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
6YJ0 2020-04-02T00:00:00+0000 0 Solution NMR structure of titin N2A region Ig domain I83
1BPV 1998-08-11T00:00:00+0000 0 TITIN MODULE A71 FROM HUMAN CARDIAC MUSCLE, NMR, 50 STRUCTURES
1G1C 2000-10-11T00:00:00+0000 2.1 I1 DOMAIN FROM TITIN
1NCT 1996-08-13T00:00:00+0000 0 TITIN MODULE M5, N-TERMINALLY EXTENDED, NMR
1NCU 1996-08-13T00:00:00+0000 0 Titin Module M5, N-terminally Extended, NMR
1TIT 1996-02-02T00:00:00+0000 0 TITIN, IG REPEAT 27, NMR, MINIMIZED AVERAGE STRUCTURE
1TIU 1996-02-02T00:00:00+0000 0 TITIN, IG REPEAT 27, NMR, 24 STRUCTURES
1TKI 1998-05-29T00:00:00+0000 2.0 AUTOINHIBITED SERINE KINASE DOMAIN OF THE GIANT MUSCLE PROTEIN TITIN
1TNM 1995-01-17T00:00:00+0000 0 TERTIARY STRUCTURE OF AN IMMUNOGLOBULIN-LIKE DOMAIN FROM THE GIANT MUSCLE PROTEIN TITIN: A NEW MEMBER OF THE I SET
1TNN 1995-01-17T00:00:00+0000 0 Tertiary structure of an immunoglobulin-like domain from the giant muscle protein titin: a new member of the I set

Relevant UniProtKB Entries

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