Muteins, i.e. proteins altered by mutation of their genes, of interleukin 2 (112) were generated by oligonucleotide-directed mutagenesis in vitro. All acidic and basic residues conserved between man and mouse were exchanged as well as four lipophilic residues contained within four hydrophobic segments of the protein.The muteins were produced in Escherichia coli and submitted to a renaturation and purification protocol, before bioactivity and receptor binding of each of them was determined.All muteins besides two (K44/T125 and QIlO/T125) could be renatured and purified. One mutein (K94/T12.5) exhibited a more than tenfold-improved renaturation yield.One amino exchange (Asp-20 to Asn) resulted in an about 20-fold reduction in proliferative activity and highaffinity receptor binding. The binding to the low-affinity 112-binding protein (Tac antigen) was unimpaired.A second exchange (Arg-38 to Gln) had no effect on proliferative activity. The binding to both the high-and the low-affinity receptor, however, was reduced about 20-fold.Preliminary trials on the stability of these muteins by guanidinium hydrochloride denaturation studies detected no differences between wild-type interleukin 2 and muteins.It is suggested that Asp-20 forms part of the binding site for the large receptor subunit whereas Arg-38 is involved in the contact site to the small subunit.The transient synthesis of interleukin 2 (112) and its receptor by activated T lymphocytes apparently represents an essential step in the elaboration of the cellular immune response (for recent reviews see [I -31). The binding of 112 to a highaffinity receptor (& = 10 pM) provides the growth-signal leading to the clonal expansion of antigen-specific T cells.Interestingly, the high-affinity receptor has been established to be assembled from a low-affinity (Kd z 10 nM) 112- large extent preliminary, however, since the proteins were not renatured and purified before measuring proliferative or receptor-binding activity. Thus, effects of a certain mutation on the folding or on the receptor-binding sites could not be clearly differentiated.During the studies presented in this paper a series of charged residue positions and of hydrophobic positions were exchanged which cover more or less the whole surface of 112. The muteins were all submitted to a renaturation and purification protocol before analysing biological activities. Two muteins appeared which define positions involved in high-or in low-affinity receptor binding. Surprisingly, one mutein was obtained exhibiting a large increase in yield during renaturation. METHODS Construction of mutant strainsA cDNA coding for the mature part of human I12 [8] plus an initiator methionine [I91 was submitted to oligonucleotidedirected mutagenesis by the gapped duplex DNA approach [20, 211. The synthetic oligonucleotides comprised 6-16 residues before and after the nucleotide(s) to be exchanged, and they were provided by Dr H. Blocker (GBF, Braunschweig) or synthesised on a DNA synthesiser (Applied Biosystems, model 380). Mutation...

ABSTRACT-Mutants of lactose permease of Escherichia coli with amino acid changes (Gly-24 -> Glu; Gly-24 --Arg; Pro-28 --Ser; Gly-24, Pro-28 -> Glu-Ser and Gly-24, -> Arg-Ser) within a putative membrane-spanning a-helix 12 24(Phe-Gly-Leu-Phe-Phe-Phe-Phe-Tyr-Phe-Phe-Ile-Met-GlyAla-Tyr-Phe-Pro-Phe-Phe-Pro-Ile) are incorporated into the cytoplasmic membrane. The mutant proteins retain the ability to bind galactosides, and the affinity for several substrates is actually increased. However, the rate of active transport is decreased to 0.01% of the wild-type rate in the mutants carrying Arg-24 or Arg-24, Ser-28. Kinetic analysis demonstrates that the two mutants require 10 min to cause occupied binding sites for galactoside and HI to change their exposure from the periplasm to the cytoplasm as compared to 50 ms in the wild type. The effect is less pronounced when these sites are unoccupied.

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