Secondary structure changes stabilize the reactive-centre cleaved form of SERPINs. A study by 1H nuclear magnetic resonance and Fourier transform infrared spectroscopy.
Proteinase inhibitor members of the SERPIN superfamily are characterized by the presence of a proteolytically sensitive reactive-site loop. Cleavage within this region results in a conformational transition from an unstable "stressed" native protein to a more stable "relaxed" cleaved molecule. In order to identify the principal molecular aspects of this transition, 1H nuclear magnetic resonance (n.m.r.) and FT-IR spectroscopy were applied to the study of four SERPINs. 1H n.m.r. spectra of approximately 20 high-field ring-current-shifted methyl signals exhibited slightly different chemical shifts in the native and cleaved forms of alpha 1-antitrypsin (alpha 1-AT), alpha 1-antichymotrypsin (alpha 1-ACT) and C1 inhibitor (C1-INH), but not ovalbumin, between 20 degrees C and 90 degrees C. Ring current calculations based on crystal co-ordinates for cleaved alpha 1-AT and alpha 1-ACT and native ovalbumin showed that these signals originate from highly localized interactions between different buried residues corresponding to alpha-helix and beta-sheet segments of the SERPIN fold. The small shift changes correspond to small relative conformational side-chain rearrangements of about 0.01 nm to 0.05 nm in the protein hydrophobic core, i.e. the tertiary structure interactions in the two forms of the SERPIN fold are well-preserved, and changes in this appear unimportant for the stabilization found after reactive centre cleavage. Fourier transform infrared (FT-IR) spectroscopic studies of the amide I band showed that the native and cleaved forms of alpha 1-AT, alpha 1-ACT and C1-INH contain 28% to 36% alpha-helix and 38% to 44% beta-sheet. Second derivative FT-IR spectra using H2O and 2H2O buffers revealed very large differences in the amide I band between the native and cleaved forms of alpha 1-AT, alpha 1-ACT and C1-INH, but not for ovalbumin. The alpha-helix band was most sensitive to 1H-2H exchange, while the beta-sheet bands were not, and greater amounts of antiparallel beta-sheet were detected in the cleaved form. 1H n.m.r. showed that polypeptide amide 1H-2H exchange was greater in the native forms of alpha 1-AT, alpha 1-ACT and C1-INH than in their cleaved forms, whereas for ovalbumin it was unchanged. The FT-IR and 1H-2H exchange data show that alterations in the secondary structure are central to the stabilization of the cleaved SERPIN structure.(ABSTRACT TRUNCATED AT 400 WORDS)
Perkins
S J
SJ
Department of Biochemistry and Chemistry, Royal Free Hospital School of Medicine, London, U.K.
Smith
K F
KF
Nealis
A S
AS
Haris
P I
PI
Chapman
D
D
Bauer
C J
CJ
Harrison
R A
RA
D003174:Complement C1 Inactivator Proteins / Q000737:chemistry / Q000378:metabolism; D005583:Fourier Analysis; D006859:Hydrogen / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010047:Ovalbumin / Q000737:chemistry; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011522:Protons; D012697:Serine Endopeptidases / Q000378:metabolism*; D015843:Serpins / Q000737:chemistry* / Q000378:metabolism; D013055:Spectrophotometry, Infrared; D000514:alpha 1-Antichymotrypsin / Q000737:chemistry / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000737:chemistry / Q000378:metabolism
D003174:Complement C1 Inactivator Proteins; D010446:Peptide Fragments; D011522:Protons; D015843:Serpins; D000514:alpha 1-Antichymotrypsin; D000515:alpha 1-Antitrypsin; C038141:plakalbumin; D006859:Hydrogen; D010047:Ovalbumin; D012697:Serine Endopeptidases