Calmodulin interacts with a C-terminus peptide from the lens membrane protein MIP26.
Lens fiber cells are coupled by communicating junctions that comprise over 50% of their appositional surfaces. The main intrinsic protein (MIP26) of lens fibers is a 28.2 kDa protein that forms large gap junction-like channels in reconstituted systems. Previously, we have shown that Ca(++)-activated calmodulin (CaM) regulates the permeability of reconstituted MIP26 channels and changes the conformation of MIP26, as measured by intrinsic fluorescence and circular dichroism spectroscopy. Examination of the MIP26 amino acid sequence has revealed a basic amphiphilic alpha-helical segment (Pep C) on the C-terminus with residue distribution similar to that found in other CaM binding proteins. To test the interaction between the amphiphilic segment and CaM, both a 20-mer peptide and trp-substituted fluorescent analog have been synthesized and purified by HPLC. Evidence from spectrofluorometric titration shows that the Pep C binds with CaM in 1:1 stoichiometry and with a kd of approximately 10 nM. Neither Ca++ nor H+ alone affects the conformation of the Pep C. However, when mixed with CaM the Pep C undergoes both a dramatic blue-shift in tryptophan fluorescence emission, indicative of strong hydrophobic interaction, and an increase in circular dichroism absorption in the alpha-helical region. Additional fluorescence blue-shift and alpha-helical content occur when Ca++ is added to the CaM:Pep C complex.
Girsch
S J
SJ
Department of Physiology, University of Rochester, School of Medicine and Dentistry, NY 14642.
Peracchia
C
C
D000595:Amino Acid Sequence; D020346:Aquaporins; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000737:chemistry*; D002942:Circular Dichroism; D005136:Eye Proteins / Q000138:chemical synthesis / Q000737:chemistry*; D007908:Lens, Crystalline / Q000737:chemistry*; D008562:Membrane Glycoproteins / Q000138:chemical synthesis / Q000737:chemistry; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D010750:Phosphoproteins / Q000138:chemical synthesis / Q000737:chemistry; D011487:Protein Conformation; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence
D020346:Aquaporins; D002147:Calmodulin; D005136:Eye Proteins; D008562:Membrane Glycoproteins; D010455:Peptides; D010750:Phosphoproteins; C030328:aquaporin 0; D002118:Calcium