Domain identification of hormone-sensitive lipase by circular dichroism and fluorescence spectroscopy, limited proteolysis, and mass spectrometry.
Structure-function relationship analyses of hormone-sensitive lipase (HSL) have suggested that this metabolically important enzyme consists of several functional and at least two structural domains (Osterlund, T., Danielsson, B., Degerman, E., Contreras, J. A., Edgren, G., Davis, R. C., Schotz, M. C., and Holm, C. (1996) Biochem. J. 319, 411-420; Contreras, J. A., Karlsson, M., Osterlund, T., Laurell, H., Svensson, A., and Holm, C. (1996) J. Biol. Chem. 271, 31426-31430). To analyze the structural domain composition of HSL in more detail, we applied biophysical methods. Denaturation of HSL was followed by circular dichroism measurements and fluorescence spectroscopy, revealing that the unfolding of HSL is a two-step event. Using limited proteolysis in combination with mass spectrometry, several proteolytic fragments of HSL were identified, including one corresponding exactly to the proposed N-terminal domain. Major cleavage sites were found in the predicted hinge region between the two domains and in the regulatory module of the C-terminal, catalytic domain. Analyses of a hinge region cleavage mutant and calculations of the hydropathic pattern of HSL further suggest that the hinge region and regulatory module are exposed parts of HSL. Together, these data support our previous hypothesis that HSL consists of two major structural domains, encoded by exons 1-4 and 5-9, respectively, of which the latter contains an exposed regulatory module outside the catalytic alpha/beta-hydrolase fold core.
Osterlund
T
T
Department of Cell and Molecular Biology, Lund University, S-221 00 Lund, Sweden.
Beussman
D J
DJ
Julenius
K
K
Poon
P H
PH
Linse
S
S
Shabanowitz
J
J
Hunt
D F
DF
Schotz
M C
MC
Derewenda
Z S
ZS
Holm
C
C
D000818:Animals; D002942:Circular Dichroism; D010450:Endopeptidases; D004795:Enzyme Stability; D005170:Factor X; D019791:Guanidine / Q000494:pharmacology; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D051381:Rats; D013050:Spectrometry, Fluorescence; D002787:Sterol Esterase / Q000737:chemistry*; D013696:Temperature; D014461:Ultracentrifugation
D010446:Peptide Fragments; D005170:Factor X; D002787:Sterol Esterase; D010450:Endopeptidases; D019791:Guanidine