research papers
Comparison of two crystal polymorphs of NowGFP reveals a new conformational state trapped by crystal packing
aDepartment of Physics, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea, bDepartment of Chemistry, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea, and cDepartment of Biotechnology and Bioinformatics, Korea University, Sejong 30019, Republic of Korea
*Correspondence e-mail: dymin@unist.ac.kr, cukim@unist.ac.kr
Crystal C2) and a newly discovered orthorhombic form (space group 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 of the orthorhombic crystal form. This new conformational state captured by orthorhombic crystal packing exhibits two unique features: a conformational shift of the β-barrel scaffold and a restriction of pH-dependent shifts of the key residue Lys61, which is crucial for the pH-dependent spectral shift of this protein. These findings demonstrate a clear connection between crystal packing and alternative conformational states of proteins, providing insights into how structural variations influence the function of fluorescent proteins.
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, two distinct crystal forms of a green fluorescent protein variant, NowGFP, are compared: a previously identified monoclinic form (space groupKeywords: fluorescent proteins; NowGFP; crystal polymorphism; protein crystal packing; conformational flexibility; protein crystallization.
1. Introduction
Proteins can be crystallized in multiple forms, a phenomenon known as crystal et al., 2001). The primary interest in discovering crystal polymorphs of proteins lies in finding a superior crystal form that enables the acquisition of detailed structural information through X-ray crystallography. This crystal form may either have high crystallinity for improved diffraction (Yamada et al., 2017) or possess enhanced thermal and chemical durability (Gerlits et al., 2019). Additionally, different crystal forms can reveal different conformation states of protein molecules which are trapped by packing (Jiang et al., 2013). The availability of different crystal forms provides insight into the conformational flexibility of protein molecules and a detailed understanding of individual conformational states (Zhang et al., 1995). In this study, we demonstrate a new case of protein crystal with NowGFP, a variant of green fluorescent protein (GFP).
A well-known example of protein crystal is lysozyme, which exhibits six crystal polymorphs (VaneyFluorescent proteins have become essential tools in cell biology and biomedicine, serving as non-invasive methods for visualizing and tracking cellular and organism-wide processes (Chudakov et al., 2010). Most of these fluorescent proteins are characterized by the tyrosine (Tyr) at the centre of the three residues responsible for forming the chromophore, resulting in fluorescence that spans from green to far-red wavelengths. Recently, a fluorescent protein based on an anionic tryptophan (Trp) was developed and was named WasCFP (Sarkisyan et al., 2012). Derived from the cyan-fluorescent mCerulean (Rizzo et al., 2004), it was engineered by introducing a critical Val61Lys substitution and four additional mutations. WasCFP was further improved to enhance its stability across a broad range of pH levels and temperatures, incorporating 13 additional mutations. This improved variant, which exhibits strong green fluorescence under physiological conditions, has been named NowGFP (Sarkisyan et al., 2015).
Spectral and structural studies of NowGFP consistently reveal pH-dependent changes. Spectrally, NowGFP exhibits green fluorescence (λex/λem = 493/502 nm) at physiological and higher pH levels, contrasting with cyan fluorescence (λex/λem = 429/475 nm) under acidic conditions (pH < 6). The intensity ratio between green and cyan fluorescence is highly sensitive to pH conditions. Structurally, NowGFP adopts two distinct pH-dependent conformations at pH 4.8 and pH 9.0. These conformations involve the chromophore and the key residue Lys61, which is believed to play a central role in chromophore ionization and the resultant shifts in the fluorescence spectrum (Pletnev et al., 2015).
We present three crystal structures of NowGFP obtained under various pH conditions from two distinct crystal forms: a monoclinic 8xh0, 1.45 Å resolution) and orthorhombic crystal structures at pH 9.0 (PDB entry 8xh1, 1.7 Å resolution) and pH 6.0 (PDB entry 8xh2, 1.8 Å resolution). Our study begins with a detailed comparative analysis of crystal contacts and stacking interactions between these two crystal forms. Following this, we identify major differences in the crystal contacts across the two crystal forms and determine how the crystal packing may alter the NowGFP structure. Lastly, we investigate the alternative conformations trapped by the orthorhombic crystal form by examining five NowGFP molecules: one from the monoclinic form at pH 4.8, two from the orthorhombic form at pH 9.0 and two from the orthorhombic form at pH 6.0. This comparison aims to deepen our understanding of how crystal packing influences the pH-dependent conformational changes in NowGFP.
