Limited proteolysis reveals conformational changes in uncoupling protein-1 from brown adipose tissue mitochondria.
Limited proteolytic digestion of uncoupling protein-1 (UCP1) from hamster brown adipose tissue mitochondria was studied. Under optimal conditions, trypsin and chymotrypsin cleave at Lys-292 and at Phe-102, yielding major products 31-kDa T1 and 22-kDa Ch1. Both T1 and Ch1 remained dimers, as in UCP1. Using fluorescent nucleotide derivative 2'-O-dansyl GTP, it is shown that T1 retains the nucleotide binding affinity (K(D)=1 microM for dansyl GTP) while Ch1 does not bind nucleotide. Previously kinetic binding and H(+) transport studies [Biochemistry 35 (1996) 7846] have shown that UCP1 forms tight complexes to varying degrees with nucleotides and their derivatives. Nucleotides strongly protect against tryptic digestion but less against chymotryptic digestion, because the chymotryptic product Ch1 does not bind nucleotide. The nucleotides and derivatives show the same potency profile in protecting against both trypsinolysis and chymotryptic digestion, suggesting that UCP1 undergoes a major conformational change upon nucleotide binding from an initial loose complex into a tight complex, in which the cleavage sites become masked from proteolysis.
Huang
Shu-Gui
SG
Institute of Physical Biochemistry, University of Munich, Schillerstrasse 44, D-80336 Munich, Germany. fhuang@agyinc.com
D017667:Adipocytes / Q000737:chemistry / Q000378:metabolism; D002001:Adipose Tissue, Brown; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002478:Cells, Cultured; D002918:Chymotrypsin / Q000737:chemistry*; D006224:Cricetinae; D006868:Hydrolysis; D007473:Ion Channels; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008928:Mitochondria / Q000737:chemistry* / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000378:metabolism; D008956:Models, Chemical; D009711:Nucleotides / Q000737:chemistry*; D010447:Peptide Hydrolases / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D014357:Trypsin / Q000737:chemistry*; D000071256:Uncoupling Protein 1
D002352:Carrier Proteins; D007473:Ion Channels; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D009711:Nucleotides; D010455:Peptides; D000071256:Uncoupling Protein 1; D010447:Peptide Hydrolases; D002918:Chymotrypsin; D014357:Trypsin