* The additional destabilization of the variants probably results from the repulsion of the three Glu residues at position 3, 46 and 66.

* In the presence of 2M NaCl the destabilization caused by the R3E/L66E double mutation was reduced to -9.6kJ/mol(Debye-Huckel screening!), which is very close to the sum of the destabilizations measured for the two individual variants R3E and L66E at 2M NaCl(-4.2 and -5.3 kJ/mol).

* Arg3 to Glu46 is 2.7 Angstrom in conformer A, 4.6 Angstrom in comformer B.

* "Ion-pair R3-E46" might not be involved in this stabilization.