at pH 4.8 (PDB entry2. Materials and methods
2.1. Macromolecule production
The fragment encoding NowGFP with an N-terminal His tag and TEV protease recognition sequence site was cloned into the pET-24a(+) vector and transformed into Escherichia coli strain BL21(DE3) (Invitrogen, USA). Bacterial cultures were grown overnight at 16°C. Isopropyl β-D-1-thiogalactopyranoside (IPTG) induction was necessary for effective protein expression. The cells were pelleted by centrifugation, resuspended in phosphate-buffered saline (PBS) pH 7.4, 1 mM PMSF, 1 mM TCEP and lysed by an EmulsiFlex-C3 at 103–117 MPa. NowGFP was purified by immobilized metal-ion using His60 Ni Superflow Resin (Clontech, USA) and then buffer-exchanged for cleavage by TEV protease on a Cytiva PD-10 DG column (1× TEV protease buffer: 25 mM Tris–HCl, 150 mM NaCl, 1 mM TCEP). TEV protease was applied (10 U µl−1, 1:100 ratio) and incubated at 30°C for 1 h. After incubation, further purification was achieved by using FPLC (ÄKTApure 25) with a HiLoad 16/600 Superdex 75 pg column (Cytiva, USA). The purity of the sample was then confirmed by SDS–PAGE analysis (Supplementary Fig. S1). The purified NowGFP protein was concentrated using an Amicon Ultra-15 centrifugal filter unit. The amino-acid sequence and other details are given in Table 1.
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2.2. Crystallization
Crystallization screening was conducted to identify optimal conditions for the formation of NowGFP crystals. Single crystals in a monoclinic C2) were obtained using KH2PO4 and PEG 3350. Additionally, single crystals in an orthorhombic (P212121) were obtained using sodium citrate and PEG 4000. Details of the crystallization screening, including other conditions that did not yield diffraction-quality single crystals, are provided in Supplementary Table S1.
(To obtain monoclinic crystals, NowGFP was transferred to 20 mM Tris pH 8.0, 200 mM NaCl buffer and concentrated to 12 mg ml−1. Crystals suitable for data collection were obtained by the hanging-drop vapour-diffusion method (McPherson, 1982). Typically, 2 µl protein solution was mixed with an equal amount of reservoir solution and incubated at 4°C for a week. The best crystal was obtained from 16 mM KH2PO4 pH 4.8, 20%(w/v) PEG 3350.
To obtain orthorhombic crystals, NowGFP was transferred to 20 mM Tris pH 8.0, 200 mM NaCl buffer and concentrated to 17 mg ml−1. Crystals suitable for data collection were obtained by the hanging-drop vapour-diffusion method. Typically, 2 µl protein solution was mixed with an equal amount of reservoir solution and incubated at 4°C for a week. The best crystal was obtained from 100 mM sodium citrate pH 6.0, 25%(w/v) PEG 4000. To obtain structures of NowGFP at pH 9.0, the crystal was transferred to 100 mM ammonium sulfate, 50 mM Tris–HCl pH 9.0, 17.5%(w/v) PEG 4000 and incubated for a week. Details of protein crystallization and photographic images of crystals are given in Table 2 and Supplementary Fig. S2, respectively.
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2.3. Data collection and processing
X-ray diffraction data were collected on Pohang Light Source-II (PLS-II) beamline 7A at the Pohang Accelerator Laboratory, Pohang, Republic of Korea. Prior to data collection, the monoclinic crystals were briefly soaked in a cryoprotectant solution consisting of 30%(v/v) glycerol and 70%(v/v) reservoir, while the orthorhombic crystals were soaked in a cryoprotectant solution consisting of 20%(v/v) glycerol and 80%(v/v) reservoir, and then flash-cooled in a 100 K nitrogen stream. A total of 360 images with 1° oscillation angles were collected with sample-to-detector distances of 150 and 175 mm for monoclinic and orthorhombic crystals, respectively. All diffraction images were processed with HKL-2000 (Otwinowski & Minor, 1997). The absorbed X-ray dose for a single data set was less than 5 × 105 Gy, which is much lower than the Henderson dose limit of 1.45 × 107 Gy (Henderson, 1990). Data-processing statistics and diffraction images are given in Table 3 and Supplementary Fig. S3, respectively.
