Probing the conformational state of apomyoglobin by limited proteolysis.
We show here that limited proteolysis can probe the structural and dynamic differences between the holo and apo form of horse myoglobin (Mb). Initial nicking of the polypeptide chain of apoMb (153 amino acid residues, no disulfide bonds) by several proteases (subtilisin, thermolysin, chymotrypsin and trypsin) occurs at the level of chain segment 89-96. In contrast, holoMb is resistant to proteolytic digestion when reacted under identical experimental conditions. Such selective proteolysis implies that the F-helix of native holoMb (residues 82 to 97) is disordered in apoMb, thus enabling binding and adaptation of this chain segment at the active site of the proteolytic enzymes for an efficient peptide bond fission. That essentially only the F-helix in apoMb is largely disrupted was earlier inferred from spectroscopic measurements and molecular dynamics simulations. The results of this study provide direct experimental evidence for this and emphasize therefore that limited proteolysis is a useful and reliable method for probing structure and dynamics of proteins, complementing other experimental techniques such as NMR and X-ray crystallography.
Fontana
A
A
CRIBI Biotechnology Centre, University of Padua, Italy.
Zambonin
M
M
Polverino de Laureto
P
P
De Filippis
V
V
Clementi
A
A
Scaramella
E
E
D000595:Amino Acid Sequence; D001059:Apoproteins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D013379:Substrate Specificity; D013381:Subtilisins / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D013820:Thermolysin / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
D001059:Apoproteins; D009211:Myoglobin; C010857:apomyoglobin; D013381:Subtilisins; D002918:Chymotrypsin; D014357:Trypsin; D013820:Thermolysin