利用多晶體分群分析法解析光敏微生物視紫紅質在環境光下的構型異質性

Abstract

Haloquadratum walsbyi 是一種嗜鹽古菌，其表現的微生物視紫紅質（HwMR）在序列上介於細菌視紫紅質與感光視紫紅質之間。由於其晶體體積小且可能存在內在構型異質性，使用傳統單晶 X 光晶體學技術只能獲得低解析度且不完整的繞射圖譜。為了解決此問題，我們採用多晶體策略，探討是否可藉由合併多個微晶體揭示潛在的構型異質性。 我們自超過 100 顆 HwMR 微晶體收集 X 光繞射圖譜，並以小角度策略進行資料擷取。完成初步處理後，採用兩種方式進行階層式叢集分析（HCA）：一是依據晶胞參數、二是根據繞射圖樣的皮爾森相關係數。分析後將資料分群，並分別進行合併與結構精修，進一步比較不同分群策略的資料品質與結構差異。 兩種分群方式皆可產生可用於結構解析的資料簇，解析度可達約 2.5–2.7 Å。值得注意的是，相關係數為基礎的分群法提供了更細緻的分類，揭示了 HwMR 的不同構型狀態。部分資料簇所對應的結構模型中，視黃醛為全反式構型；另一些簇則須建模為 13-順式構型。這些視黃醛異構化的差異伴隨七條跨膜螺旋的明顯重排。在細胞外側視角中，C 到 G 螺旋在 13-順式構型中向外位移；在細胞質側，B、F 與 G 螺旋亦出現類似的外移現象。這些結果顯示 HwMR 在視黃醛異構化過程中會產生大尺度的構型轉變，反映其功能上的異質性。 為了證實這些構型差異並非由環境因素（如環境光）所致，我們以結構穩定、非光敏性的蛋白質 Lysozyme 為對照組進行負控制組實驗。利用相同的多晶體策略，我們共收集了 346 組 Lysozyme 的繞射圖譜，並進行分群與結構比對。所有解析出的結構之間差異極小（RMSD < 0.3 Å），顯示 HwMR 中觀察到的構型異質性確實源自於蛋白質本身的構象變化，而非實驗上的偏差或光照所致。

Haloquadratum walsbyi is a halophilic archaeon that expresses a microbial rhodopsin (HwMR) whose sequence lies between bacteriorhodopsin and sensory rhodopsin. Due to small crystal sizes and intrinsic conformational heterogeneity, HwMR crystals yielded only partial, low-resolution diffraction when analyzed by conventional single-crystal X-ray crystallography. To address this, we employed a multi-crystal strategy to assess whether structural heterogeneity could be detected by combining many micro-crystals. We collected X-ray diffraction data from over 100 HwMR micro-crystals using a small-wedge synchrotron approach. After indexing and scaling, hierarchical clustering analysis (HCA) was performed using two approaches: unit-cell-based and correlation coefficient-based methods. The resulting dataset clusters were individually merged and used for structure refinement. This allowed us to compare clustering strategies and examine possible structural differences between clusters. Both clustering strategies produced clusters suitable for structure determination, with diffraction data reaching ~2.5–2.7 Å resolution. Notably, correlation coefficient-based clustering offered finer classification, revealing distinct conformational states of HwMR. In particular, structural models showed that certain clusters contained retinal in the all-trans form, while others exhibited a 13-cis retinal configuration. These isomerization differences were associated with prominent rearrangements in the seven-transmembrane helices. From the extracellular view, helices C through G were observed to shift outward in the 13-cis form. From the cytoplasmic view, helices B, F, and G also showed outward displacements. These findings demonstrate that HwMR undergoes large-scale structural transitions upon retinal isomerization, capturing its functional heterogeneity. To confirm that these observed conformational differences were not caused by environmental conditions (e.g., ambient light), we conducted a negative control using lysozyme, a non-photosensitive protein with a stable conformation. Using the same multi-crystal protocol, we collected 346 lysozyme datasets and performed clustering and structure comparison. All resolved structures showed negligible differences (RMSD < 0.3 Å), confirming that the conformational heterogeneity observed in HwMR originates from intrinsic structural states rather than experimental artifacts.