Details

Autor(en) / Beteiligte

• Dong, Wen-Ji
• Chandra, Murali
• Xing, Jun
• She, Mingda
• Solaro, R. John
• Cheung, Herbert C

Titel

Phosphorylation-Induced Distance Change in a Cardiac Muscle Troponin I Mutant

Ist Teil von

• Biochemistry (Easton), 1997-06, Vol.36 (22), p.6754-6761

Ort / Verlag

United States: American Chemical Society

Erscheinungsjahr

1997

Quelle

MEDLINE

Beschreibungen/Notizen

• Phosphorylation of two adjacent serine residues in the unique N-terminal extension of cardiac muscle troponin I (cTnI) is known to decrease the Ca2+-sensitivity of cardiac myofilaments. To probe the structural significance of the N-terminal extension, we have constructed two cTnI mutants each containing a single cysteine:  (1) a full-length cTnI mutant (S5C/C81I/C98S) and (2) a truncated cTnI mutant (S9C/C50I/C67S) in which the N-terminal 32 amino acid residues were deleted. We determined the apparent binding constants for the complex formation between IAANS-labeled cardiac troponin C (cTnC) and the two cTnI mutants. The affinities of the cTnC for the truncated cTnI mutant were:  (1) 1.5 × 106 M-1 in EGTA, (2) 28.9 × 106 M-1 in Mg2+, and (3) 87.5 × 106 M-1 in Mg2+ + Ca2+. These binding constants were approximately 1.4-fold smaller than the corresponding values obtained with the full-length cTnI mutant, suggesting a very small contribution of the N-terminal extension to the binding of cTnI to cTnC. Cys-5 in the full-length cTnI mutant was labeled with IAANS, and the distribution of the separation between this site and Trp-192 was determined by analysis of the efficiency of fluorescence resonance energy transfer from Trp-192 to IAANS. The following mean distances were obtained with the unphosphorylated full-length mutant:  44.4 Å (cTnI alone), 48.3 Å (cTnI + cTnC), 46.3 Å (cTnI + cTnC in Mg2+), and 51.6 Å (cTnI + cTnC in Mg2+ + Ca2+). The corresponding values of the mean distance determined with the phosphorylated full-length cTnI mutant were 35.8, 36.6, 34.8, and 37.3 Å. The phosphorylation of cTnI reduced the half-width of the distribution from 9.5 to 3.7 Å. Similar but less pronounced decreases of the half-widths were also observed with the phosphorylated cTnI complexed with cTnC in different ionic conditions. Thus, phosphorylation of cTnI resulted in a decrease of 9−12 Å in the mean distance between the sites located at the N- and C-terminal portion of cTnI. Our results indicate that phosphorylation elicits a change in the conformation of cTnI which underlies the basis of the phosphorylation-induced modulation of cTnI activity.