Probing the relationship between .alpha.-helix formation and calcium affinity in troponin C: proton NMR studies of calcium binding to synthetic and variant site III helix-loop-helix peptides

Biochemistry ◽  
1991 ◽  
Vol 30 (34) ◽  
pp. 8339-8347 ◽  
Author(s):  
Gary S. Shaw ◽  
Robert S. Hodges ◽  
Brian D. Sykes
Keyword(s):  
Calcium Binding ◽  
Proton Nmr ◽  
Alpha Helix ◽  
Troponin C ◽  
Nmr Studies ◽  
Helix Loop Helix ◽  
Helix Formation ◽  
Calcium Affinity ◽  
The Relationship

Calcium binding proteins. Elucidating the contributions to calcium affinity from an analysis of species variants and peptide fragments

1990 ◽  
Vol 68 (3) ◽  
pp. 587-601 ◽  
Author(s):  
Brian J. Marsden ◽  
Gary S. Shaw ◽  
Brian D. Sykes
Keyword(s):  
Binding Affinity ◽  
Sequence Homology ◽  
Calcium Binding ◽  
Binding Proteins ◽  
Troponin C ◽  
Calcium Binding Proteins ◽  
Intact Protein ◽  
Peptide Fragments ◽  
Helix Loop Helix ◽  
Calcium Affinity

This paper describes the sequence homology of calcium-binding proteins belonging to the troponin C superfamily. Specifically, this similarity has been examined for 276 twelve-residue calcium-binding loops. It has been found that, in the calcium-binding loop, several residues appear invariant, regardless of the species of origin or the affinity of the protein. These residues are Asp at position 1 (+ X of the coordinating position of the calcium), Asp or Asn at position 3 (+ Y), Gly at position 6, Ile at position 8, and Glu at position 12 (− Z). It has also been found that conservation of certain residues can vary in similar sites in similar proteins. For example, position 3 (+ Y) in site 3 of troponin C is always an Asn, whereas in calmodulin the residue is always Asp. This study also examined the calcium-binding affinities of peptide fragments comprising the loop, helix–loop, loop–helix, and helix–loop–helix. These were compared with larger enzymatic or chemically generated protein fragments in an effort to understand the various contributions to the calcium-binding affinity of a single-site versus a two-site domain as found in troponin C and calmodulin. Based on free energy differences, it was found that a 34-residue helix–loop–helix peptide represents about 60% of the binding affinity found in the intact protein. Cooperativity with a second calcium binding site accounted for the remaining 40% of the affinity.Key words: calcium-binding proteins, sequence homology, peptide fragments, species variants, calcium affinity.

scholarly journals X-ROS Signaling Depends on Length-Dependent Calcium Buffering by Troponin

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1189
Author(s):  
Sarita Limbu ◽  
Benjamin L. Prosser ◽  
William J. Lederer ◽  
Christopher W. Ward ◽  
Mohsin S. Jafri
Keyword(s):  
Calcium Binding ◽  
Calcium Release ◽  
Calcium Transient ◽  
Troponin C ◽  
Buffering Capacity ◽  
Oxygen Species ◽  
Open Probability ◽  
Ros Signaling ◽  
Calcium Buffering ◽  
Calcium Affinity

The stretching of a cardiomyocyte leads to the increased production of reactive oxygen species that increases ryanodine receptor open probability through a process termed X-ROS signaling. The stretching of the myocyte also increases the calcium affinity of myofilament Troponin C, which increases its calcium buffering capacity. Here, an integrative experimental and modeling study is pursued to explain the interplay of length-dependent changes in calcium buffering by troponin and stretch-activated X-ROS calcium signaling. Using this combination, we show that the troponin C-dependent increase in myoplasmic calcium buffering during myocyte stretching largely offsets the X-ROS-dependent increase in calcium release from the sarcoplasmic reticulum. The combination of modeling and experiment are further informed by the elimination of length-dependent changes to troponin C calcium binding in the presence of blebbistatin. Here, the model suggests that it is the X-ROS signaling-dependent Ca2+ release increase that serves to maintain free myoplasmic calcium concentrations during a change in myocyte length. Together, our experimental and modeling approaches have further defined the relative contributions of X-ROS signaling and the length-dependent calcium buffering by troponin in shaping the myoplasmic calcium transient.

