Investigations of Sso7d catalytic residues by NMR titration shifts and electrostatic calculations.
Sso7d is a small basic protein consisting of 62 amino acids isolated from the thermoacidophilic archeobacterium Sulfolobus solfataricus. The protein is endowed with DNA binding properties, RNase activity, and the capability of rescuing aggregated proteins in the presence of ATP. In this study, the electrostatic properties of Sso7d are investigated by using the Poisson-Boltzmann calculation of the surface potential distribution and following by NMR spectroscopy the proton chemical shift pH titration of acidic residues. Although the details of the catalytic mechanism still have to be defined, the results from NMR experiments confirm the possible involvement of Glu35 as the proton acceptor in the catalytic reaction, as seen by its abnormally high pK(a) value. Poisson-Boltzmann calculations and NMR titration shifts suggest the presence of a possible hydrogen bond between Glu35 and Tyr33, with a consequent rather rigid arrangement at these positions. Comparison with RNase T1 suggests that Tyr7 may be a good candidate for acting as a proton donor in the active site of Sso7d as shown by its low phenolic pK(a) of approximately 9.3. Titration experiments performed with the UpA, a RNA dinucleotide model, showed that the protein residues affected by the interaction are mainly located in a different region with respect to the surface affected by DNA recognition, in good agreement with the surface potential distribution found with electrostatic calculations.
Consonni
Roberto
R
Istituto per lo Studio delle Macromolecole, Lab. NMR, CNR, v. Ampère 56, I-20131 Milan, Italy. roberto@icmnmr.mi.cnr.it
Arosio
Ivana
I
Belloni
Barbara
B
Fogolari
Federico
F
Fusi
Paola
P
Shehi
Erlet
E
Zetta
Lucia
L
D019843:Archaeal Proteins / Q000737:chemistry*; D001224:Aspartic Acid / Q000737:chemistry; D020134:Catalytic Domain; D004268:DNA-Binding Proteins / Q000737:chemistry*; D015226:Dinucleoside Phosphates / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010446:Peptide Fragments / Q000737:chemistry; D010777:Photochemistry / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry; D006163:Ribonuclease T1 / Q000737:chemistry; D012259:Ribonuclease, Pancreatic / Q000737:chemistry; D055672:Static Electricity; D016944:Sulfolobus / Q000201:enzymology*; D013816:Thermodynamics; D017186:Titrimetry
D019843:Archaeal Proteins; D004268:DNA-Binding Proteins; D015226:Dinucleoside Phosphates; D010446:Peptide Fragments; D011994:Recombinant Proteins; C093228:Sso7d protein, Sulfolobus; D001224:Aspartic Acid; C009628:uridylyl-(3'-5')-adenosine; D018698:Glutamic Acid; D006163:Ribonuclease T1; D012259:Ribonuclease, Pancreatic