Ligand binding and thermodynamic stability of a multidomain protein, calmodulin.


Abstract

Chemical and thermal denaturation of calmodulin has been monitored spectroscopically to determine the stability for the intact protein and its two isolated domains as a function of binding of Ca2+ or Mg2+. The reversible urea unfolding of either isolated apo-domain follows a two-state mechanism with relatively low deltaG(o)20 values of approximately 2.7 (N-domain) and approximately 1.9 kcal/mol (C-domain). The apo-C-domain is significantly unfolded at normal temperatures (20-25 degrees C). The greater affinity of the C-domain for Ca2+ causes it to be more stable than the N-domain at [Ca2+] > or = 0.3 mM. By contrast, Mg2+ causes a greater stabilization of the N- rather than the C-domain, consistent with measured Mg2+ affinities. For the intact protein (+/-Ca2+), the bimodal denaturation profiles can be analyzed to give two deltaG(o)20 values, which differ significantly from those of the isolated domains, with one domain being less stable and one domain more stable. The observed stability of the domains is strongly dependent on solution conditions such as ionic strength, as well as specific effects due to metal ion binding. In the intact protein, different folding intermediates are observed, depending on the ionic composition. The results illustrate that a protein of low intrinsic stability is liable to major perturbation of its unfolding properties by environmental conditions and liganding processes and, by extension, mutation. Hence, the observed stability of an isolated domain may differ significantly from the stability of the same structure in a multidomain protein. These results address questions involved in manipulating the stability of a protein or its domains by site directed mutagenesis and protein engineering. Study holds ProTherm entries: 9571, 9572, 9573, 9574, 9575, 9576, 9577, 9578, 9579, 9580, 9581, 9583, 9584, 9585, 9586, 9587, 9588, 9589, 9590, 9591, 9592, 9593, 9594, 9595, 9596, 9597, 9598, 9599, 9600, 9601, 9602, 9603, 9604, 9605, 9606, 9607, 9608, 9611, 9612, 9615, 9616, 9617, 9618, 9619, 9620, 9621, 9622, 9623, 9624, 9625, 9626, 9627, 9628, 9629, 9630, 9631, 9632, 9633, 9634, 9635, 9636, 9637, 9638, 9639, 9640, 9641, 9642, 9643, 9644, 9645, 9646, 9647, 9648 Extra Details: transition Tr1C; N-terminal tryptic fragments of calmodulin,apo form calcium binding; calmodulin; chemical denaturation;,circular dichroism; thermal denaturation

Submission Details

ID: FNdzbT973

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:38 p.m.

Version: 1

Publication Details
Masino L;Martin SR;Bayley PM,Protein Sci. (2000) Ligand binding and thermodynamic stability of a multidomain protein, calmodulin. PMID:10975573
Additional Information

Study Summary

Number of data points 121
Proteins Calmodulin ; Calmodulin
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: Cm ionic:MgCl2: 100 mM ; Experimental Assay: dG_H2O ionic:MgCl2: 100 mM ; Experimental Assay: Cm ionic:MgCl2: 30 mM ; Experimental Assay: dG_H2O ionic:MgCl2: 30 mM ; Experimental Assay: dG ; Experimental Assay: dG ; Experimental Assay: dG ; Experimental Assay: dG ; Experimental Assay: dG ; Experimental Assay: Cm ionic:KCl: 200 M ; Experimental Assay: dG_H2O ionic:KCl: 200 M ; Experimental Assay: Cm ionic:KCl: 100 M ; Experimental Assay: dG_H2O ionic:KCl: 100 M ; Experimental Assay: Cm ionic:KCl: 30 M ; Experimental Assay: dG_H2O ionic:KCl: 30 M ; Experimental Assay: dG pH:7.5, buffers:HEPES: 10 mM, ionic:KCl: 0.2 M ; Experimental Assay: dG pH:7.5, ionic:KCl: 0.1 M, buffers:HEPES: 10 mM ; Experimental Assay: dG pH:7.5, ionic:: , buffers:HEPES: 10 mM ; Experimental Assay: Tm pH:7.5, buffers:HEPES: 10 mM, ionic:KCl: 0.2 M ; Experimental Assay: dHvH pH:7.5, buffers:HEPES: 10 mM, ionic:KCl: 0.2 M ; Experimental Assay: Tm ionic:KCl: 0.1 M, pH:7.5, buffers:HEPES: 10 mM ; Experimental Assay: dHvH ionic:KCl: 0.1 M, pH:7.5, buffers:HEPES: 10 mM ; Experimental Assay: Tm pH:7.5, ionic:: , buffers:HEPES: 10 mM ; Experimental Assay: dHvH pH:7.5, ionic:: , buffers:HEPES: 10 mM ; Experimental Assay: dG temp:37.0 C ; Experimental Assay: dG ; Experimental Assay: Tm ionic:KCl: 100 mM, buffers:Tris: 25 mM, pH:8.0 ; Experimental Assay: dHvH ionic:KCl: 100 mM, buffers:Tris: 25 mM, pH:8.0 ; Experimental Assay: Cm ionic:KCl: 100 mM ; Experimental Assay: dG_H2O ionic:KCl: 100 mM ; Experimental Assay: Cm ionic:KCl: 100 mM ; Experimental Assay: dG_H2O ionic:KCl: 100 mM ; Experimental Assay: Cm ionic:KCl: 100 mM ; Experimental Assay: dG_H2O ionic:KCl: 100 mM ; Experimental Assay: Cm ionic:KCl: 100 mM ; Experimental Assay: dG_H2O ionic:KCl: 100 mM ; Experimental Assay: Cm ionic:KCl: 100 mM ; Experimental Assay: dG_H2O ionic:KCl: 100 mM ; Experimental Assay: Cm ionic:KCl: 100 mM ; Experimental Assay: dG_H2O ionic:KCl: 100 mM
Libraries Mutations for sequence A:ADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGFISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVTMMTSK/B:KRRWKKNFIAVSAANRFKKISSSGAL

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