Proton-NMR studies of the solution conformations of vitamin-D-induced bovine intestinal calcium-binding protein

1983 ◽  
Vol 137 (3) ◽  
pp. 523-529 ◽  
Author(s):  
David C. DALGARNO ◽  
Barry A. LEVINE ◽  
Robert J. P. WILLIAMS ◽  
Curtis S. FULLMER ◽  
Robert H. WASSERMAN
Keyword(s):  
Vitamin D ◽  
Calcium Binding ◽  
Binding Protein ◽  
Proton Nmr ◽  
Calcium Binding Protein ◽  
Nmr Studies ◽  
Solution Conformations

Interaction of troponin I and troponin C: 19F NMR studies of the binding of the inhibitory troponin I peptide to turkey skeletal troponin C

1991 ◽  
Vol 69 (9) ◽  
pp. 674-681 ◽  
Author(s):  
A. Patricia Campbell ◽  
Paul J. Cachia ◽  
Brian D. Sykes
Keyword(s):  
Calcium Binding ◽  
Troponin I ◽  
Dissociation Constants ◽  
Chemical Shifts ◽  
Troponin C ◽  
Resonance Spectroscopy ◽  
Nmr Studies ◽  
Binding Model ◽  
Peptide Binding Site ◽  
Titration Data

We have used 19F nuclear magnetic resonance spectroscopy to study the interaction of the inhibitory region of troponin (TnI) with apo- and calcium(II)-saturated turkey skeletal troponin C (TnC), using the synthetic TnI analogue Nα-acetyl[19FPhe106]TnI(104–115)amide. Dissociation constants of Kd = (3.7 ± 3.1) × 10−5 M for the apo interaction and Kd = (4.8 ± 1.8) × 10−5 M for the calcium(II)-saturated interaction were obtained using a 1:1 binding model of peptide to protein. The 19F NMR chemical shifts for the F-phenylalanine of the bound peptide are different from the apo- and calcium-saturated protein, indicating a different environment for the bound peptide. The possibility of 2:1 binding of the peptide to Ca(II)-saturated TnC was tested by calculating the fit of the experimental titration data to a series of theoretical binding curves in which the dissociation constants for the two hypothetical binding sites were varied. We obtained the best fit for 0.056 mM ≤ Kd1 ≤ 0.071 mM and 0.5 mM ≤ Kd2 ≤ 2.0 mM. These results allow the possibility of a second peptide binding site on calcium(II)-saturated TnC with an affinity 10- to 20-fold weaker than that of the first site.Key words: tropinin C, tropinin I, calcium binding, NMR studies, muscle proteins.

Physico-Chemical Properties of Recombinant Desulphatohirudin

1990 ◽  
Vol 63 (03) ◽  
pp. 499-504 ◽  
Author(s):  
A Electricwala ◽  
L Irons ◽  
R Wait ◽  
R J G Carr ◽  
R J Ling ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Chemical Properties ◽  
Alpha Helix ◽  
Apparent Molecular Weight ◽  
Correlation Spectroscopy ◽  
Nmr Studies ◽  
Recombinant Hirudin ◽  
Physico Chemical ◽  
Recombinant Molecule

SummaryPhysico-chemical properties of recombinant desulphatohirudin expressed in yeast (CIBA GEIGY code No. CGP 39393) were reinvestigated. As previously reported for natural hirudin, the recombinant molecule exhibited abnormal behaviour by gel filtration with an apparent molecular weight greater than that based on the primary structure. However, molecular weight estimation by SDS gel electrophoresis, FAB-mass spectrometry and Photon Correlation Spectroscopy were in agreement with the theoretical molecular weight, with little suggestion of dimer or aggregate formation. Circular dichroism studies of the recombinant molecule show similar spectra at different pH values but are markedly different from that reported by Konno et al. (13) for a natural hirudin-variant. Our CD studies indicate the presence of about 60% beta sheet and the absence of alpha helix in the secondary structure of recombinant hirudin, in agreement with the conformation determined by NMR studies (17)

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