Thermal stability of calmodulin and mutants studied by (1)H-(15)N HSQC NMR measurements of selectively labeled [(15)N]Ile proteins.


Abstract

Calmodulin, the Ca(2+)-dependent activator of many cellular processes, contains two well-defined structural domains, each of which binds two Ca(2+) ions. In its Ca(2+)-free (apo) form, it provides an attractive model for studying mechanisms of protein unfolding, exhibiting two separable, reversible processes, indicating two structurally autonomous folding units. (1)H-(15)N HSQC NMR in principle offers a detailed picture of the behavior of individual residues during protein unfolding transitions, but is limited by the lack of dispersion of resonances in the unfolded state. In this work, we have used selective [(15)N]Ile labeling of four distinctive positions in each calmodulin domain to monitor the relative thermal stability of the folding units in wild-type apocalmodulin and in mutants in which either the N- or C-domain is destabilized. These mutations lead to a characteristic perturbation of the stability (T(m)) of the nonmutated domain relative to that of wild-type apocalmodulin. The ability to monitor specific (15)N-labeled residues, well-distributed throughout the domain, provides strong evidence for the autonomy of a given folding unit, as well as providing accurate measurements of the unfolding parameters T(m) and DeltaH(m). The thermodynamic parameters are interpreted in terms of interactions between one folded and one unfolded domain of apocalmodulin, where stabilization on the order of a few kilocalories per mole is sufficient to cause significant changes in the observed unfolding behavior of a given folding unit. The selective (15)N labeling approach is thus a general method that can provide detailed information about structural intermediates populated in complex protein unfolding processes. Study holds ProTherm entries: 15195, 15196, 15197, 15198, 15199, 15200, 15201, 15202, 15203, 15204, 15205, 15206, 15207, 15208, 15209, 15210, 15211, 15212, 15213, 15214, 15215, 15216, 15217, 15218, 15219, 15220 Extra Details: N-Domain; Thermal unfolding was monitored using 1H at Ile9 Ca(2+)-dependent activator; structural domains; thermal stability; structural intermediate

Submission Details

ID: MJAt3Eer3

Submitter: Connie Wang

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

Version: 1

Publication Details
Biekofsky RR;Martin SR;McCormick JE;Masino L;Fefeu S;Bayley PM;Feeney J,Biochemistry (2002) Thermal stability of calmodulin and mutants studied by (1)H-(15)N HSQC NMR measurements of selectively labeled [(15)N]Ile proteins. PMID:12022890
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
4QNH 2014-06-17T00:00:00+0000 2.02 Calcium-calmodulin (T79D) complexed with the calmodulin binding domain from a small conductance potassium channel SK2-a
6ALE 2017-08-07T00:00:00+0000 2.5 A V-to-F substitution in SK2 channels causes Ca2+ hypersensitivity and improves locomotion in a C. elegans ALS model
1OOJ 2003-03-03T00:00:00+0000 2.11 Structural genomics of Caenorhabditis elegans : Calmodulin
2LV6 2012-06-29T00:00:00+0000 0 The complex between Ca-Calmodulin and skeletal muscle myosin light chain kinase from combination of NMR and aqueous and contrast-matched SAXS data
2MES 2013-09-26T00:00:00+0000 0 Backbone 1H, 13C, 15N resonance assignments of calcium-bound calmodulin in complex with PSD95 N-terminal peptide
2RRT 2011-04-27T00:00:00+0000 0 Solution structure of Magnesium-bound form of calmodulin C-domain E104D/E140D mutant
3KF9 2009-10-27T00:00:00+0000 2.6 Crystal structure of the SdCen/skMLCK complex
5H7D 2016-11-17T00:00:00+0000 2.57 Crystal structure of the YgjG-protein A-Zpa963-calmodulin complex
2BBM 1992-07-16T00:00:00+0000 0 SOLUTION STRUCTURE OF A CALMODULIN-TARGET PEPTIDE COMPLEX BY MULTIDIMENSIONAL NMR
2BBN 1992-07-16T00:00:00+0000 0 SOLUTION STRUCTURE OF A CALMODULIN-TARGET PEPTIDE COMPLEX BY MULTIDIMENSIONAL NMR

