Rice M S, Dahlquist F W
Institute of Molecular Biology, University of Oregon, Eugene 97403.
J Biol Chem. 1991 May 25;266(15):9746-53.
The sensory transducer proteins in bacterial chemotaxis undergo two covalent modifications, deamidation and reversible methylation, in response to attractants and repellents. Oligonucleotide-directed mutagenesis was used to alter putative methylation and deamidation sites in one of the transducers to further define these sites and their role in chemotaxis. The mutations, in combination with peptide maps and Edman analysis, have clarified the sites of covalent modification in Tsr. Tsr contains six specific glutamates and glutamines that serve as methyl-accepting sites. An arginine-containing tryptic peptide (R1) has two sites, one at glutamate 493 and a newly located site at glutamate 502. A lysine-containing peptide (K1) has four methyl-accepting sites. Two of the lysine peptide sites are glutamates and can accept methyl groups without deamidation. The other two sites are glutamines and two methyl-accepting sites are created by two distinct deamidations. Both deamidations can occur on the same polypeptide chain. Single glutamate mutants have shown that one deamidation (at glutamine 311) proceeds rapidly, while the other deamidation (at glutamine 297) has a half-life of approximately 60 min under our experimental conditions.
细菌趋化作用中的感觉转导蛋白会响应引诱剂和驱避剂发生两种共价修饰，即脱酰胺作用和可逆甲基化作用。利用寡核苷酸定向诱变来改变其中一种转导蛋白中假定的甲基化和脱酰胺位点，以进一步确定这些位点及其在趋化作用中的作用。这些突变与肽图和埃德曼分析相结合，阐明了Tsr中共价修饰的位点。Tsr含有六个特定的谷氨酸和谷氨酰胺，它们作为甲基接受位点。一个含精氨酸的胰蛋白酶肽（R1）有两个位点，一个在谷氨酸493处，另一个新定位的位点在谷氨酸502处。一个含赖氨酸的肽（K1）有四个甲基接受位点。赖氨酸肽的两个位点是谷氨酸，可以在不脱酰胺的情况下接受甲基基团。另外两个位点是谷氨酰胺，两个甲基接受位点是由两种不同的脱酰胺作用产生的。两种脱酰胺作用都可以在同一条多肽链上发生。单个谷氨酸突变体表明，一种脱酰胺作用（在谷氨酰胺311处）进行得很快，而另一种脱酰胺作用（在谷氨酰胺297处）在我们的实验条件下半衰期约为60分钟。
