為甚麼Taq可以耐高溫工作
為甚麼肉類要低溫保鮮
你認為教授想聽的重點在哪
蛋白質的結構跟環境條件的關係
蛋白質的結構怎樣造成兩者的差別
用二級結構以上的構成去解析
題目鎖定在Taq酶跟肉類保鮮
引注資料
Structure of Taq polymerase with DNA at the polymerase active site
https://www.nature.com/articles/382278a0
A Study on the Effect of Surface Lysine to Arginine Mutagenesis on Protein Stability and Structure Using Green Fluorescent Protein
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3392243/
Thermodynamic effects of proline introduction on protein stability.
https://www.ncbi.nlm.nih.gov/pubmed/17034035
Protease structure
https://goo.gl/HVhspf
Biochemistry: Why is Taq polymerase so thermostable?
https://goo.gl/DfvMQz
Special cases: Histidine, proline, glycine, cysteine
https://goo.gl/7jpdhA
Engineering protein stability with atomic precision in a monomeric miniprotein
https://goo.gl/WpYjvc
Thermodynamic effects of proline introduction on protein stability.
https://goo.gl/vBt2oX
Definitions of Def-L
https://goo.gl/eCGMsJ
Crystal structure of the large fragment of Thermus aquaticus DNA polymerase I at 2.5-A resolution: structural basis for thermostability
https://goo.gl/4ELvRd
1TAQ-STRUCTURE OF TAQ DNA POLYMERASE
https://goo.gl/tS8Aq4
為何Taq可以耐高溫
結構
目前應用的現況
催化核苷酸沿 5'→3'方向進行聚合反應
Primer extension
DNA sequencing
跟Ecoli同源性很高
序列：
TP has more arginines instead of lysines, more glycines instead of alanines, more prolines instead of alanines, and a slight increase in hydrophobic residue content.
三個domain：
– Polymerase Activity
– 3’ to 5’ Exonuclease - Fix errors
– 5’ to 3’ Exonuclease – Remove RNA or DNA in “front” of primer
The fact that there is significant similarity in native states but dissimilarity in stability, points to the involvement of denatured states.
The DNA polymerase from Thermus aquaticus (Taq polymerase) is homologous to Escherichia coli DNA polymerase I (Pol I) and likewise has domains responsible for DNA polymerase and 5' nuclease activities
Taq酶跟E.coli的pol同源，都有domain可以去做5'的活性
glycine: 沒有支鏈，具有較大彈性
proline:被用作提高蛋白質穩定度的胺基酸
Thermodynamic effects of proline introduction on protein stability.
https://goo.gl/vBt2oX
arginine:形成salt bridge
A Study on the Effect of Surface Lysine to Arginine Mutagenesis on Protein Stability and Structure Using Green Fluorescent Protein
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3392243/
本身活性
74℃ 複製 DNA，在 95℃ 的半衰期為 40 分鐘 ⭐
鎂離子存在下可催化核苷酸沿 5'→3'方向進行聚合反應，產生雙股 DNA；
同時它還具有 5' →3' 外切酶活性 (exonuclease activity)
Taq DNA Polymerase 產生的PCR產物在 3' 端會多加一個 “A”，可用於TA cloning。
Taq酶可在72°C時於10秒內複製一含有1000個鹼基對的DNA
缺點之一是缺乏從3'端到5'端的外切酶校正活性，從而導致Taq在複製時保真度不高。原先測得的錯誤率為每9000個核苷酸中出現1次錯誤
Pfu是具有3'至5'端外切酵素特性的聚合酶，出錯機率約為1/2.6x1000000。但相較於Taq聚合酶，Pfu合成DNA的速度較慢
使合成的產物「末端帶有一個A鹼基」，TA克隆即是利用Taq聚合酶此特性，Taq的PCR產物在3'末端會多出一個A，此時只須有一個與其互補的T表現在質體上，即能彼此靠近，藉由接合酶連接起來。透過此方式，可省去限制酶剪切的時間，直接利用PCR產物與質體彼此有互補兩端的特性，可快速黏合起來。
DNA polymerases from Pyrococcus furiosus (open circles), Thermus aquaticus (closed circles), and Bacillus caldotenax (open squares)
https://www.frontiersin.org/files/Articles/108662/fmicb-05-00465-HTML/image_m/fmicb-05-00465-g001.jpg
在高熱度下面還是很穩定
Crystal structure of Taq has more stable interactions (or less unfavorable interactions) compared E. coli Pol I polymerase, nothing significant to explain thermostability
Folding enthalpy of Taq polymerase is less than that of E. coli Pol I polymerase. Taq is more stable due to reduced entropic penalty of folding
Many thermophilic proteins are stable at high temperatures due to enthalpic contributions. That is, more hydrogen bonds/salt bridges/non-covalent interactions
氫鍵在一定的溫度下就會有機率被打斷，氫鍵的形成則是依照原子間的距離而生成，所以重點應該在整個骨架
Taq跟pol的結構非常像，而差別是在序列上的不同
你要怎樣呈現簡報？
Taq
1.用影片開場：間歇泉，帶入Taq
2.介紹Taq基本性質，作為後面深入探討的起點
3.先複習蛋白質結構，不然等一下要講的時候聽眾會跟不上，重點是要說出CBK想聽的，蛋白質的結構是依靠非共價鍵的弱力組成
4.切入重點：介紹Taq跟Ecoli同源性很高的情況下，到底是哪裡的差別
蛋白質就是看結構，但是兩者好像？
明明都是依靠氫鍵、雙硫鍵、鹽橋，為何Taq就是比較耐高溫？
回去看序列
序列：
TP has more arginines instead of lysines, more glycines instead of alanines, more prolines instead of alanines, and a slight increase in hydrophobic residue content.
glycine: 沒有支鏈，具有較大彈性
proline:被用作提高蛋白質穩定度的胺基酸
Thermodynamic effects of proline introduction on protein stability.
https://goo.gl/vBt2oX
arginine:形成salt bridge
A Study on the Effect of Surface Lysine to Arginine Mutagenesis on Protein Stability and Structure Using Green Fluorescent Protein
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3392243/
肉類
1.從我們知道的開始下手：肉裡面就有protease、細菌也有
2.開始介紹Protease
3.在介紹一下，如果只是變成胺基酸，為何會有臭味？