pH dependent thermodynamic and amide exchange studies of the C-terminal domain of the ribosomal protein L9: implications for unfolded state structure.


Abstract

It is now recognized that unfolded states of globular proteins are not random coils but instead can contain significant amounts of residual structure. Here, we combine amide H/D exchange studies and thermodynamic measurements to probe pH dependent structure in the unfolded state of the small, mixed alpha-beta protein CTL9. The m value measured by urea denaturation is strongly dependent upon pD, increasing by 40% from pD 7.5 to 4.85. Likewise, the change in heat capacity upon unfolding, deltaCp(o), increases significantly from pD 7.5 to 5.5. These studies argue that the unfolded state contains interactions, presumably hydrophobic in nature, that lead to a more compact state at high pH. The expansion at lower pH correlates with the estimated unfolded state pKa values of the three histidines in CTL9 with additional contributions from acid side chains at the lower pH. Amide H/D exchange studies were conducted at pD 5.0, 6.0, and 7.0. At pD 5.0, the exchange rates could be measured for 44 residues, 29 of which exchanged by global unfolding. No evidence was found for any super protected sites, that is, sites that exchange at rates slower than those expected for global exchange. The estimated precision for the experiments limits detection to residues that are protected 2.3-fold above the intrinsic exchange rate. Thirty-seven residues could be followed at pD 6 and 27 residues at pD 7. Again no evidence for a significant super protected structure was observed. The properties of CTL9(11) are compared to other structured denatured states. Study holds ProTherm entries: 20112, 20113, 20114, 20115 Extra Details: C-Terminal Domain globular proteins, thermodynamic measurements, pH dependent, urea denaturation, hydrophobic, unfolded state.

Submission Details

ID: 6ZZdxRM33

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:52 p.m.

Version: 1

Publication Details
Li Y;Horng JC;Raleigh DP,Biochemistry (2006) pH dependent thermodynamic and amide exchange studies of the C-terminal domain of the ribosomal protein L9: implications for unfolded state structure. PMID:16834323
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1CQU 2002-04-27 SOLUTION STRUCTURE OF THE N-TERMINAL DOMAIN OF RIBOSOMAL PROTEIN L9
2HBA 2007-05-29 1.25 Crystal Structure of N-terminal Domain of Ribosomal Protein L9 (NTL9) K12M
2HVF 2007-06-12 1.57 Crystal Structure of N-terminal Domain of Ribosomal Protein L9 (NTL9), G34dA
2HBB 2007-05-29 1.9 Crystal Structure of the N-terminal Domain of Ribosomal Protein L9 (NTL9)
1DIV 1997-01-11 2.6 RIBOSOMAL PROTEIN L9
487D 2000-04-10 7.5 SEVEN RIBOSOMAL PROTEINS FITTED TO A CRYO-ELECTRON MICROSCOPIC MAP OF THE LARGE 50S SUBUNIT AT 7.5 ANGSTROMS RESOLUTION

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
95.3 50S ribosomal protein L9 Q5KU74 RL9_GEOKA
96.6 50S ribosomal protein L9 A4ITV1 RL9_GEOTN
100.0 50S ribosomal protein L9 P02417 RL9_GEOSE