The structural basis of the thermostability of SP1, a novel plant (Populus tremula) boiling stable protein.
We previously reported on a new boiling stable protein isolated from aspen plants (Populus tremula), which we named SP1. SP1 is a stress-related protein with no significant sequence homology to other stress-related proteins. It is a 108-amino-acid hydrophilic polypeptide with a molecular mass of 12.4 kDa (Wang, W. X., Pelah, D., Alergand, T., Shoseyov, O., and Altman, A. (2002) Plant Physiol. 130, 865-875) and is found in an oligomeric form. Preliminary electron microscopy studies and matrix-assisted laser desorption ionization time-of-flight mass spectrometry experiments showed that SP1 is a dodecamer composed of two stacking hexamers. We performed a SDS-PAGE analysis, a differential scanning calorimetric study, and crystal structure determination to further characterize SP1. SDS-PAGE indicated a spontaneous assembly of SP1 to one stable oligomeric form, a dodecamer. Differential scanning calorimetric showed that SP1 has high thermostability i.e. Tm of 107 degrees C (at pH 7.8). The crystal structure of SP1 was initially determined to 2.4 A resolution by multi-wavelength anomalous dispersion method from a crystal belonging to the space group I422. The phases were extended to 1.8 A resolution using data from a different crystal form (P21). The final refined molecule includes 106 of the 108 residues and 132 water molecules (on average for each chain). The R-free is 20.1%. The crystal structure indicated that the SP1 molecule has a ferredoxin-like fold. Strong interactions between each two molecules create a stable dimer. Six dimers associate to form a ring-like-shaped dodecamer strongly resembling the particle visualized in the electron microscopy studies. No structural similarity was found between the crystal structure of SP1 and the crystal structure of other stress-related proteins such as small heat shock proteins, whose structure has been already determined. This structural study further supports our previous report that SP1 may represent a new family of stress-related proteins with high thermostability and oligomerization.
Dgany
Or
O
Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University, Beer-Sheva 84105, Israel.
Gonzalez
Ana
A
Sofer
Oshrat
O
Wang
Wangxia
W
Zolotnitsky
Gennady
G
Wolf
Amnon
A
Shoham
Yuval
Y
Altman
Arie
A
Wolf
Sharon G
SG
Shoseyov
Oded
O
Almog
Orna
O
D000595:Amino Acid Sequence; D002152:Calorimetry, Differential Scanning; D018360:Crystallography, X-Ray; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D018698:Glutamic Acid / Q000737:chemistry; D006360:Heat-Shock Proteins / Q000737:chemistry*; D008854:Microscopy, Electron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry*; D032107:Populus / Q000378:metabolism*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
D006360:Heat-Shock Proteins; D010455:Peptides; D010940:Plant Proteins; D018698:Glutamic Acid