蛋白質(zhì)熱穩(wěn)定性的計(jì)算機(jī)模擬.pdf

1、我們采用并行計(jì)算的分子動(dòng)力學(xué)研究了蛋白質(zhì)的熱穩(wěn)定性,利用蛋白質(zhì)敲除的技術(shù)研究了局部的結(jié)構(gòu)對(duì)蛋白質(zhì)整體結(jié)構(gòu)的熱穩(wěn)定性的影響,另外還通過全電子結(jié)構(gòu)計(jì)算來研究蛋白質(zhì)分子的前線軌道和蛋白質(zhì)活性和熱穩(wěn)定性的聯(lián)系.我們的研究工作主要包括三個(gè)方面的內(nèi)容:1.用并行計(jì)算分子動(dòng)力學(xué)模擬耐熱性鄰苯二酚雙加氧酶(TC23O)的熱穩(wěn)定性.蛋白質(zhì)的動(dòng)力學(xué)轉(zhuǎn)變溫度的差異,說明了TC23O與同源蛋白1MPY相比有較高的熱穩(wěn)定性.通過分析與中子散射譜相關(guān)的指數(shù)β和定壓

2、熱容量Cp,揭示了蛋白質(zhì)中的228位的脯氨酸殘基、鹽橋Lys188N-Glu291OE1、金屬離子和水溶劑對(duì)蛋白質(zhì)熱穩(wěn)定性的影響存在一定的差異,其中突變體Pro228Ser和Glu291Gly的動(dòng)力學(xué)相變溫度分別降低了10℃和19℃,四種金屬離子的替換后發(fā)現(xiàn)沒有顯著地影響蛋白質(zhì)的熱穩(wěn)定性,而水溶劑通過在一定程度上影響蛋白質(zhì)可能構(gòu)象的改變過程來改變蛋白質(zhì)的熱穩(wěn)定性.而所有的結(jié)果和已知實(shí)驗(yàn)得到的結(jié)果有很好的吻合.2.用敲除蛋白質(zhì)分子的部分片

3、斷來研究相應(yīng)突變體的熱穩(wěn)定性.我們選擇的敲除主要有尾部的敲除、無規(guī)則卷曲的敲除和α-helix的去除,其中的突變體mut1在尾部切除了從300-319的9個(gè)殘基,突變體mut2從61-66的無規(guī)則卷曲上切除了6個(gè)殘基,突變體mut3從21-26的包含有部分α-helix的片段上切除6個(gè)殘基.3.用蛋白質(zhì)的全電子結(jié)構(gòu)計(jì)算研究了前線軌道與蛋白質(zhì)TC23O和突變體P228S的生物活性以及穩(wěn)定性的聯(lián)系.分析前線軌道發(fā)現(xiàn),在野生型突變體P228S

4、中出現(xiàn)的活性位點(diǎn)中心的殘基及其能級(jí)為TRP_196-10.63526和-9.98105,而突變體P228S中TYR_262-10.66049和TRP_196-9.98779.可以看出在蛋白質(zhì)的活性中心位點(diǎn),盡管二者有差距,但能級(jí)差距很小.也可能正因?yàn)槿绱?二者在常溫下并沒有特別明顯的酶催化活性的差別.另外在前線軌道里面出現(xiàn)了大量的參與維持蛋白質(zhì)穩(wěn)定的鹽橋的氨基酸殘基,在野生型和突變體P228的前線軌道中出現(xiàn)比例為1/6,說明前線軌道的成

5、分,除了酶活性中心位點(diǎn)的一些殘基以外,還有大量的形成鹽橋的殘基.關(guān)鍵詞:并行計(jì)算分子動(dòng)力學(xué),蛋白質(zhì)敲除,酶活性中心,鹽橋,定壓熱容量,動(dòng)力學(xué)相變溫度,全電子結(jié)構(gòu)計(jì)算,前線軌道AbstractThe thermostability of protein thermostable cathechol 2,3-dixoygenase (TC23O)and mutants has been studied by the parallel mol

6、ecular dynamics simulations, farther the different fragments are knocked out to learn how the fragment influence the thermostability of TC23O, finally the linkage of active center and thermostability of protein TC23O and

7、 mutant P228S with the frontier orbital energy levels using the method of full electronic calculation.1.The thermostability of protein thermostable cathechol 2,3-dixoygenase (TC23O)has been studied by the parallel molecu

8、lar dynamics simulations. By analysis ofthe exponent β which is related of scattering spectrum and constant-pressure heatcapacity Cp, we reveal the respective contribution of a specific residue 228proline, a specific sal

9、t bridge Arg186NH1-Glu291OE2, four ions and differentwater environment to the thermostability of TC23O. The dynamic transition temperature of the mutants Pro228Ser, Glu291Gly of the TC23O was decreased about 10℃ and 19℃

10、respectively. The displacement of the four ions we tried have no significant change of the thermostability of TC23O. Water affects the thermostability by influencing the changes of accessible conformation in certain exte

11、nt. All these results agree with the known experimental results.2.The thermostability of tentative mutants of protein thermostable cathechol2,3-dixoygenase (TC23O) which generated by protein knockout has been studied by

12、the parallel molecular dynamics simulations. The targets of the knockout include C-end fragment, random coil fragment of 61-66 and part of a α -helix fragment of 21-26. By analysis the amplitude of the root mean squared

13、(rms)amplitude of motions〈△U2〉 change with temperature, we find that below the room temperature all of the tentative fragment do not significantly influence the thermostability of protein thermostable cathechol 2,3-dixoy

14、genase (TC23O),C-end fragment does not ,as expected, remarkably influence the thermostability of TC23O with the increasing temperature, which is contrary to the fragment of61-66, the fragment 21-26 only significantly eff

15、ect the thermostability of TC23O in the temperature range of 330-430K.3.We investigate the linkage of active center and thermostability of protein TC23Oand mutant P228S with the frontier orbital energy levels using the m

16、ethod of fullelectronic calculation. The result indicated that the frontier orbital energy levelsare sensitive to the change of the primary sequence and advanced conformationof protein. We could find some residues of the

17、 active center present in the frontierorbits, and about 1/6 molecular orbits are composed of residues which areelements of salt bridge.Key words: parallel molecular dynamics, protein knockout, active center of enzyme,sal