Exploring local and non-local interactions for protein stability by structural motif engineering.


Abstract

In order to probe the relative contribution of local and non-local interactions to the thermodynamic stability of proteins, we have devised an experimental approach based on a combination of motif engineering and sequence shuffling. Candidate chain segments in an immunoglobulin V(L) domain were identified whose conformation is proposed to be dominated by non-local interactions. Locally interacting structural motifs of a different conformation were then constructed as replacements, by introducing motif consensus sequences. We find that all nine replacements we constructed systematically reduce the folding cooperativity. By comparing this destabilising effect with the folding transitions of shuffled sequences for three of these motifs, we estimate the contribution of local, native interactions to the free energy of folding. Our results suggest that local and non-local interactions contribute to stability by an approximately equal amount, but that local interactions stabilise by increasing the resistance to denaturation while non-local interactions increase folding cooperativity. The systematic loss of stability by sequence shuffling in these host-guest experiments suggests that the designed interactions indeed are present in the native state, thus consensus sequence engineering may be a useful tool in structure design, but non-local interactions must be taken into account for global stability engineering. Statistical approaches are powerful tools for engineering protein structure and stability, but an analysis based on local sequence propensities alone does not adequately represent the balance of sequence and context in protein structures. Study holds ProTherm entries: 8269, 8270, 8271, 8272, 8273, 8274, 8275, 8276, 8277, 8278 Extra Details: protein engineering; protein stability; protein folding; structural motifs;,immunoglobulin variable domains

Submission Details

ID: 7Zb5oGmx

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:35 p.m.

Version: 1

Publication Details
Niggemann M;Steipe B,J. Mol. Biol. (2000) Exploring local and non-local interactions for protein stability by structural motif engineering. PMID:10656826
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
2Q20 2008-04-08 1.3 Structure of the germline Vk1 O18/O8 light chain variable domain homodimer
1EEQ 2001-02-01 1.5 M4L/Y(27D)D/T94H Mutant of LEN
3CDC 2008-09-02 1.53 kI O18/O8 N34I/Y87H immunoglobulin light chain variable domain
3CDF 2008-09-02 1.53 kI O18/O8 Y87H immunoglobulin light chain variable domain
1EFQ 2001-02-09 1.6 Q38D mutant of LEN
1EEU 2001-02-03 1.6 M4L/Y(27D)D/Q89D/T94H mutant of LEN
4L1H 2014-06-04 1.68 Bence-Jones immunoglobulin REI variable portion with seven point mutations
1BWW 1998-10-07 1.7 BENCE-JONES IMMUNOGLOBULIN REI VARIABLE PORTION, T39K MUTANT
1QAC 2000-02-23 1.8 CHANGE IN DIMERIZATION MODE BY REMOVAL OF A SINGLE UNSATISFIED POLAR RESIDUE
1B0W 1998-11-16 1.8 Structural comparison of amyloidogenic light chain dimer in two crystal forms with nonamyloidogenic counterparts
1EK3 2001-03-06 1.9 KAPPA-4 IMMUNOGLOBULIN VL, REC
1WTL 1994-11-01 1.9 COMPARISON OF CRYSTAL STRUCTURES OF TWO HOMOLOGOUS PROTEINS: STRUCTURAL ORIGIN OF ALTERED DOMAIN INTERACTIONS IN IMMUNOGLOBULIN LIGHT CHAIN DIMERS
1LVE 1998-01-21 1.95 STRUCTURE OF THE VARIABLE DOMAIN OF HUMAN IMMUNOGLOBULIN K-4 LIGHT CHAIN LEN
3LVE 1999-05-18 2.0 LEN Q38E MUTANT: A DOMAIN FLIP FROM A SINGLE AMINO ACID SUBSTITUTION
1REI 1976-05-19 2.0 THE MOLECULAR STRUCTURE OF A DIMER COMPOSED OF THE VARIABLE PORTIONS OF THE BENCE-JONES PROTEIN REI REFINED AT 2.0 ANGSTROMS RESOLUTION
5LVE 2000-02-18 2.0 STRUCTURE OF THE VARIABLE DOMAIN OF HUMAN IMMUNOGLOBULIN K-4 LIGHT CHAIN LEN
1QP1 1999-06-17 2.06 KAPPA VARIABLE LIGHT CHAIN
1JV5 2002-01-09 2.2 Anti-blood group A Fv
4LVE 1999-05-18 2.3 LEN K30T MUTANT: A DOMAIN FLIP AS A RESULT OF A SINGLE AMINO ACID SUBSTITUTION
3CDY 2008-09-02 2.43 AL-09 H87Y, immunoglobulin light chain variable domain
2LVE 1999-05-18 2.7 RECOMBINANT LEN
1AR2 1997-11-12 2.8 DISULFIDE-FREE IMMUNOGLOBULIN FRAGMENT
4K07 2013-10-30 2.83 Crystal structure of the amyloid-forming immunoglobulin AL-103 cis-proline 95 mutant
5XP1 2017-08-02 2.88 Structure of monomeric mutant of REI immunoglobulin light chain variable domain crystallized at pH 6

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Immunoglobulin kappa variable 1D-33 P01593 KVD33_HUMAN
100.0 Immunoglobulin kappa variable 1D-33 P01594 KV133_HUMAN
99.0 Immunoglobulin kappa variable 4-1 P06312 KV401_HUMAN