The effect of core destabilization on the mechanical resistance of I27.


Abstract

It is still unclear whether mechanical unfolding probes the same pathways as chemical denaturation. To address this point, we have constructed a concatamer of five mutant I27 domains (denoted (I27)(5)*) and used it for mechanical unfolding studies. This protein consists of four copies of the mutant C47S, C63S I27 and a single copy of C63S I27. These mutations severely destabilize I27 (DeltaDeltaG(UN) = 8.7 and 17.9 kJ mol(-1) for C63S I27 and C47S, C63S I27, respectively). Both mutations maintain the hydrogen bond network between the A' and G strands postulated to be the major region of mechanical resistance for I27. Measuring the speed dependence of the force required to unfold (I27)(5)* in triplicate using the atomic force microscope allowed a reliable assessment of the intrinsic unfolding rate constant of the protein to be obtained (2.0 x 10(-3) s(-1)). The rate constant of unfolding measured by chemical denaturation is over fivefold faster (1.1 x 10(-2) s(-1)), suggesting that these techniques probe different unfolding pathways. Also, by comparing the parameters obtained from the mechanical unfolding of a wild-type I27 concatamer with that of (I27)(5)*, we show that although the observed forces are considerably lower, core destabilization has little effect on determining the mechanical sensitivity of this domain. Study holds ProTherm entries: 15280, 15281 Extra Details: 1 mM EDTA and 2 mM dithiothreitol were added in the experiment mechanical unfolding; unfolding rate constant; unfolding pathways; core destabilization

Submission Details

ID: PhmSRkct3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:46 p.m.

Version: 1

Publication Details
Brockwell DJ;Beddard GS;Clarkson J;Zinober RC;Blake AW;Trinick J;Olmsted PD;Smith DA;Radford SE,Biophys. J. (2002) The effect of core destabilization on the mechanical resistance of I27. PMID:12080133
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
1NCT 1996-11-08 TITIN MODULE M5, N-TERMINALLY EXTENDED, NMR
1TIU 1996-07-11 TITIN, IG REPEAT 27, NMR, 24 STRUCTURES
1TNN 1995-04-20 Tertiary structure of an immunoglobulin-like domain from the giant muscle protein titin: a new member of the I set
1TIT 1996-07-11 TITIN, IG REPEAT 27, NMR, MINIMIZED AVERAGE STRUCTURE
1TNM 1995-04-20 TERTIARY STRUCTURE OF AN IMMUNOGLOBULIN-LIKE DOMAIN FROM THE GIANT MUSCLE PROTEIN TITIN: A NEW MEMBER OF THE I SET
1NCU 1996-11-08 Titin Module M5, N-terminally Extended, NMR
1BPV 1999-08-12 TITIN MODULE A71 FROM HUMAN CARDIAC MUSCLE, NMR, 50 STRUCTURES
2RQ8 2010-02-02 Solution NMR structure of titin I27 domain mutant
3PUC 2011-12-21 0.96 Atomic resolution structure of titin domain M7
3KNB 2009-12-01 1.4 Crystal structure of the titin C-terminus in complex with obscurin-like 1
2WP3 2010-02-16 1.48 Crystal structure of the Titin M10-Obscurin like 1 Ig complex
5JDD 2016-09-14 1.53 Crystal structure of I9-I11 tandem from titin (P212121)
3LPW 2010-09-08 1.65 Crystal structure of the FnIII-tandem A77-A78 from the A-band of titin
2BK8 2006-08-09 1.69 M1 domain from titin
2WWK 2010-02-16 1.7 Crystal structure of the Titin M10-Obscurin like 1 Ig F17R mutant complex
5JDJ 2016-08-17 1.74 Crystal structure of domain I10 from titin in space group P212121
1WAA 2006-07-05 1.8 IG27 protein domain
4O00 2014-12-24 1.85 Crystal structure of the Titin A-band domain A3
2Y9R 2011-03-02 1.9 Crystal structure of the M10 domain of Titin
5JDE 2016-09-14 1.9 Crystal structure of I9-I11 tandem from titin (P1)
4C4K 2014-09-24 1.95 Crystal structure of the titin M10-Obscurin Ig domain 1 complex
2J8H 2007-08-21 1.99 Structure of the immunoglobulin tandem repeat A168-A169 of titin
2A38 2006-06-24 2.0 Crystal structure of the N-Terminus of titin
1TKI 1999-06-08 2.0 AUTOINHIBITED SERINE KINASE DOMAIN OF THE GIANT MUSCLE PROTEIN TITIN
3QP3 2012-02-15 2.0 Crystal structure of titin domain M4, tetragonal form
4QEG 2015-07-22 2.0 Crystal structure of domain I10 from titin (space group P41)
5JOE 2016-08-17 2.0 Crystal structure of I81 from titin
3Q5O 2012-02-01 2.05 Crystal structure of human titin domain M10
4JNW 2014-05-14 2.06 Bacterially expressed Titin Kinase
1G1C 2001-10-12 2.1 I1 DOMAIN FROM TITIN
2ILL 2007-02-20 2.2 Anomalous substructure of Titin-A168169
2WWM 2010-02-16 2.3 Crystal structure of the Titin M10-Obscurin like 1 Ig complex in space group P1
5BS0 2016-03-02 2.4 MAGE-A3 Reactive TCR in complex with Titin Epitope in HLA-A1
1YA5 2005-12-20 2.44 Crystal structure of the titin domains z1z2 in complex with telethonin
2J8O 2007-08-21 2.49 Structure of the immunoglobulin tandem repeat of titin A168-A169
3LCY 2011-05-25 2.5 Titin Ig tandem domains A164-A165
2F8V 2006-06-27 2.75 Structure of full length telethonin in complex with the N-terminus of titin
2NZI 2007-02-13 2.9 Crystal structure of domains A168-A170 from titin
6FWX 2019-03-27 3.0 Chimeric titin Z1Z2-Z1Z2 tandem (Z1212) functionalized with a GRGDS exogenous peptide from fibronectin
6I0Y 2018-12-05 3.2 TnaC-stalled ribosome complex with the titin I27 domain folding close to the ribosomal exit tunnel
4UOW 2014-12-17 3.3 Crystal structure of the titin M10-Obscurin Ig domain 1 complex

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
90.9 TITIN A2ASS6 TITIN_MOUSE
100.0 Titin Q8WZ42 TITIN_HUMAN