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 B Calmodulin P20689 MYLK2_RAT
100.0 B Calmodulin P07313 MYLK2_RABIT
100.0 B Calmodulin Q8VCR8 MYLK2_MOUSE
100.0 B Calmodulin Q9H1R3 MYLK2_HUMAN
100.0 B Calmodulin A4IFM7 MYLK2_BOVIN
93.6 Calmodulin P18061 CALM_TRYCR
93.6 Calmodulin P69098 CALM_TRYBG
93.6 Calmodulin P69097 CALM_TRYBB
100.0 A Calmodulin P62184 CALM_RENRE
100.0 A Calmodulin P11121 CALM_PYUSP
100.0 A Calmodulin P02595 CALM_PATSP
92.3 A Calmodulin P53440 CALMF_NAEGR
94.3 Calmodulin P11118 CALM_EUGGR
100.0 A Calmodulin P02594 CALM_ELEEL
91.4 A Calmodulin P05932 CALMB_ARBPU
96.5 A Calmodulin Q9HFY6 CALM_BLAEM
100.0 A Calmodulin Q40302 CALM_MACPY
91.9 A Calmodulin P62150 CALM_ORYLA
96.6 Calmodulin O02367 CALM_CIOIN
91.2 A Calmodulin P27166 CALM_STYLE
91.3 Calmodulin O97341 CALM_SUBDO
91.3 Calmodulin A8I1Q0 CALM_HETTR
92.6 A Calmodulin P02598 CALM_TETPY
92.6 A Calmodulin A3E4D8 CALM_PROMN
92.6 A Calmodulin A3E3H0 CALM_PFIPI
92.6 A Calmodulin A3E4F9 CALM_KARVE
92.6 A Calmodulin A4UHC0 CALM_ALEFU
92.6 A Calmodulin O96081 CALMB_HALRO
92.6 A Calmodulin P62146 CALMA_ARBPU
99.1 Calmodulin Q95NR9 CALM_METSE
98.3 Calmodulin Q7T3T2 CALM_EPIAK
99.1 Calmodulin P0DP35 CAM2B_XENLA
99.1 Calmodulin P0DP34 CAM2A_XENLA
99.1 Calmodulin P62151 CALM_TETCF
99.1 Calmodulin P21251 CALM_STIJA
99.1 Calmodulin Q6YNX6 CALM_SHEEP
99.1 Calmodulin P62160 CALM_RABIT
99.1 Calmodulin Q5RAD2 CALM_PONAB
99.1 Calmodulin Q71UH6 CALM_PERFV
99.1 Calmodulin P62156 CALM_ONCSP
99.1 Calmodulin Q6PI52 CALM_DANRE
99.1 Calmodulin Q6IT78 CALM_CTEID
99.1 Calmodulin P62149 CALM_CHICK
99.1 Calmodulin P62157 CALM_BOVIN
99.1 Calmodulin P62144 CALM_ANAPL
99.1 Calmodulin P0DP31 CALM3_RAT
99.1 Calmodulin P0DP28 CALM3_MOUSE
99.1 Calmodulin P0DP25 CALM3_HUMAN
99.1 Calmodulin P0DP30 CALM2_RAT
99.1 Calmodulin P0DP27 CALM2_MOUSE
99.1 Calmodulin P0DP24 CALM2_HUMAN
100.0 Calmodulin Q9UB37 CALM2_BRALA
99.1 Calmodulin P0DP33 CALM1_XENLA
99.1 Calmodulin P0DP29 CALM1_RAT
99.1 Calmodulin P0DP26 CALM1_MOUSE
99.1 Calmodulin P0DP23 CALM1_HUMAN
98.6 Calmodulin Q8STF0 CALM_STRIE
100.0 Calmodulin P62154 CALM_LOCMI
100.0 Calmodulin P62152 CALM_DROME
100.0 Calmodulin P62145 CALM_APLCA
100.0 Calmodulin P62153 CALMA_HALRO
100.0 Calmodulin P62148 CALM1_BRALA
100.0 Calmodulin P62147 CALM1_BRAFL
98.6 A Calmodulin O16305 CALM_CAEEL