Purification, characterization, and structural analysis of a plant low-temperature-induced protein.
We have purified to near homogeneity a recombinant form of the protein BN28 (rBN28), expressed in response to low temperature in Brassica napus plants, and we have determined its solution structure. Antibodies raised against rBN28 were used to characterize the recombinant and native proteins. Similar to many other low-temperature-induced proteins, BN28 is extremely hydrophilic, such that it remains soluble following boiling. Immunoblot analysis of subcellular fractions indicated that BN28 was not strongly associated with cellular membranes and was localized exclusively within the soluble fraction of the cell. Contrary to predicted secondary structure that suggested significant helical content, circular dichroism analysis revealed that rBN28 existed in aqueous solution largely as a random coil. However, the helical propensity of the protein could be demonstrated in the presence of trifluoroethanol. Nuclear magnetic resonance analysis further showed that rBN28 was in fact completely unstructured (100% coil) in aqueous solution. Although it had earlier been speculated that BN28-like proteins from Arabidopsis thaliana might possess antifreeze protein activity (S. Kurkela and M. Franck [1990] Plant Mol Biol 15: 137-144), no such activity could be detected in ice recrystallization assays with rBN28.
Boothe
J G
JG
Department of Agriculture, Food and Nutritional Science, University of Alberta, Edmonton, Canada.
Sönnichsen
F D
FD
de Beus
M D
MD
Johnson-Flanagan
A M
AM
D000595:Amino Acid Sequence; D001937:Brassica / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003080:Cold Temperature; D015870:Gene Expression; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D007053:Ice; D015151:Immunoblotting; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010940:Plant Proteins; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000737:chemistry
C104898:BN28 protein, Brassica napus; D006360:Heat-Shock Proteins; D007053:Ice; D010940:Plant Proteins; D011994:Recombinant Proteins