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2.4. Structure solution and model refinement
The structures of NowGFP from both crystal forms were determined using the CCP4 suite (Agirre et al., 2023). The crystal structures were solved by the molecular-replacement method with MOLREP (Vagin & Teplyakov, 2010) using the previously solved structure of NowGFP (PDB entry 4rtc; Pletnev et al., 2015) as a model. Crystallographic was performed with REFMAC5 (Murshudov et al., 2011), alternating with manual revision of the model with Coot (Emsley et al., 2010). The location of water molecules and structure validation were performed with Coot. The monoclinic (C2) of NowGFP at pH 4.8 was determined at 1.45 Å resolution and orthorhombic crystal structures (P212121) of NowGFP at pH 9.0 and 6.0 were solved at 1.7 and 1.8 Å resolution, respectively. The coordinates and structure factors for NowGFP in the monoclinic form at pH 4.8, the orthorhombic form at pH 9.0 and the orthorhombic form at pH 6.0 were deposited in the Protein Data Bank under accession codes 8xh0, 8xh1 and 8xh2, respectively. Structure-solution and data-refinement statistics are given in Table 4. All structural figures were rendered with PyMOL (Schrödinger).
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3. Results and discussion
3.1. Crystal structures of two crystal polymorphs
The assemblies of protein molecules within the unit cells of both orthorhombic and monoclinic crystals are illustrated in Fig. 1. For the comparative study of the two crystal forms, we chose the orthorhombic obtained at pH 9.0 for comparison with the monoclinic structure since it has a higher resolution (1.7 Å). Comparison between the lower resolution orthorhombic obtained at pH 6.0 (1.8 Å) and the monoclinic structure revealed nearly identical results to the higher pH (pH 9.0) counterpart. This comparison is detailed in Supplementary Figs. S4–S7 and Supplementary Tables S2 and S3.
In the case of the monoclinic (C2) crystal form there are four molecules inside the and one molecule is in the (chain A). We refer to four molecules, which are chains A from symmetry operations (x, y, z), (−x, y, −z), (x + 1/2, y + 1/2, z) and (−x + 1/2, y + 1/2, −z), as molecules 1, 2, 3 and 4, respectively (Figs. 1a–1c).
In the case of the orthorhombic (P212121) crystal form there are eight molecules inside the and two molecules are in the (chains A and B). We refer to four molecules, which are chains A from symmetry operations (x, y, z), (x + 1/2, −y + 1/2, −z), (−x, y + 1/2, −z + 1/2) and (−x + 1/2, −y, z + 1/2), as molecules 1A, 2A, 3A and 4A, respectively. Similarly, we refer to four molecules, which are chains B from symmetry operations (x, y, z), (x + 1/2, −y + 1/2, −z) (−x, y + 1/2, −z + 1/2) and (−x + 1/2, −y, z + 1/2) as molecules 1B, 2B, 3B and 4B, respectively (Figs. 1d–1f).
For simplicity, we refer to chain A of the NowGFP structure in the monoclinic form as Mono. Similarly, we refer to chains A and B in the orthorhombic form as Orth(A) and Orth(B), respectively. Comparative analysis of Mono, Orth(A) and Orth(B) indicates that there are no significant differences in the overall protein structures, with the Cα–Cα r.m.s.d. being less than 0.4 Å. However, partial differences are observed in the r.m.s.d. from ideal angles, B factors and solvation free energy (Tables 4 and 5). These differences will be discussed in detail in Section 3.3.
‡Solvation energy gain upon protein folding. |
3.2. Crystal contacts and related crystal packing
The term `crystal contacts' denotes the interactions between protein molecules that emerge through the crystallization process (Janin & Rodier, 1995; Dasgupta et al., 1997). In this section, the crystal contacts between NowGFP molecules are considered using PISA analysis (Krissinel & Henrick, 2005, 2007) and the protein crystal packing is discussed. All crystal contacts from each molecule are listed in Table 6.
