CRALBP function, we have characterized human recombinant CRALBP containing the disease causing mutations R233W or M225K. Protein structures were verified by amino acid analysis and mass spectrometry, retinoid binding properties were evaluated by UV-visible and fluorescence spectroscopy and substrate carrier functions were assayed for recombinant 11-cis-retinol dehydrogenase (rRDH5). The M225K mutant was less soluble than the R233W mutant and lacked retinoid binding capability and substrate carrier function. In contrast, the R233W mutant exhibited solubility comparable to wild type rCRALBP and bound stoichiometric amounts of 11-cis- and 9-cis-retinal with at least 2-fold higher affinity than wild type rCRALBP. Holo-R233W significantly decreased the apparent affinity of rRDH5 for 11-cis-retinoid relative to wild type rCRALBP. Analyses by heteronuclear single quantum correlation NMR demonstrated that the R233W protein exhibits a different conformation than wild type rCRALBP, including a different retinoid-binding pocket conformation. The R233W mutant also undergoes less extensive structural changes upon photoisomerization of bound ligand, suggesting a more constrained structure than that of the wild type protein. Overall, the results show that the M225K mutation abolishes and the R233W mutation tightens retinoid binding and both impair CRALBP function in the visual cycle as an 11-cis-retinol acceptor and as a substrate carrier.
| Original language | English |
|---|---|
| Pages (from-to) | 12397-12402 |
| Number of pages | 6 |
| Journal | Journal of Biological Chemistry |
| Volume | 278 |
| Issue number | 14 |
| DOIs | |
| State | Published - Apr 4 2003 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
Cite this
Disease-causing mutations in the cellular retinaldehyde binding protein tighten and abolish ligand interactions. / Golovleva, Irina; Bhattacharya, Sanjoy K; Wu, Zhiping; Shaw, Natacha; Yang, Yanwu; Andrabi, Khurshid; West, Karen A.; Burstedt, Marie S I; Forsman, Kristina; Holmgren, Gösta; Sandgren, Ola; Noy, Noa; Qin, Jun; Crabb, John W.
In: Journal of Biological Chemistry, Vol. 278, No. 14, 04.04.2003, p. 12397-12402.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Disease-causing mutations in the cellular retinaldehyde binding protein tighten and abolish ligand interactions
AU - Golovleva, Irina
AU - Bhattacharya, Sanjoy K
AU - Wu, Zhiping
AU - Shaw, Natacha
AU - Yang, Yanwu
AU - Andrabi, Khurshid
AU - West, Karen A.
AU - Burstedt, Marie S I
AU - Forsman, Kristina
AU - Holmgren, Gösta
AU - Sandgren, Ola
AU - Noy, Noa
AU - Qin, Jun
AU - Crabb, John W.
PY - 2003/4/4
Y1 - 2003/4/4
N2 - Mutations in the human cellular retinaldehyde binding protein (CRALBP) gene cause retinal pathology. To understand the molecular basis of impaired CRALBP function, we have characterized human recombinant CRALBP containing the disease causing mutations R233W or M225K. Protein structures were verified by amino acid analysis and mass spectrometry, retinoid binding properties were evaluated by UV-visible and fluorescence spectroscopy and substrate carrier functions were assayed for recombinant 11-cis-retinol dehydrogenase (rRDH5). The M225K mutant was less soluble than the R233W mutant and lacked retinoid binding capability and substrate carrier function. In contrast, the R233W mutant exhibited solubility comparable to wild type rCRALBP and bound stoichiometric amounts of 11-cis- and 9-cis-retinal with at least 2-fold higher affinity than wild type rCRALBP. Holo-R233W significantly decreased the apparent affinity of rRDH5 for 11-cis-retinoid relative to wild type rCRALBP. Analyses by heteronuclear single quantum correlation NMR demonstrated that the R233W protein exhibits a different conformation than wild type rCRALBP, including a different retinoid-binding pocket conformation. The R233W mutant also undergoes less extensive structural changes upon photoisomerization of bound ligand, suggesting a more constrained structure than that of the wild type protein. Overall, the results show that the M225K mutation abolishes and the R233W mutation tightens retinoid binding and both impair CRALBP function in the visual cycle as an 11-cis-retinol acceptor and as a substrate carrier.
AB - Mutations in the human cellular retinaldehyde binding protein (CRALBP) gene cause retinal pathology. To understand the molecular basis of impaired CRALBP function, we have characterized human recombinant CRALBP containing the disease causing mutations R233W or M225K. Protein structures were verified by amino acid analysis and mass spectrometry, retinoid binding properties were evaluated by UV-visible and fluorescence spectroscopy and substrate carrier functions were assayed for recombinant 11-cis-retinol dehydrogenase (rRDH5). The M225K mutant was less soluble than the R233W mutant and lacked retinoid binding capability and substrate carrier function. In contrast, the R233W mutant exhibited solubility comparable to wild type rCRALBP and bound stoichiometric amounts of 11-cis- and 9-cis-retinal with at least 2-fold higher affinity than wild type rCRALBP. Holo-R233W significantly decreased the apparent affinity of rRDH5 for 11-cis-retinoid relative to wild type rCRALBP. Analyses by heteronuclear single quantum correlation NMR demonstrated that the R233W protein exhibits a different conformation than wild type rCRALBP, including a different retinoid-binding pocket conformation. The R233W mutant also undergoes less extensive structural changes upon photoisomerization of bound ligand, suggesting a more constrained structure than that of the wild type protein. Overall, the results show that the M225K mutation abolishes and the R233W mutation tightens retinoid binding and both impair CRALBP function in the visual cycle as an 11-cis-retinol acceptor and as a substrate carrier.
UR - http://www.scopus.com/inward/record.url?scp=0038701035&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0038701035&partnerID=8YFLogxK
U2 - 10.1074/jbc.M207300200
DO - 10.1074/jbc.M207300200
M3 - Article
C2 - 12536144
AN - SCOPUS:0038701035
VL - 278
SP - 12397
EP - 12402
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 14
ER -