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.
Villemain
J L
JL
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
Giedroc
D P
DP
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