‡Solvation energy gain upon formation of the interface. This value does not include the effect of satisfied hydrogen bonds and salt bridges across the interface. |
In the monoclinic (C2) crystal form, the crystal contacts are categorized into three types, denoted as contacts I, II and III. Contact I is between molecules 1 and 4 (or 2 and 3) (Figs. 2a and 2b), which has the largest contact area for Mono. More specifically, contact I comes from two different symmetry-related molecules (Mono) at (−x + 1/2, y − 1/2, −z − 1) and (−x + 1/2, y + 1/2, −z − 1) and connects molecules 1 and 4 (or 2 and 3) to form a zigzag linear assembly along the b axis (Fig. 2d). Contact II is between molecules 1 and 2 (or 3 and 4) (Figs. 2a and 2c). More specifically, contact II comes from symmetry-related molecules at (−x, y, −z − 1) and connects two types of linear assemblies (1–4 and 2–3) to form layers in the ab plane (Fig. 2d). The between two neighbouring linear assemblies is (−x, y, −z), which is the same as the between molecules 1 and 2 or 3 and 4 (represented as opposite white arrows in Fig. 2d). Contact III is between molecules 1 and 3 (or 2 and 4), which has the smallest contact area (Figs. 2a and 2b). More specifically, contact III comes from two different symmetry-related molecules at (−x + 1/2, y − 1/2, −z) and (−x + 1/2, y + 1/2, −z) and results from the packing of sheets along the c axis (Fig. 2d).
In the orthorhombic (P212121) crystal form, the crystal contacts are categorized into six types: contacts IA, IB, IIAB, IIIAB, IVAB and VB. Contact IA is between molecules 1A and 2A (or 3A and 4A) (Figs. 3a and 3c), which has the largest contact area for Orth(A). More specifically, contact IA comes from symmetry-related molecules chain A [Orth(A)] at (x − 1/2, −y + 1/2, −z) and (x + 1/2, −y + 1/2, −z) and connects molecules 1A and 2A (or 3A and 4A) to form a zigzag linear assembly along the a axis (Fig. 3e). Contact IB is between molecules 1B and 3B (or 2B and 4B) (Figs. 3b and 3c), which has the largest contact area for Orth(B). More specifically, contact IB comes from symmetry-related molecules chain B [Orth (B)] at (−x + 1, y − 1/2, −z + 1/2) and (−x + 1, y + 1/2, −z + 1/2) to form a zigzag linear assembly along the b axis (Fig. 3e). Contacts IIAB and IIIAB are between molecules 1A and 1B (or 2A and 2B, 3A and 3B, or 4A and 4B) (Fig. 3d). More specifically, contact IIAB comes from chain A and chain B in the same while contact IIIAB comes from a symmetry-related chain B (x, y + 1, z) for chain A (x, y, z). Contact IVAB is between molecules 1A and 3B (or 2A and 4B, 3A and 1B, or 4A and 2B) (Fig. 3d). More specifically, contact IVAB comes from a symmetry-related chain B (−x, y + 1/2, −z + 1/2) for chain A (x, y, z). Contacts IIAB, IIIAB and IVAB connect two types of linear assemblies which are orthogonal to each other (assembly of molecules 1A–2A and 1B–3B, 1A–2A and 2B–4B, 3A–4A and 1B–3B, or 3A–4A and 2B–4B; Fig. 3e), and IIAB has the largest contact area among them. The between two linear assemblies made of the same chains (chain A or B) is (−x + 1/2, −y, z + 1/2). This is the same as the between molecules 1A and 4A (or 1B or 4B, 2A and 3A, or 2B and 3B) and is represented as opposite white arrows in Fig. 3(e). Contact VB is between molecules 1B and 3B (or 2B and 4B), which has the smallest contact area (Fig. 3b). More specifically, contact VB comes from a symmetry-related chain B at (−x, y − 1/2, −z + 1/2) and (−x, y + 1/2, −z + 1/2).
