Overexpression, biophysical characterization, and crystallization of ribonuclease I from Escherichia coli, a broad-specificity enzyme in the RNase T2 family.
We have constructed a strain that overproduces ribonuclease I of Escherichia coli and we have purified large quantities of the enzyme. Data from fluorescence, CD, and DSC measurements showed that it was a very stable protein. The conformation energy determined from urea and guanidine hydrochloride denaturation experiments was 11.5 kcal mol(-1) at pH 7.5. Thermal denaturation studies indicated that it had a T(m) of 64 degrees C at pH 4.0. RNase I belongs to the RNase T2/S-RNase group of endoribonucleases, but near the amino terminus it has an unusually long hydrophilic segment. Part of this was removed in the deletion construct, RNase I Delta(26-38). We have obtained crystals of both RNase I and of an enzyme-G2'p5'G complex in the P2(1) space group and oligonucleotide complexes with both wild type and mutant enzymes. The current study lays the groundwork for extensive investigation into the structure, function, and physical properties of this widely distributed group of ribonucleases.
Padmanabhan
S
S
Department of Medical Biochemistry, Southern Illinois University, Carbondale, Illinois 62901, USA. savita_paddy@hotmail.com
Zhou
K
K
Chu
C Y
CY
Lim
R W
RW
Lim
L W
LW
D000595:Amino Acid Sequence; D001483:Base Sequence; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003001:Cloning, Molecular; D003460:Crystallization; D017931:DNA Primers / Q000235:genetics; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013816:Thermodynamics
D017931:DNA Primers; D004722:Endoribonucleases; C022543:ribonuclease T(2); D012259:Ribonuclease, Pancreatic