Mutational analysis demonstrates that specific electrostatic interactions can play a key role in the denatured state ensemble of proteins.


Abstract

The nature of the denatured state ensemble has been controversial for decades owing, in large part, to the difficulty in characterizing the structure and energetics of denatured state interactions. There is increasing evidence for relatively non-specific hydrophobic clustering in the denatured states of some proteins but other types of interactions are much less well characterized. Here, we report the characterization of highly specific electrostatic interactions in the denatured state of a small alpha-beta protein, the N-terminal domain of the ribosomal protein L9 (NTL9). Mutation of Lys12 to Met has been shown to increase the stability of NTL9 significantly through the disruption of denatured state interactions. Here, we describe the analysis of the pH-dependent stability of 13 mutants designed to probe the nature of the Lys12 denatured state interaction. Lys12 is located in a lysine-rich region of the protein but analysis of a set of Lys to Met mutants shows that it plays a unique role in the denatured state. Analysis of mutants of all of the acidic residues in NTL9 shows that Lys12 forms a specific non-native electrostatic interaction with Asp8 in the denatured state ensemble. Thus the distribution of charge-charge interactions in the denatured state ensemble of NTL9 appears to be biased by few key interactions and is very different from that expected in a random coil. We propose that these interactions are not encoded by local sequence effects but rather reflect interactions among residues more distant in sequence. These results demonstrate that electrostatic as well as hydrophobic interactions can play an important role in the denatured state ensemble. Study holds ProTherm entries: 19719, 19720, 19721, 19722, 19723, 19724, 19725, 19726, 19727, 19728, 19729, 19730 Extra Details: denatured state; pKa perturbation; electrostatic interaction; pH-dependent stability change; protein folding

Submission Details

ID: 86NQuDjZ3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:51 p.m.

Version: 1

Publication Details
Cho JH;Raleigh DP,J. Mol. Biol. (2005) Mutational analysis demonstrates that specific electrostatic interactions can play a key role in the denatured state ensemble of proteins. PMID:16165156
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