Role of hydration in the closed-to-open transition involved in Ca2+ binding by troponin C.
Troponin C (TnC) is the Ca(2+)-binding subunit of the troponin complex of vertebrate skeletal muscle. It consists of two structurally homologous domains, N and C, connected by an exposed alpha-helix. The C-domain has two high-affinity sites for Ca(2+) that also bind Mg(2+), whereas the N-domain has two low-affinity sites for Ca(2+). Previous studies using isolated N- and C-domains showed that the C-domain apo form was less stable than the N-domain. Here we analyzed the stability of isolated N-domain (F29W/N-domain) against urea and pressure denaturation in the absence and in the presence of glycerol using fluorescence spectroscopy. Increasing the glycerol concentration promoted an increase in the stability of the protein to urea (0-8 M) in the absence of Ca(2+). Furthermore, the ability to expose hydrophobic surfaces normally promoted by Ca(2+) binding or low temperature under pressure was partially lost in the presence of 20% (v/v) glycerol. Glycerol also led to a decrease in the Ca(2+) affinity of the N-domain in solution. From the ln K(obs) versus ln a(H)2(O), we obtained the number of water molecules (63.5 +/- 8.7) involved in the transition N <=>N:Ca(2) that corresponds to an increase in the exposed surface area of 571.5 +/- 78.3 A(2). In skinned fibers, the affinity for Ca(2+) was also reduced by glycerol, although the effect was much less pronounced than in solution. Our results demonstrate quantitatively that the stability of this protein and its affinity for Ca(2+) are critically dependent on protein hydration.
Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Rio de Janeiro, RJ, Brazil.
Marisa C
MC
Suarez
Cosme José V
CJ
Machado
Luís Maurício T R
LM
Lima
Lawrence B
LB
Smillie
Joyce R
JR
Pearlstone
Jerson L
JL
Silva
Martha M
MM
Sorenson
Débora
D
Foguel
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002645:Chickens; D002942:Circular Dichroism; D018482:Muscle, Skeletal / Q000378:metabolism*; D009997:Osmotic Pressure; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D019209:Troponin C / Q000378:metabolism*; D014508:Urea / Q000494:pharmacology; D014867:Water / Q000378:metabolism*
D019209:Troponin C; D014867:Water; D014508:Urea; D002118:Calcium