3.3. The structural alteration of NowGFP results from crystal contacts
Comparison of the crystal contacts of three types of molecules [Mono, Orth(A) and Orth(B)] from two different crystal forms shows that contacts I, IA and IB are similar in structure and sequence (blue and yellow in Fig. 4). Notably, linear assemblies derived from contacts I, IA and IB are also almost identical (Figs. 2a, 2b and 3a–3c). However, there is a significant difference between contacts II and IIAB (red in Fig. 4). This difference is responsible for the packing mode of the linear assemblies: parallel for the monoclinic form and perpendicular for the orthorhombic form. There are minor differences in the other contacts, including contacts III, IIIAB, IVAB and VB, but those have a much smaller contact area compared with contacts I, IA, IB, II and IIAB (Table 6). In addition, the histogram of the distances between atom pairs for each crystal contact indicates that contacts I, IA, IB, II and IIAB are more closely interacting compared with others (Figs. 4e and 4f). We thus focused on analysing the differences between contacts II and IIAB as represented in Fig. 5.
For contact II in the monoclinic crystal form, there are no atom pairs closer than 3.0 Å (Table 6). Phe99, Tyr182, Gln157 and Lys156 from two molecules contact each other without any water molecule in between. Gln157 also contacts the main chain of Arg96 and Gln183. Additionally, residues corresponding to contact II show no significant deviation from Mono to Orth(A) and Orth(B) (Fig. 5a). This indicates that contact II does not significantly influence the overall protein structure.
For contact IIAB in the orthorhombic crystal form, there are four atom pairs closer than 3.0 Å (Table 6). Two of them come from the arginine–aspartic acid salt bridge between Asp180 of Orth(A) and Arg168 of Orth(B). The other two are from Tyr182 and Tyr164 of Orth(A), which are connected to the main chain of Val176 and Asp173 of Orth(B) through hydrogen bonding. Compared with Mono, a significant shift of the main chain is observed for residues 170–176 of Orth(B), which mainly involves contact IIAB and the loop between the β8 and β9 strands (Figs. 5b and 5c). This indicates that contact IIAB might be responsible for the structural shifts in Orth(B). Additionally, this shift causes residues 141–147, which are part of the β7 strand, to move away from the β10 strand (Fig. 5). Specifically, the Cα–Cα distances between Asn144 in the β7 strand and Gln207 in the β10 strand are 6.3, 6.1 and 6.7 Å for Mono, Orth(A) and Orth(B), respectively.
The evidence that Orth(B) has a more unstable structure compared with Orth(A) or Mono is that the r.m.s.d. from ideal angles, average B factor and solvation free energy all show the highest values for Orth(B). In detail, the r.m.s.d.s from ideal angles for Mono, Orth(A) and Orth(B) are ∼2.0°, ∼1.9° and ∼2.1°, respectively, the average B factors of the main chain from Mono, Orth(A) and Orth(B) are ∼22.7, ∼26.8 and 31.0 Å2, respectively, and the solvation free energy from the isolated structure of each molecule is ∼−215 kcal mol−1 for Orth(A) and Mono and ∼−209 kcal mol−1 for Orth(B) (Tables 4 and 5). These results indicate that Orth(A) exhibits similar conformational behaviour to Mono, while Orth(B) represents a relatively unstable conformational state.
Moreover, protein crystals are highly hydrated, with those with lower solvent content typically having better diffraction quality (Matthews, 1968; Zhang et al., 1995). However, solvent-content estimation reveals that the orthorhombic crystal has less solvent (∼50%) yet results in a lower resolution (1.7 Å) compared with the monoclinic crystal, which contains more solvent (∼58%) but diffracts to a higher resolution (1.45 Å) (Table 3). This discrepancy could be additional evidence that the crystallinity of the orthorhombic crystal is poorer due to the instability of Orth(B).
3.4. New conformational state of NowGFP trapped by packing
In a previous study by Pletnev and coworkers, it was revealed that the key residue Lys61 plays a central role in the ionization process of the chromophore, showing significant pH-dependent conformational changes. Specifically, at high pH the Nζ atom of Lys61 makes two hydrogen bonds: one to the Nɛ atom of Trp66 and the other to the Oɛ1 atom of Glu222 (k1 conformation). This dual bonding helps to form a non';covalent connection and promotes the deprotonation of Trp66. In contrast, at lower pH the orientation of Lys61 shifts away from Trp66, instead forming a hydrogen bond to the Nɛ2 atom of Gln207 (k2 conformation; Pletnev et al., 2015).
