PubMed · 5573765
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J Ninio. 1971-02-28. Codon-anticodon recognition: the missing triplet hypothesis.. https://doi.org/10.1016/0022-2836(71)90084-2
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Alanine scanning mutagenesis has been used to identify specific side chains of insulin which strongly influence binding to the insulin receptor. A total of 21 new insulin analog constructs were made, and in addition 7 high pressure liquid chromatography-purified analogs were tested, covering alanine substitutions in positions B1, B2, B3, B4, B8, B9, B10, B11, B12, B13, B16, B17, B18, B20, B21, B22, B26, A4, A8, A9, A12, A13, A14, A15, A16, A17, A19, and A21. Binding data on the analogs revealed that the alanine mutations that were most disruptive for binding were at positions TyrA19, GlyB8, LeuB11, and GluB13, resulting in decreases in affinity of 1,000-, 33-, 14-, and 8-fold, respectively, relative to wild-type insulin. In contrast, alanine substitutions at positions GlyB20, ArgB22, and SerA9 resulted in an increase in affinity for the insulin receptor. The most striking finding is that B20Ala insulin retains high affinity binding to the receptor. GlyB20 is conserved in insulins from different species, and in the structure of the B-chain it appears to be essential for the shift from the alpha-helix B8-B19 to the beta-turn B20-B22. Thus, replacing GlyB20 with alanine most likely modifies the structure of the B-chain in this region, but this structural change appears to enhance binding to the insulin receptor.
Alanine-scanning mutants of the primer grip region of human immunodeficiency virus type 1 reverse transcriptase were tested for their ability to extend RNA and DNA versions of the polypurine tract primer, and an oligonucleotide representing the 18-nucleotide sequence at the 3' end of tRNALys3. A majority of the mutant enzymes were either completely or severely deficient in RNA priming activity, but, with only one exception, were able to efficiently extend DNA versions of the same primers. The mutant enzymes were able to bind to RNA primers, indicating that the defect in RNA priming was not simply a loss of binding activity. Mutations at positions 229, 233, and 235 dramatically reduced the amount of specific RNase H cleavage at the 3' terminus of the polypurine tract, which is required for primer removal. An alanine substitution at position 232 led to loss of cleavage specificity, although total activity was close to the wild-type level. Taken together, these results demonstrate for the first time that there are residues in human immunodeficiency virus type 1 reverse transcriptase which are specifically involved in protein-nucleic acid interactions with RNA primers.
Physiological and pharmacological properties of possible subtypes of the native glycine receptor were investigated in retinal neurons using whole-cell voltage-clamp techniques. Two discrete inhibitory glycine responses were identified in ganglion cells. The responses could be distinguished pharmacologically: one was sensitive to strychnine and the other to 5,7-dichlorokynurenic acid. The two responses had different kinetics: the former had a fast onset and fast desensitization, whereas the latter had a slower onset and was much more sustained. The physiological and pharmacological distinctions suggest that the responses are mediated by different receptors. These receptors transduce glycinergic synaptic signals to ganglion cells, where they serve as low- and high-pass filters, respectively, of EPSPs.