Comparison of two crystal polymorphs of NowGFP reveals a new conformational state trapped by crystal packing
Dr Jin Kyun Kim [1]
Molecular Tour
Crystal polymorphism serves as a strategy to study the conformational flexibility of proteins. However, the relationship between protein crystal packing and protein conformation often remains elusive. In this study, we compare two distinct crystal forms of a green fluorescent protein variant, NowGFP: a previously identified (C2) and a newly discovered (P212121). Comparative analysis reveals that both crystal forms exhibit nearly identical linear assemblies of NowGFP molecules interconnected through similar crystal contacts. However, a notable difference lies in the stacking of these assemblies: parallel in the monoclinic form and perpendicular in the orthorhombic form. This distinct mode of stacking leads to different crystal contacts and induces structural alteration in one of the two molecules within the asymmetry unit of the orthorhombic crystal form. This new conformational state captured by orthorhombic crystal packing exhibits two unique features: a conformational shift of the beta-barrel scaffold and restriction of pH-dependent shifts of the key residue Lys61, which is crucial for pH-dependent spectral shift of this protein. These findings demonstrate a clear connection between crystal packing and the alternative conformational states of proteins, providing insights into how structural variations influence the function of fluorescent proteins.
In this study, we report the discovery of a novel orthorhombic crystal form of NowGFP and conduct a detailed comparison with the known monoclinic crystal form. Our investigations primarily focused on the crystal contacts, revealing that both forms exhibit similar zig-zag linear assemblies of protein molecules, resulting from crystal contact I. The key distinction between the two forms lies in their stacking modes: parallel stacking for the monoclinic form and perpendicular stacking for the orthorhombic form. This difference in packing correlates with a specific crystal contact, referred to as crystal contact II (or IIAB), and results in an alteration of one molecule in the symmetry unit of the orthorhombic crystal form, designated as Orth(B). Given that these structural shifts are predominantly concentrated between β-strands 7 to 10, which are known for their partial flexibility, we propose that this altered molecule represents an alternative conformational state of NowGFP. In contrast, the other molecule in the orthorhombic form, Orth(A), remains unchanged and is similar to that found in the monoclinic form.
Significantly, this new conformational state of NowGFP captured in the orthorhombic crystal packing exhibits different functional behavior: the key residue Lys61, which is known for its pH-dependent shifts from k1 to k2 conformations, appears locked in a k1 configuration regardless of pH conditions. Specifically, this can be seen at pH 9.0, that Lys-61 in has two alternative conformations (80% in k1 and 20% in k2) while Lys-61 in is seen to only be in the k1 conformation.
At ph 6.0 has two alternative conformations (50% in k1 and 50% in k2) while is seen to only be in the k1 conformation. The form, at pH 6.0, has only one molecule in the asymmetric unit, with Lys-61 showing two conformations has (20% in k1 and 80% in k2).
This contrasts with the unaltered molecule, in which Lys61 exhibits the pH-dependent movement as expected. These observations provide valuable insights into how crystal lattice packing influences the conformational states of protein molecules, enhancing our understanding of protein structure's conformational flexibility.
References
- ↑ Kim JK, Jeong H, Seo J, Kim S, Kim KH, Min D, Kim CU. Comparison of two crystal polymorphs of NowGFP reveals a new conformational state trapped by crystal packing. Acta Crystallogr D Struct Biol. 2024 Sep 1. PMID:39222305 doi:10.1107/S2059798324008246
