Comparison of native and recombinant non-phosphorylated human beta-casein: further evidence for a unique beta-casein folding pattern.
Recombinant wild-type non-phosphorylated human beta-casein was obtained from Escherichia coli. Turbidity vs. temperature (T) without Ca(2+) showed wild-type self-association like native except for irreversibility upon T-cycling with the original pattern re-established after concentrated urea/dialysis. With Ca(2+), wild-type was more native-like. Intrinsic Trp fluorescence spectra were similar but with lowered intensity for the wild-type protein. Changes in extrinsic ANS fluorescence from 4 to 37 degrees C showed less exposure of hydrophobic surface for wild-type than native. Trp to ANS fluorescence resonance energy transfer was higher for wild-type than native at 4 degrees C but 2- to 3-fold lower at 37 degrees C. The native protein must be directed by the environment and/or a chaperone to fold into a unique, somewhat flexible, conformation, unaltered by urea during purification. Wild-type protein, with many native properties, does not spontaneously fold to the native conformation, even after solubilization with urea. T-cycling gives a stable conformation that is different from the native.
Bu
Hongyin
H
Department of Biochemistry, School of Medicine, Loma Linda University, Loma Linda, CA 92350, USA.
Hu
Yilin
Y
Sood
Satish M
SM
Slattery
Charles W
CW
D002364:Caseins / Q000096:biosynthesis / Q000737:chemistry* / Q000145:classification* / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D008895:Milk, Human / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009391:Nephelometry and Turbidimetry; D010766:Phosphorylation; D011487:Protein Conformation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D002364:Caseins; D011993:Recombinant Fusion Proteins