Characterization of a cooperativity domain mutant Lys3 --> Ala (K3A) T4 gene 32 protein.
The N-terminal "B" domain of T4 gene 32 protein contains a Lys3-Arg4-Lys5 sequence that has been postulated to provide a major determinant for cooperative binding. In this report, the equilibrium binding properties of a Lys3 --> Ala substitution mutant of gp32 (K3A gp32) and described and compared to a set of substitution mutants of Arg4 previously described (Villemain, J. L., and Giedroc, D. P. (1993) Biochemistry 32, 11235-11246) and further characterized here. K3A gp32 exhibits binding behavior which mirrors that of R4Q gp32. Despite an 6-8-fold decrease in overall binding affinity (Kapp = Kint x omega) at pH 8.1, 0.20 M NaCl, 20 degrees C, the magnitude of the cooperativity parameter is at most 2-3-fold smaller than that of the wild-type protein. The magnitude of omega is independent of salt concentration and type over the range in [NaCl] from 0.125 to 0. 225 M and [NaF] between 0.20 and 0.32 M (log omega = 2.86 +/- 0.19). For comparison, log omega for wild-type gp32 is 2.91 (+/- 0.21) resolved at 0.275 M NaCl and 3.39 +/- 0.11 in [NaF] between 0.40 and 0.45 M. In contrast to omega, the [NaCl] dependence of Kapp is large and markedly nonlinear for both wild-type and K3A gp32s over a [NaCl] range extending from 0.05 M to 0.40 M NaCl. Modeling of the complete salt dependence of Kapp for wild-type, K3A, and R4T gp32s in NaCl and NaF with a simple ion-exchange model suggests that substitutions within the basic Lys3-Arg4-Lys5 sequence do not strongly modulate the net displacement of cations and anions upon poly(A) complex formation by gp32.
Department of Biochemistry and Biophysics, Center for Macromolecular Design, Institute of Biosciences and Technology, Texas A&M University, College Station, Texas 77843-2128, USA. giedroc@bioch.tamu.edu
J L
JL
Villemain
D P
DP
Giedroc
D000409:Alanine; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008239:Lysine; D008956:Models, Chemical; D017354:Point Mutation; D011061:Poly A / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D012969:Sodium Fluoride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism*
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4; D011061:Poly A; D012965:Sodium Chloride; D012969:Sodium Fluoride; D008239:Lysine; D000409:Alanine