Solution NMR evidence for a cis Tyr-Ala peptide group in the structure of [Pro93Ala] bovine pancreatic ribonuclease A.
Proline peptide group isomerization can result in kinetic barriers in protein folding. In particular, the cis proline peptide conformation at Tyr92-Pro93 of bovine pancreatic ribonuclease A (RNase A) has been proposed to be crucial for chain folding initiation. Mutation of this proline-93 to alanine results in an RNase A molecule, P93A, that exhibits unfolding/refolding kinetics consistent with a cis Tyr92-Ala93 peptide group conformation in the folded structure (Dodge RW, Scheraga HA, 1996, Biochemistry 35:1548-1559). Here, we describe the analysis of backbone proton resonance assignments for P93A together with nuclear Overhauser effect data that provide spectroscopic evidence for a type VI beta-bend conformation with a cis Tyr92-Ala93 peptide group in the folded structure. This is in contrast to the reported X-ray crystal structure of [Pro93Gly]-RNase A (Schultz LW, Hargraves SR, Klink TA, Raines RT, 1998, Protein Sci 7:1620-1625), in which Tyr92-Gly93 forms a type-II beta-bend with a trans peptide group conformation. While a glycine residue at position 93 accommodates a type-II bend (with a positive value of phi93), RNase A molecules with either proline or alanine residues at this position appear to require a cis peptide group with a type-VI beta-bend for proper folding. These results support the view that a cis Pro93 conformation is crucial for proper folding of wild-type RNase A.
Xiong
Y
Y
Center for Advanced Biotechnology and Medicine and Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08854-5638, USA.
Juminaga
D
D
Swapna
G V
GV
Wedemeyer
W J
WJ
Scheraga
H A
HA
Montelione
G T
GT
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010179:Pancreas / Q000201:enzymology; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics; D012996:Solutions
D011994:Recombinant Proteins; D012996:Solutions; D011392:Proline; D012259:Ribonuclease, Pancreatic