7SPF

Crystal structure of sperm whale myoglobin variant sMb5(pCaaF)

7SPF の概要

• エントリーDOI: 10.2210/pdb7spf/pdb
• 分子名称: Myoglobin, NITRATE ION, PROTOPORPHYRIN IX CONTAINING FE, ... (6 entities in total)
• 機能のキーワード: heme, metalloprotein, thioether, myoglobin, oxygen storage
• 由来する生物種: Physeter catodon (sperm whale)
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 18624.13

構造登録者

Bacik, J.P.,Fasan, R.,Ando, N. (登録日: 2021-11-02, 公開日: 2022-06-01, 最終更新日: 2024-10-16)

主引用文献

Iannuzzelli, J.A.,Bacik, J.P.,Moore, E.J.,Shen, Z.,Irving, E.M.,Vargas, D.A.,Khare, S.D.,Ando, N.,Fasan, R.
Tuning Enzyme Thermostability via Computationally Guided Covalent Stapling and Structural Basis of Enhanced Stabilization.
Biochemistry, 61:1041-1054, 2022

PubMed Abstract: Enhancing the thermostability of enzymes without impacting their catalytic function represents an important yet challenging goal in protein engineering and biocatalysis. We recently introduced a novel method for enzyme thermostabilization that relies on the computationally guided installation of genetically encoded thioether "staples" into a protein via cysteine alkylation with the noncanonical amino acid -2-bromoethyl tyrosine (O2beY). Here, we demonstrate the functionality of an expanded set of electrophilic amino acids featuring chloroacetamido, acrylamido, and vinylsulfonamido side-chain groups for protein stapling using this strategy. Using a myoglobin-based cyclopropanase as a model enzyme, our studies show that covalent stapling with -chloroacetamido-phenylalanine (pCaaF) provides higher stapling efficiency and enhanced stability (thermodynamic and kinetic) compared to the other stapled variants and the parent protein. Interestingly, molecular simulations of conformational flexibility of the cross-links show that the pCaaF staple allows fewer energetically feasible conformers than the other staples, and this property may be a broader indicator of stability enhancement. Using this strategy, pCaaF-stapled variants with significantly enhanced stability against thermal denaturation (Δ' = +27 °C) and temperature-induced heme loss (Δ = +30 °C) were obtained while maintaining high levels of catalytic activity and stereoselectivity. Crystallographic analyses of singly and doubly stapled variants provide key insights into the structural basis for stabilization, which includes both direct interactions of the staples with protein residues and indirect interactions through adjacent residues involved in heme binding. This work expands the toolbox of protein stapling strategies available for protein stabilization.

PubMed: 35612958
DOI: 10.1021/acs.biochem.2c00033

実験手法

X-RAY DIFFRACTION (1.17 Å)