阿拉伯芥穿膜蛋白HHP1之N端結構解析

Abstract

Heptahelical proteins 1 (HHP1) 是一個阿拉伯芥的穿膜蛋白，主要參與ABA和滲透壓信號的調控。HHP1的N端片段 (第1至第96個胺基酸) 被推測包含固有無序區域 (Intrinsically disordered regions, IDRs)。我們先前的研究發現，HHP1的N端可能與冷逆境調節因子ICE1 產生交互作用。由於ICE1是阿拉伯芥對抗冷逆境的關鍵調節因子，我們推測 HHP1 的 N 端可能在冷訊號及滲透壓訊號傳遞中扮演一定的角色。為了研究HHP1 N 端片段的生物物理及生化特性，我們使用E coli以及pET16b質體建構了重組蛋白生產系統，並表現重組蛋白10His_nHHP1 (13.96kDa)。最佳的表現條件是在25°C下，以0.2 mM IPTG誘導E. coli Rosetta (DE3)，並經由LC-MS/MS確認為目標蛋白。利用圓二色光譜 (Circular Dichroism, CD) 進行二級結構分析，我們發現10His_nHHP1在加熱至95°C後，原本呈現的random coils結構部分轉變為α-helices結構。根據這一觀察，我們初步推測，可能是樣品中的雜蛋白干擾了nHHP1的實際構像，導致其光譜特徵顯示為random coils。因此，我們計劃在純化前進行熱處理，藉此降解雜蛋白以驗證此假設。然而，經過熱處理後的10His_nHHP1，並未顯現出有序結構的特徵。我們推測，這可能與環境因素改變有關，不僅僅是因為溫度，還可能包括鹽濃度和 pH 的變化。基於這些結果，我們推論nHHP1的結構轉變可能需要其他因子的協助。總而言之，我們成功建立了 nHHP1 的重組蛋白生產系統，並初步揭示了其在各種環境條件下的結構動態特性。

Heptahelical Proteins 1 (HHP1) is a transmembrane protein in Arabidopsis thaliana, primarily involved in regulating ABA and osmotic stress signaling. The N-terminal of HHP1 (1 A.A to 96 A.A) is predicted to be intrinsically disordered regions. Our previous research found that the N-terminal of HHP1 may interact with the cold stress regulator ICE1. Given that ICE1 is a key factor in Arabidopsis cold stress response, we hypothesize that the N-terminal of HHP1 may play a role in cold and osmotic stress pathways. In order to study the biophysical and biochemical properties of this fragment, we established a recombinant protein production system using E. coli and pET16b plasmid to express 10His_nHHP1 (13.96 kDa). Optimal expression was achieved in E. coli Rosetta (DE3) with 0.2 mM IPTG at 25°C, and soluble protein was confirmed by LC-MS/MS. CD spectroscopy revealed that nHHP1 undergoes a structural transition from random coils to partially α-helices upon heating to 95°C. This observation led us to hypothesize that other proteins might influence the actual conformation of nHHP1, resulting in spectral characteristics resembling random coils. To test this hypothesis, we implemented heat treatment prior to purification to degrade other proteins. However, following heat treatment, 10His_nHHP1 did not exhibit features of ordered structure. These findings suggest that the observed effects may be influenced not only by temperature but also by other environmental factors, such as variations in salt concentration and pH. In summary, we successfully established a recombinant protein production system for nHHP1 and gained preliminary insights into its structural dynamics under diverse environmental conditions.