Mutation of Arg55/56 to Leu55/Ala56 in insulin-like growth factor-I results in two forms different in disulfide structure and native conformation but similar under reverse-phase conditions.
Folding of recombinant human insulin-like growth factor-I (IGF-I) results in two distinct species as resolved by reversed-phase high-performance liquid chromatography (RP-HPLC). The earlier eluting peak (PI) has a nonnative disulfide structure, while the later eluting peak (PII) assumes the native disulfide structure. This folding problem causes a lower yield and requires expensive RP-HPLC separation. In contrast, IGF-II folds mainly into a single form with all three disulfide bonds correctly formed. Sequence comparison of the two molecules revealed that IGF-I has arginine at residues 55 and 56, while IGF-II has alanine and leucine, respectively, at these positions. Two analogs of IGF-I, IGF-I (Ala55/Leu56) and IGF-I (Leu56), behave similarly to IGF-II upon refolding and RP-HPLC; that is, a single peak eluted from the RP-HPLC column. However, when the peaks isolated by RP-HPLC were subjected to hydrophobic interaction chromatography, circular dichroism, and peptide mapping, they were found to be a mixture of PI and PII. It was then concluded that factors other than just these two residues contribute to correct folding of IGF-II and that the PI and PII of the above two IGF-I mutants assume different conformation at neutral pH but similar conformation under the RP-HPLC condition.
Amgen Inc., Amgen Center, Thousand Oaks, California 91320.
R D
RD
Rosenfeld
N M
NM
Noone
S L
SL
Lauren
M F
MF
Rohde
L O
LO
Narhi
T
T
Arakawa
D000409:Alanine / Q000737:chemistry*; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D007334:Insulin-Like Growth Factor I / Q000737:chemistry* / Q000235:genetics*; D007930:Leucine / Q000737:chemistry*; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology*; D010449:Peptide Mapping; D011487:Protein Conformation; D017510:Protein Folding; D017386:Sequence Homology, Amino Acid
D004220:Disulfides; D007334:Insulin-Like Growth Factor I; D001120:Arginine; D007930:Leucine; D000409:Alanine