Fine structure analysis of a protein folding transition state; distinguishing between hydrophobic stabilization and specific packing.


Abstract

Developing a detailed understanding of the structure and energetics of protein folding transition states is a key step in describing the folding process. The phi-value analysis approach allows the energetic contribution of side-chains to be mapped out by comparing wild-type with individual mutants where conservative changes are introduced. Studies where multiple substitutions are made at individual sites are much rarer but are potentially very useful for understanding the contribution of each element of a side-chain to transition state formation, and for distinguishing the relative importance of specific packing versus hydrophobic interactions. We have made a series of conservative mutations at multiple buried sites in the N-terminal domain of L9 in order to assess the relative importance of specific side-chain packing versus less specific hydrophobic stabilization of the transition state. A total of 28 variants were prepared using both naturally occurring and non-naturally occurring amino acids at six sites. Analysis of the mutants by NMR and CD showed no perturbation of the structure. There is no correlation between changes in hydrophobicity and changes in stability. In contrast, there is excellent linear correlation between the hydrophobicity of a side-chain and the log of the folding rate, ln(k(f)). The correlation between ln(k(f)) and the change in hydrophobicity holds even for substitutions that change the shape and/or size of a side-chain significantly. For most sites, the correlation with the logarithm of the unfolding rate, ln(k(u)), is much worse. Mutants with more hydrophobic amino acid substitutions fold faster, and those with less hydrophobic amino acid substitutions fold slower. The results show that hydrophobic interactions amongst core residues are an important driving force for forming the transition state, and are more important than specific tight packing interactions. Finally, a number of substitutions lead to negative phi-values and the origin of these effects are described. Study holds ProTherm entries: 18827, 18828, 18829, 18830, 18831, 18832, 18833, 18834, 18835, 18836, 18837, 18838, 18839, 18840, 18841, 18842 Extra Details: N-terminal domain (residues 1-56) protein folding; phi-values; ribosomal protein L9; unnatural amino acids; transition state

Submission Details

ID: ZFHhdzCd

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:50 p.m.

Version: 1

Publication Details
Anil B;Sato S;Cho JH;Raleigh DP,J. Mol. Biol. (2005) Fine structure analysis of a protein folding transition state; distinguishing between hydrophobic stabilization and specific packing. PMID:16246369
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