Thompson T M, Mark B L, Gray C W, Terwilliger T C, Sreerama N, Woody R W, Gray D M
Department of Molecular and Cell Biology, The University of Texas at Dallas, Richardson 75083-0688, USA.
Biochemistry. 1998 May 19;37(20):7463-77. doi: 10.1021/bi972545k.
A core Y61F mutant of the gene 5 single-stranded DNA-binding protein (g5p) of f1 bacterial virus aggregated when expressed from a plasmid, but, after refolding in vitro, it behaved much like wild-type and may be a stability or folding mutant. Circular dichroism (CD) titrations showed the same cooperative polynucleotide binding modes for Y61F and wild-type g5p. There are n = 4 and n congruent with 2.5 modes for binding to poly[d(A)] at low ionic strengths, but n = 4, n = 3, and n congruent with 2-2.5 modes for binding to fd single-stranded viral DNA (fd ssDNA), where n is the number of nucleotides occluded by each bound g5p monomer in a given mode. Y61F g5p has slightly reduced affinity in the n = 4 mode. Electron microscopy showed that Y61F g5p forms left-handed nucleoprotein superhelices indistinguishable from wild-type. Progression from binding to fd ssDNA in the n = 4 to n = 3 to n congruent with 2-2.5 mode is accompanied by an increase in the number of helical turns, an increase from (7.7 +/- 0.3) to (9.5 +/- 0.3) to ( approximately 10-13) g5p dimers per turn, and a decrease in the number of DNA nucleotides per turn. From CD spectra for four of five possible Y --> F g5p mutants, we infer that the fifth tyrosine, Tyr 56, contributes strongly to the CD. Retention of a strong 229 nm CD band in all mutants indicates that all retain elements of the native structure. Spectra of Y26F, Y34F, and Y61F g5p imply limited mobility of the replacement Phe. Comparison of measured with calculated CD spectra also suggests limited mobility for Tyr 26 and Tyr 34 in g5p in solution, and provides new information that the g5p structure in solution may be dominated by Tyr 41 rotamers differing from that stabilized in the crystal.
F1 细菌病毒基因 5 单链 DNA 结合蛋白(g5p)的核心 Y61F 突变体在从质粒表达时会发生聚集,但在体外重折叠后,其行为与野生型非常相似,可能是一个稳定性或折叠突变体。圆二色性(CD)滴定显示 Y61F 和野生型 g5p 具有相同的协同多核苷酸结合模式。在低离子强度下与聚[d(A)]结合时存在 n = 4 和 n ≈ 2.5 模式,但与 fd 单链病毒 DNA(fd ssDNA)结合时存在 n = 4、n = 3 和 n ≈ 2 - 2.5 模式,其中 n 是在给定模式下每个结合的 g5p 单体所占据的核苷酸数。Y61F g5p 在 n = 4 模式下的亲和力略有降低。电子显微镜显示 Y61F g5p 形成与野生型无法区分的左手核蛋白超螺旋。从与 fd ssDNA 结合的 n = 4 模式转变为 n = 3 模式再到 n ≈ 2 - 2.5 模式,伴随着螺旋圈数的增加,每圈 g5p 二聚体数量从(7.7 ± 0.3)增加到(9.5 ± 0.3)再到(约 10 - 13),以及每圈 DNA 核苷酸数量的减少。从五个可能的 Y → F g5p 突变体中的四个的 CD 光谱,我们推断第五个酪氨酸 Tyr 56 对 CD 有很大贡献。所有突变体中 229 nm 处强 CD 带的保留表明所有突变体都保留了天然结构的元素。Y26F、Y34F 和 Y61F g5p 的光谱表明替代苯丙氨酸的移动性有限。实测 CD 光谱与计算光谱的比较还表明溶液中 g5p 中 Tyr 26 和 Tyr 34 的移动性有限,并提供了新的信息,即溶液中 g5p 的结构可能由与晶体中稳定的构象不同的 Tyr 41 旋转异构体主导。