Hastings C A, Barton J K
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena 91125, USA.
Biochemistry. 1999 Aug 3;38(31):10042-51. doi: 10.1021/bi982039a.
Metallointercalator-peptide conjugates that provide small molecular mimics to explore peptide-nucleic acid recognition have been prepared. Specifically, a family of peptide conjugates of Rh(phi)(2)(phen') [where phi = 9,10-phenanthrenequinone diimine and phen' = 5-(amidoglutaryl)-1,10-phenanthroline] has been synthesized and their DNA-binding characteristics examined. Single amino acid modifications were made from the parent metallointercalator-peptide conjugate Rh(phi)(2)(phen')-AANVAIAAWERAA-CONH(2), which targets 5'-CCA-3' site-specifically. Moving the glutamate at position 10 in the sequence of the appended peptide to position 6 {Rh(phi)(2)(phen')-AANVAEAAWARAA-CONH(2)} changed the sequence preference of the metallointercalator-peptide conjugate to 5'-ACA-3'. Subsequent mutation of the glutamate at position 6 to arginine {Rh(phi)(2)(phen')-AANVARAAWARAA-CONH(2)} caused more complex changes in DNA recognition. Thermodynamic dissociation constants were determined for these metallointercalator-peptide conjugates by photoactivated DNA cleavage assays with the rhodium intercalators. At 55 degrees C in the presence of 5 mM MnCl(2), Rh(phi)(2)(phen')-AANVAIAAWERAA-CONH(2) binds to a 5'-CCA-3' site with K(d) = 5.7 x 10(-)(8) M, whereas Rh(phi)(2)(phen')-AANVAEAAWARAA-CONH(2) binds to its target 5'-ACA-3' site with K(d) = 9.9 x 10(-8) M. The dissociation constant for Rh(phi)(2)(phen') with random-sequence DNA is 7.0 x 10(-7) M. Structural models have been developed and refined to account for the observed sequence specificities. As with much larger DNA-binding proteins, with these metal-peptide conjugate mimics, single amino acid changes can lead to single or multiple base changes in the DNA site targeted.
已制备出金属嵌入剂 - 肽缀合物，其可作为小分子模拟物用于探索肽 - 核酸识别。具体而言，已合成了一系列Rh(phi)(2)(phen')的肽缀合物（其中phi = 9,10 - 菲醌二亚胺，phen' = 5 -（氨基戊二酰基）-1,10 - 菲咯啉），并检测了它们的DNA结合特性。从母体金属嵌入剂 - 肽缀合物Rh(phi)(2)(phen')-AANVAIAAWERAA - CONH(2)进行单氨基酸修饰，该母体缀合物特异性靶向5'-CCA-3'位点。将附加肽序列中第10位的谷氨酸移至第6位{Rh(phi)(2)(phen')-AANVAEAAWARAA - CONH(2)}，改变了金属嵌入剂 - 肽缀合物对5'-ACA-3'的序列偏好。随后将第6位的谷氨酸突变为精氨酸{Rh(phi)(2)(phen')-AANVARAAWARAA - CONH(2)}，导致DNA识别发生更复杂的变化。通过使用铑嵌入剂的光活化DNA切割测定法，确定了这些金属嵌入剂 - 肽缀合物的热力学解离常数。在55℃、5 mM MnCl(2)存在下，Rh(phi)(2)(phen')-AANVAIAAWERAA - CONH(2)以K(d)=5.7×10(-8) M的解离常数结合到5'-CCA-3'位点，而Rh(phi)(2)(phen')-AANVAEAAWARAA - CONH(2)以K(d)=9.9×10(-8) M的解离常数结合到其靶标5'-ACA-3'位点。Rh(phi)(2)(phen')与随机序列DNA的解离常数为7.0×10(-7) M。已开发并完善了结构模型以解释观察到的序列特异性。与大得多的DNA结合蛋白一样，使用这些金属 - 肽缀合物模拟物时，单个氨基酸变化可导致靶向DNA位点出现单个或多个碱基变化。
