The effect of protein concentration on ion binding.
The concentration of protein in a solution has been found to have a significant effect on ion binding affinity. It is well known that an increase in ionic strength of the solvent medium by addition of salt modulates the ion-binding affinity of a charged protein due to electrostatic screening. In recent Monte Carlo simulations, a similar screening has been detected to arise from an increase in the concentration of the protein itself. Experimental results are presented here that verify the theoretical predictions; high concentrations of the negatively charged proteins calbindin D9k and calmodulin are found to reduce their affinity for divalent cations. The Ca(2+)-binding constant of the C-terminal site in the Asn-56 --> Ala mutant of calbindin D9k has been measured at seven different protein concentrations ranging from 27 microM to 7.35 mM by using 1H NMR. A 94% reduction in affinity is observed when going from the lowest to the highest protein concentration. For calmodulin, we have measured the average Mg(2+)-binding constant of sites I and II at 0.325, 1.08, and 3.25 mM protein and find a 13-fold difference between the two extremes. Monte Carlo calculations have been performed for the two cases described above to provide a direct comparison of the experimental and simulated effects of protein concentration on metal ion affinities. The overall agreement between theory and experiment is good. The results have important implications for all biological systems involving interactions between charged species.
Linse
S
S
Chemical Center, Lund University, Sweden.
Jönsson
B
B
Chazin
W J
WJ
D000409:Alanine; D000818:Animals; D001216:Asparagine; D001665:Binding Sites; D064026:Calbindins; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D005813:Genes, Synthetic; D007477:Ions; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008715:Methionine; D009010:Monte Carlo Method; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000737:chemistry; D011392:Proline; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D064030:S100 Calcium Binding Protein G / Q000737:chemistry* / Q000378:metabolism*
D064026:Calbindins; D007477:Ions; D009419:Nerve Tissue Proteins; D011506:Proteins; D011994:Recombinant Proteins; D064030:S100 Calcium Binding Protein G; D001216:Asparagine; D011392:Proline; D008715:Methionine; D008274:Magnesium; D000409:Alanine; D002118:Calcium