PDBsum entry: 1j81

Hydrolase

PDB id: 1j81

Contents

Protein chains

16 a.a.

101 a.a. *

Ligands

SO4

Waters ×39

* Residue conservation analysis

PDB id:

1j81

Name:

Hydrolase

Title:

Osmolyte stabilization of rnase

Structure:

Ribonuclease pancreatic. Chain: a. Fragment: s peptide. Synonym: rnase s. Engineered: yes. Ribonuclease pancreatic. Chain: b. Fragment: s protein. Synonym: rnase s.

Source:

Synthetic: yes. Other_details: this peptide was chemically synthesized. It is naturally found in bos taurus. Bos taurus. Cattle. Organism_taxid: 9913. Organ: pancreas

Biol. unit:

Dimer (from PQS)

Resolution:

2.20Å R-factor: 0.210 R-free: 0.250

Authors:

G.S.Ratnaparkhi,R.Varadarajan

Key ref:

G.S.Ratnaparkhi and R.Varadarajan (2001). Osmolytes stabilize ribonuclease S by stabilizing its fragments S protein and S peptide to compact folding-competent states. J Biol Chem, 276, 28789-28798. PubMed id: 11373282 DOI: 10.1074/jbc.M101906200

Date:

19-May-01 Release date: 06-Jun-01

Protein chain

P61823  (RNAS1_BOVIN) -  Ribonuclease pancreatic

Seq: 150 a.a.

Struc: 16 a.a.*

Protein chain

P61823  (RNAS1_BOVIN) -  Ribonuclease pancreatic

Seq: 150 a.a.

Struc: 101 a.a.

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: Chains A, B: E.C.3.1.27.5 - Pancreatic ribonuclease.
• Reaction: Endonucleolytic cleavage to nucleoside 3'-phosphates and 3'-phosphooligonucleotides ending in C-P or U-P with 2',3'-cyclic phosphate intermediates.

 Gene Ontology (GO) functional annotation 

• Biochemical function: nucleic acid binding (2 terms)

DOI no: 10.1074/jbc.M101906200

J Biol Chem 276:28789-28798 (2001)

PubMed id: 11373282

Osmolytes stabilize ribonuclease S by stabilizing its fragments S protein and S peptide to compact folding-competent states.

G.S.Ratnaparkhi, R.Varadarajan.

ABSTRACT

Osmolytes stabilize proteins to thermal and chemical denaturation. We have studied the effects of the osmolytes sarcosine, betaine, trimethylamine-N-oxide, and taurine on the structure and stability of the protein.peptide complex RNase S using x-ray crystallography and titration calorimetry, respectively. The largest degree of stabilization is achieved with 6 m sarcosine, which increases the denaturation temperatures of RNase S and S pro by 24.6 and 17.4 degrees C, respectively, at pH 5 and protects both proteins against tryptic cleavage. Four crystal structures of RNase S in the presence of different osmolytes do not offer any evidence for osmolyte binding to the folded state of the protein or any perturbation in the water structure surrounding the protein. The degree of stabilization in 6 m sarcosine increases with temperature, ranging from -0.52 kcal mol(-1) at 20 degrees C to -5.4 kcal mol(-1) at 60 degrees C. The data support the thesis that osmolytes that stabilize proteins, do so by perturbing unfolded states, which change conformation to a compact, folding competent state in the presence of osmolyte. The increased stabilization thus results from a decrease in conformational entropy of the unfolded state.

Selected figure(s)

Figure 3.
Fig. 3. Effect of sarcosine on the tryptic cleavage of S pro. A, tryptic cleavage of S pro in the absence (lanes 1-5) and presence (lanes 6-10) of 6 M sarcosine. The time points are 0, 10, 20, 30, and 60 min for each case. The arrows indicate the intact S pro and a protected fragment (S pro-(38-124)) in the presence of sarcosine. B, the effect of 6 M sarcosine on the cleavage of a fluorescent substrate of trypsin, BMAAC at pH 8 ( squares) and pH 7 (circles). In the presence of 6 M sarcosine (filled symbols), the rate of trypsin cleavage is half of that in its absence. The rate of cleavage of substrate by trypsin is equal in the absence and presence of 6 M sarcosine if twice the concentration of trypsin is used in the presence of 6 M sarcosine. C, potential sites for tryptic cleavage of S pro (indicated by arrows). The shaded residues indicate the fragment protected (S pro-(38-124)) in the presence of sarcosine. The residue numbering is based on the sequence of RNase A.

Figure 4.
Fig. 4. Effect of osmolytes on the crystal structure of RNase S. The background error for r.m.s.d. and B-factor plots was determined by comparison between the control structures obtained in the absence of osmolyte. A, r.m.s.d. plot for all osmolytes. MC and SC r.m.s.d. values are represented by solid and dashed lines, respectively. The large side-chain r.m.s.d. values are generally due to lack of density for side chains on the surface. The structure was superposed on its control structure before calculating the r.m.s.d. B, the restrained B-factor per residue for the control structure was subtracted from the corresponding B-factor of the structure of interest (osmolyte control) to get the B-factor plot. The filled bars indicate the MC B-factors and the lines represent the SC B-factors. The secondary structure representation on top of the panel indicates helices ( circle ), -strand ( ), and loops/turns ( ). C, r.m.s.d.; D, B-factor plot for the crystallographic water molecules surrounding the osmolyte-soaked proteins. The r.m.s.d. and B-factors were calculated as described above.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2001, 276, 28789-28798) copyright 2001.

Figures were selected by the author.