To elucidate the alternative conformation state trapped by the orthorhombic
a total of five NowGFP molecules are compared (one molecule from the monoclinic at pH 4.8 and four molecules from the orthorhombic crystal structures at pH 6.0 and pH 9.0).In the case of the Orth(A) and Mono structures, the key residue Lys61 has ∼80% k1 conformation and ∼20% k2 conformation under a high-pH condition (pH 9.0). This shifts to ∼50% k1 conformation and ∼50% k2 conformation as the pH decreases to 6.0, and finally to ∼20% k1 conformation and ∼80% k2 conformation under an acidic pH condition (pH 4.8) (Figs. 6a, 6c and 6e). This trend of conformational shifts from k1 to k2 as the pH decreases is consistent with previously published structures: the monoclinic structure at pH 9.0 (PDB entry 4rtc, 1.18 Å resolution) and pH 4.8 (PDB entry 4rys, 1.35 Å resolution) (Supplementary Fig. S8; Pletnev et al., 2015). Even though the high-resolution structure of NowGFP shows a partial trans conformation of the chromophore at pH 4.8, this is difficult to observe in our moderate-resolution structure (1.45 Å).
In the case of the Orth(B) structure, however, the conformation of Lys61 is almost 100% k1 at both pH 9.0 and pH 6.0, and the distance between the Nɛ atoms of the Trp66 indole and the Nζ atom of Lys61 in the k1 conformation is ∼0.3 Å closer compared with the Orth(A) structure (Figs. 6a–6d). It appears that strong hydrogen bonding between the Trp66 indole and the k1 conformation of Lys61 is responsible for the high occupancies of the k1 conformation in the Orth(B) structure. Considering that Orth(B) has an unstable structure compared with Orth(A) and Mono, and this instability is linked to crystal contact IIAB as detailed in Section 3.3, it is evident that the alteration of the Lys61 conformation in the Orth(B) structure is a result of orthorhombic crystal packing. Table 7 shows the χ1 and χ2 angles of two alternative conformations of the key residue Lys61.
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While this alternative conformational state could be dismissed as merely an artefact of crystal packing, a previous study of crystal polymorphs of photoactive yellow protein indicates that the et al., 2000). Instead, the protein shifts along essential eigenvectors to adapt to different lattice environments. Therefore, different protein structures from crystal polymorphs may represent the inherent conformational flexibility of the molecule (van Aalten et al., 1997).
does not simply impose arbitrary conformational changes on the protein molecule (van AaltenPrevious studies, including the unfolding and backbone dynamics of GFP, reveal that β-strands 7, 8, 9 and 10 exhibit greater flexibility (Huang et al., 2007; Seifert et al., 2003). Additionally, the parental WasCFP shows conformational flexibility in the β7 and β10 strands, which opens the solvent channel between these two β-strands. This flexibility is expected to play a crucial role in managing solvent access to the chromophore (Laricheva et al., 2015). These observations are consistent with our finding that the r.m.s.d. of the main-chain atoms between two structures from crystal polymorphs shows higher values at residues corresponding to β-strands 7, 8, 9 and 10. The most significant deviations are noted in the loop between the β8 and β9 strands, resulting in opening of the solvent channel between the β7 and β10 strands (Fig. 5). Based on these correspondences, the new conformational state of NowGFP captured by crystal packing in this study is likely to represent one of the potential conformational states inherent to this protein.
4. Conclusions
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 zigzag 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 7–10, 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 a different functional behaviour: the key residue Lys61, which is known for its pH-dependent shift from the k1 to the k2 conformation, appears to be locked in the k1 configuration regardless of pH conditions. This contrasts with the unaltered molecule, in which Lys61 exhibits pH-dependent movement as expected. These observations provide valuable insights into how packing influences the conformational states of protein molecules, enhancing our understanding of the conformational flexibility of protein structures.
Supporting information
Supplementary figures and tables. DOI: https://doi.org/10.1107/S2059798324008246/jb5066sup1.pdf
Acknowledgements
The authors would like to thank the staff of beamline 7A at Pohang Light Source-II for their support during data collection. Author contributions were as follows. JKK and CUK conceived the research. HJ, JS, SK and DM ran the experiments. JKK analyzed the data and wrote the manuscript. KHK, DM and CUK contributed to the overall scientific interpretation and edited the manuscript.
