Examination of the slow unfolding of pro-nerve growth factor argues against a loop threading mechanism for nerve growth factor.
Nerve growth factor (NGF), a member of the neurotrophin family, is an all-beta-sheet protein with a characteristic structure motif, the cystine knot. Unfolding of NGF in 6 M GdnHCl has been described previously to involve an initial partial loss of structure and a subsequent very slow conversion to a second, completely unfolded state. This latter conversion was postulated to represent a back-threading of the disulfide bond that passes through the cystine knot (loop threading hypothesis). Here, this hypothesis was questioned with the pro form of the protein (proNGF). In proNGF, the mature part is preceded by the 103-amino acid pro-peptide. Consequently, loop threading of the N-terminally extended protein should be significantly delayed. However, unfolding kinetics of proNGF monitored by RP-HPLC, intrinsic fluorescence, and NMR spectroscopy were comparable to those of mature NGF. Time-resolved (1)H-(15)N HSQC spectra revealed a slow time-dependent loss of residual structure of which the kinetics correlated well with the transition observed by RP-HPLC. Refolding from the completely unfolded state led to a partial recovery of natively folded proNGF. In summary, the sequential unfolding of proNGF only marginally differed from that of mature NGF. Therefore, it is very unlikely that a loop threading mechanism is the cause of the slow unfolding step.
Institut für Biotechnologie der Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120 Halle, Germany.
Marco
M
Kliemannel
Ulrich
U
Weininger
Jochen
J
Balbach
Elisabeth
E
Schwarz
Rainer
R
Rudolph
D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003553:Cystine / Q000737:chemistry; D019277:Entropy; D019791:Guanidine / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D020932:Nerve Growth Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry*; D020673:Protein Renaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors
D010455:Peptides; D011498:Protein Precursors; D011994:Recombinant Proteins; D003553:Cystine; D020932:Nerve Growth Factor; D019791:Guanidine