Abstract
布頓氏酪胺酸激脢在B細胞的發育中扮演了很重要的角色,一旦它的 SH3區塊從 C端失去了14個胺基酸會造成X染色體之無珈瑪免疫球蛋白症。我們的實驗主要在探討布頓氏酪胺酸激脢的穩定性和他的折疊,我們選用布頓氏酪胺酸激脢的SH3區塊216-273和216-259序列,前者包含SH3區塊所有胺基酸序列,後者在 C端缺失了14個胺基酸。前者形成了一個β-barrel的折疊形式。雖然這個SH3區塊缺少雙硫鍵,但是它呈現了很強的熱穩定性,在CD實驗中在pH=6.0 情況下它的熔點高到82℃,但是相較於一些較大的單區塊蛋白他的吉伯氏自由能卻相當低只有2.6kcal/mol,這說明了 SH3區塊的低穩定性。當我們加入 500mM Na2SO4 時可以增高吉伯氏自由能到4.0kcal/mol。對另一個含44個胺基酸序列的蛋白質,它無法折疊成β-barrel,反而以 random coil 形式處在溶液中,說明了 C 端的 14 個胺基酸對於 SH3 區塊結構形成了很大的貢獻,同時表示了 BTK 的 SH3 區塊發生突變,影響到其穩定性和折疊時會造成 X 染色體之無珈瑪免疫球蛋白症的發生。Bruton's tyrosine kinase(BTK) plays an important role in B cedevelopment. Deletion of C-terminal 14 amino acids of the SH3do of BTK results in X-linked agammaglobulinmia (XLA), aninherited disease. We report here on the stability and foldingof SH3 doma of BTK. Peptides corresponding to residues216-273(58 residues) and 216-259(44 residues) of BTK SH3 domainwere synthesized by so phase methods; the first peptideconstitutes the entire SH3 domai of BTK while the latterpeptide lacks 14 amino acid residues of t C-terminal. The 58amino acid peptide forms mainly a beta - barre type foldingunit. Although small and lacking disulfide bonds, t peptide isextremely stable to thermal denaturation. Based on circulardichroism measurements, its melting temperature was foun to behigh,82℃ at pH 6.0. Howeveer,the Gibbs free energy △G(H2 ofthe intrinsic stability and thermodynamic spontaneity of unfolwere found to be low, 2.6 kcal/mol by Gdn.HCl denaturationexperi as compared to 12 kcal/mol obtained for larger singledomain prot indicating poor stability of SH3 domain. Additionof 500 mM of Na increased the free energy change △G(H2O) to4.0 kcal/mol, sugges ionic strength effect. The truncatedpeptide fails to fold corre and adopts random coil conformationin contast to 58 amino acid beta-ballel peptide, which exhibitshigh thermal stability but no or low stability at ambienttemperature. These results, to our k the ffirst to delineatethe importaance of C-terminal in structur integrity of SH3domains, inddicate also that imporper folding an poor stabilityof mutant SH3 domain in BTK likely causes XLA.