Funding information
This research was supported by the National Research Foundation of Korea (award Nos. NRF-2022R1A2C2091815 to CUK and NRF-2020R1C1C1003937 to DM).
References
Aalten, D. M. F. van, Conn, D. A., de Groot, B. L., Berendsen, H. J. C., Findlay, J. B. C. & Amadei, A. (1997). Biophys. J. 73, 2891–2896. PubMed Web of Science Google Scholar
Aalten, D. M. F. van, Crielaard, W., Hellingwerf, K. J. & Joshua-Tor, L. (2000). Protein Sci. 9, 64–72. Web of Science PubMed Google Scholar
Agirre, J., Atanasova, M., Bagdonas, H., Ballard, C. B., Baslé, A., Beilsten-Edmands, J., Borges, R. J., Brown, D. G., Burgos-Mármol, J. J., Berrisford, J. M., Bond, P. S., Caballero, I., Catapano, L., Chojnowski, G., Cook, A. G., Cowtan, K. D., Croll, T. I., Debreczeni, J. É., Devenish, N. E., Dodson, E. J., Drevon, T. R., Emsley, P., Evans, G., Evans, P. R., Fando, M., Foadi, J., Fuentes-Montero, L., Garman, E. F., Gerstel, M., Gildea, R. J., Hatti, K., Hekkelman, M. L., Heuser, P., Hoh, S. W., Hough, M. A., Jenkins, H. T., Jiménez, E., Joosten, R. P., Keegan, R. M., Keep, N., Krissinel, E. B., Kolenko, P., Kovalevskiy, O., Lamzin, V. S., Lawson, D. M., Lebedev, A. A., Leslie, A. G. W., Lohkamp, B., Long, F., Malý, M., McCoy, A. J., McNicholas, S. J., Medina, A., Millán, C., Murray, J. W., Murshudov, G. N., Nicholls, R. A., Noble, M. E. M., Oeffner, R., Pannu, N. S., Parkhurst, J. M., Pearce, N., Pereira, J., Perrakis, A., Powell, H. R., Read, R. J., Rigden, D. J., Rochira, W., Sammito, M., Sánchez Rodríguez, F., Sheldrick, G. M., Shelley, K. L., Simkovic, F., Simpkin, A. J., Skubak, P., Sobolev, E., Steiner, R. A., Stevenson, K., Tews, I., Thomas, J. M. H., Thorn, A., Valls, J. T., Uski, V., Usón, I., Vagin, A., Velankar, S., Vollmar, M., Walden, H., Waterman, D., Wilson, K. S., Winn, M. D., Winter, G., Wojdyr, M. & Yamashita, K. (2023). Acta Cryst. D79, 449–461. Web of Science CrossRef IUCr Journals Google Scholar
Chudakov, D. M., Matz, M. V., Lukyanov, S. & Lukyanov, K. A. (2010). Physiol. Rev. 90, 1103–1163. Web of Science CrossRef CAS PubMed Google Scholar
Dasgupta, S., Iyer, G. H., Bryant, S. H., Lawrence, C. E. & Bell, J. A. (1997). Proteins, 28, 494–514. CrossRef CAS PubMed Google Scholar
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. (2010). Acta Cryst. D66, 486–501. Web of Science CrossRef CAS IUCr Journals Google Scholar
Gerlits, O., Ho, K. Y., Cheng, X., Blumenthal, D., Taylor, P., Kovalevsky, A. & Radić, Z. (2019). Chem. Biol. Interact. 309, 108698. CrossRef PubMed Google Scholar
Henderson, R. (1990). Proc. R. Soc. Lond. B, 241, 6–8. CrossRef CAS Web of Science Google Scholar
Huang, J. R., Craggs, T. D., Christodoulou, J. & Jackson, S. E. (2007). J. Mol. Biol. 370, 356–371. CrossRef PubMed CAS Google Scholar
Janin, J. & Rodier, F. (1995). Proteins, 23, 580–587. CrossRef CAS PubMed Web of Science Google Scholar
