A. D. Sherry

Reductive methylation of the lysyl residues in the fd gene 5 DNA-binding protein: CD and 13C NMR results on the modified protein

The epsilon-amino groups of the six lysyl residues of the fd gene 5 DNA-binding protein have been modified by reductive methylation to form N epsilon, N epsilon-dimethyl lysyl derivatives containing 13C-labeled methyl groups. The alpha-amino terminus of the protein was not accessible to methylation. Circular dichroism studies show that the modified protein binds to fd DNA, but with a slightly reduced affinity compared with that of unmodified gene 5 protein. We also find that both the modified and unmodified proteins bind to an oligodeoxynucleotide, d(A)7, but in neither case does binding cause a decrease in the 228 nm CD band of the protein as occurs when the protein binds to long DNA polymers. 13C NMR spectra at 50.1 MHz of [13C]methylated gene 5 protein show five distinct resonances between 43.30 and 42.76 ppm originating from the six N epsilon, N epsilon-dimethyl lysyl residues. We attribute one of the resonances to two solvated lysyl residues and the other four to individual lysyl residues in different microenvironments. All four of these latter resonances are affected by the binding of d(A)7. However, since two of these resonances are similarly affected by the presence of salt in the absence of DNA, only two are uniquely affected by DNA binding.

Concanavalin a will not assume the sugar binding conformation in the complete absence of metal ions

Abstract Concanavalin A (Con A) is known to exist in two conformations that differ in their capacity to bind metals and sugars. The conformation that binds metals tightly and has a high affinity for sugars is termed the “locked” form and the conformation with low affinity for sugars and binds metals weakly is termed the “unlocked” form. It has recently been reported [Brown, et al. Biochemistry 21 , 465(1982)] that apo-Con A will form the locked conformation in the absence of metals to the extent of 12.5% when equilibrated at 25°C for one week. In this report we show that Con A will not assume the locked conformation in the complete absence of metals and that only trace amounts of Ca2+ can catalytically convert a significant amount of the protein into the locked conformation.

Interaction of Reductively Methylated Lysyl-Fd Gene 5 Protein with a Negatively Charged Lanthanide Paramagnetic Macrocyclic Chelate or with Oligonucleotides using 13-C NMR

The bacteriophage fd gene 5 protein (G5P) binds strongly to single stranded DNA. Electrostatic interactions, involving ion pairing of positively charged lysine and arginine protein side chains with the negatively charged phosphates of four nucleotidyl units of DNA, are very important in this interaction process1–3. However, implication of specific lysyl residues in the DNA-binding site is more difficult. X-ray crystal structure and modeling studies of G5P have been used4,5 to obtain a structural binding model which only includes one (Lys-46) of its six lysyl residues. However, chemical modification studies6 seem to implicate three lysyl residues (Lys-24, 46 and 69) in the nucleic acid binding site of G5P.