Biophysical comparison of BMP-2, ProBMP-2, and the free pro-peptide reveals stabilization of the pro-peptide by the mature growth factor.
Pro-forms of growth factors have received intensive scientific attention recently because in some cases different biological activities have been ascribed compared with the mature growth factors. Examples are the pro-apoptotic role of the nerve growth factor (NGF) proform (proNGF) or the latency of the transforming growth factor (TGF)-beta pro-form (proTGF-beta). To investigate a possible biological function of the pro-form of bone morphogenetic protein (BMP)-2, a member of the TGF-beta family, mature BMP-2, proBMP-2, and the isolated pro-peptide were recombinantly produced in Escherichia coli cells, and a biophysical comparison was performed. Protocols were developed that allowed efficient refolding and subsequent purification of the proteins. ProBMP-2 could be processed to an N-terminally truncated form of BMP-2, digit removed BMP-2 (drBMP-2), that possessed biological activity, i.e. it induced ectopic bone formation. Bone inducing activity was also displayed by proBMP-2. The three proteins were characterized both by fluorescence and CD spectroscopy. From these analyses, predominant beta-sheet secondary structural elements in the pro-peptide were deduced. The thermodynamic stability of the pro-peptide was determined by chemical unfolding assays. As in the case of NGF/proNGF, the mature part of BMP-2 stabilized the structure of the pro-peptide moiety. However, in contrast to NGF/proNGF, the pro-peptide did not stimulate oxidative folding of the mature part in vitro.
Hillger
Frank
F
Institut für Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120 Halle, Germany.
Herr
Gerhard
G
Rudolph
Rainer
R
Schwarz
Elisabeth
E
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006133:Growth Substances / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008297:Male; D020932:Nerve Growth Factor / Q000737:chemistry; D010100:Oxygen / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D016212:Transforming Growth Factor beta / Q000737:chemistry* / Q000378:metabolism; D014466:Ultraviolet Rays
C525720:Bmp2 protein, rat; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D006133:Growth Substances; D010455:Peptides; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D004247:DNA; D020932:Nerve Growth Factor; D010100:Oxygen