Jiang, Y., Lu, G., Trescott, L. R., Hou, Y., Guan, X., Wang, S., Stamenkovich, A., Brunzelle, J., Sirinupong, N., Li, C. & Yang, Z. (2013). PLoS One, 8, e81904. CrossRef PubMed Google Scholar
Krissinel, E. & Henrick, K. (2005). Computational Life Sciences, edited by M. R. Berthold, R. C. Glen, K. Diederichs, O. Kohlbacher & I. Fischer, pp. 163–174. Berlin, Heidelberg: Springer. Google Scholar
Krissinel, E. & Henrick, K. (2007). J. Mol. Biol. 372, 774–797. Web of Science CrossRef PubMed CAS Google Scholar
Laricheva, E. N., Goh, G. B., Dickson, A. & Brooks, C. L. III (2015). J. Am. Chem. Soc. 137, 2892–2900. CrossRef CAS PubMed Google Scholar
Matthews, B. W. (1968). J. Mol. Biol. 33, 491–497. CrossRef CAS PubMed Web of Science Google Scholar
McPherson, A. (1982). Preparation and Analysis of Protein Crystals. Chichester: John Wiley & Sons. Google Scholar
Murshudov, G. N., Skubák, P., Lebedev, A. A., Pannu, N. S., Steiner, R. A., Nicholls, R. A., Winn, M. D., Long, F. & Vagin, A. A. (2011). Acta Cryst. D67, 355–367. Web of Science CrossRef CAS IUCr Journals Google Scholar
Otwinowski, Z. & Minor, W. (1997). Methods Enzymol. 276, 307–326. CrossRef CAS PubMed Web of Science Google Scholar
Pletnev, V. Z., Pletneva, N. V., Sarkisyan, K. S., Mishin, A. S., Lukyanov, K. A., Goryacheva, E. A., Ziganshin, R. H., Dauter, Z. & Pletnev, S. (2015). Acta Cryst. D71, 1699–1707. Web of Science CrossRef IUCr Journals Google Scholar
Rizzo, M. A., Springer, G. H., Granada, B. & Piston, D. W. (2004). Nat. Biotechnol. 22, 445–449. CrossRef PubMed CAS Google Scholar
Sarkisyan, K. S., Goryashchenko, A. S., Lidsky, P. V., Gorbachev, D. A., Bozhanova, N. G., Gorokhovatsky, A. Y., Pereverzeva, A. R., Ryumina, A. P., Zherdeva, V. V., Savitsky, A. P., Solntsev, K. M., Bommarius, A. S., Sharonov, G. V., Lindquist, J. R., Drobizhev, M., Hughes, T. E., Rebane, A., Lukyanov, K. A. & Mishin, A. S. (2015). Biophys. J. 109, 380–389. CrossRef CAS PubMed Google Scholar
Sarkisyan, K. S., Yampolsky, I. V., Solntsev, K. M., Lukyanov, S. A., Lukyanov, K. A. & Mishin, A. S. (2012). Sci. Rep. 2, 608. Web of Science CrossRef PubMed Google Scholar
Seifert, M. H., Georgescu, J., Ksiazek, D., Smialowski, P., Rehm, T., Steipe, B. & Holak, T. A. (2003). Biochemistry, 42, 2500–2512. Web of Science CrossRef PubMed CAS Google Scholar
Vagin, A. & Teplyakov, A. (2010). Acta Cryst. D66, 22–25. Web of Science CrossRef CAS IUCr Journals Google Scholar
Vaney, M. C., Broutin, I., Retailleau, P., Douangamath, A., Lafont, S., Hamiaux, C., Prangé, T., Ducruix, A. & Riès-Kautt, M. (2001). Acta Cryst. D57, 929–940. Web of Science CrossRef CAS IUCr Journals Google Scholar
Yamada, K. D., Kunishima, N., Matsuura, Y., Nakai, K., Naitow, H., Fukasawa, Y. & Tomii, K. (2017). Acta Cryst. D73, 757–766. CrossRef IUCr Journals Google Scholar
Zhang, X. J., Wozniak, J. A. & Matthews, B. W. (1995). J. Mol. Biol. 250, 527–552. CrossRef CAS PubMed Web of Science Google Scholar
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