Identification and Characterization of an Inside-Out Folding Intermediate of T4 Phage Sliding Clamp  Manika Indrajit Singh, Vikas Jain  Biophysical Journal  Volume 113, Issue 8, Pages 1738-1749 (October 2017) DOI: 10.1016/j.bpj.2017.08.043 Copyright © 2017 Biophysical Society Terms and Conditions

Figure 1 W199 indicates the presence of a molten globule intermediate. (A) Average lifetime (τavg) of W92 and W199 present in gp45W92 and gp45W199, respectively, in varying urea concentrations is plotted. Each data point represents an average of at least two independent experiments with error bars as SD. (B) Graph depicts the rotational correlation time (τc) calculated for gp45W92 and gp45W199 using the equation described in Materials and Methods. Both W92 and W199 show restricted conformation in 0 M urea. Upon unfolding in presence of urea, W92 experiences conformational flexibility as observed by a decrease in τc. W199, although exposed on CTD surface, remains rigid in 0 M urea, and shows flexibility upon MG formation (at 3.5 M urea). In 8 M urea, both tryptophans show very low τc. (C) REES is plotted at each urea concentration for W92 and W199 present in gp45W92 (circle) and gp45W199 (triangle), respectively. Shaded region marks the zone of urea concentration in which gp45WT forms MG. Beginning and end of the shaded region refers here to the first and the second transitions of gp45WT unfolding, respectively. REES increases on the onset of MG formation and decreases slightly after the protein has completely unfolded. Although CTD (represented by W199 in gp45W199) unfolds at ∼5.5 M urea, the increase in REES at first transition indicates the role of CTD in MG formation. The data represent an average of at least three independent experiments. For representative raw data, see Fig. S2. (D) The 1H-15N HSQC overlaid profiles of gp45WT are plotted at different urea concentrations to monitor the change in Trp indole resonances (enlarged in inset). The data were recorded at 0 M (black), 2 M (red), 2.5 M (blue), 3 M (green), 4 M (orange), 6 M (cyan), and 8 M (purple) and were compared with HSQC profiles of gp45W92 and gp45W199 at different urea concentrations (data not shown) to confirm the chemical shift in indole resonances. For gp45W92 and gp45W199 HSQC profiles, see Fig. S3. (E) Indole resonances are plotted against the urea concentrations to map the movement of each Trp. W199 loses its resonance in MG form. To see this figure in color, go online. Biophysical Journal 2017 113, 1738-1749DOI: (10.1016/j.bpj.2017.08.043) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 2 C-terminal domain of gp45 unfolds with a dry molten globule intermediate. ANS fluorescence profiles of gp45WT, gp45S88P, gp45CTD, and gp45NTD with increasing urea concentrations are plotted. The shaded region marks the zone of urea concentration in which gp45WT forms MG. The plots are divided in three sections—a, b, and c. Section a—the pre-MG state—shows high ANS fluorescence in the case of gp45NTD, no fluorescence in gp45CTD, and intermediate values for gp45WT and gp45S88P. Section b—the MG state—shows negligible ANS fluorescence from gp45CTD. The fluorescence in all other cases reduces with increasing urea concentrations, suggesting loss of ANS binding to protein. Here, the negligible ANS fluorescence at least in the case of gp45CTD suggests DMG formation that does not allow water molecules containing dye to penetrate the core. In all other proteins, ANS fluorescence, although insignificant, occurs primarily due to the NTD. Section c—post-MG—shows almost no fluorescence in all the cases, which is primarily due to complete denaturation. The data represent an average of three independent experiments with SD. To see this figure in color, go online. Biophysical Journal 2017 113, 1738-1749DOI: (10.1016/j.bpj.2017.08.043) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 3 Trp scanning mutagenesis of gp45 surface and core residues located in the C-terminal domain. (A) Crystal structure of gp45 (PDB: 1CZD) shows the two domains of a gp45 subunit. The single subunit is enlarged and the residues that were chosen for Trp scanning mutagenesis are marked. The residues that were stably purified and used for further studies are shown as sticks. (B) Given here is a schematic representation of the gp45 CTD sequence (residues from 110 to 228) and the secondary structure (shown as arrows and cylinders). The SASA values for all residues are presented on a scale of zero (least accessible) to one (most accessible). Residues selected for Trp scanning mutagenesis are shown in red. The 25 residues, which upon mutation to Trp were successfully purified and analyzed, are marked with a dot. To see this figure in color, go online. Biophysical Journal 2017 113, 1738-1749DOI: (10.1016/j.bpj.2017.08.043) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 4 gp45 surface residues bury themselves in CTD core during DMG formation. Bimolecular quenching constants for the Trp fluorescence in gp45WT (W92 and W199; top) and Trp mutants (both top and bottom) in their fully folded (0 M urea), MG (3.5 M urea), and fully unfolded (8 M urea) states are depicted. Most of the surface residues show reduced kq values in 3.5 M urea because of burying of the side chain in the protein’s core in the MG state. The residues are finally exposed upon unfolding in 8 M urea. The data presented here are an average of three independent experiments with error bars representing the SD. To see this figure in color, go online. Biophysical Journal 2017 113, 1738-1749DOI: (10.1016/j.bpj.2017.08.043) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 5 The surface residues come in close proximity during DMG formation. (A) Crystal structure of gp45 subunit shows the arrangement of residues that were mutated to Cys and labeled with an acceptor IAEDANS to perform FRET analysis with the donor W199. All the residues are located in the CTD except S19, which lies in the NTD. (B) Normalized FRET data for the different residues that were mutated to Cys and were labeled with IAEDANS (marked with an asterisk) are plotted. In the left panels, data normalized with reference to the emission intensity of Trp at 0 M urea are plotted. Right panels show the FRET data that are plotted after normalizing the IAEDANS emission at 0 M urea equal to 1. The shaded region in the right panels marks the zone of urea concentration in which gp45WT forms MG. In the MG state, R131C∗, E144C∗, and G192C∗ come in close proximity to W199 and show increase in FRET. In contrast, C206∗ that lies in close proximity to W199 shows reduction in FRET because the two residues move apart. S19C∗ shows negligible change and was used as a negative control. The data shown are the average of at least two independent experiments with error bars representing the SD. To see this figure in color, go online. Biophysical Journal 2017 113, 1738-1749DOI: (10.1016/j.bpj.2017.08.043) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 6 Trapping the MG form of gp45 in native condition. (A) Acrylamide-mediated fluorescence quenching of T137C∗MG shows W199 to be significantly buried when compared to W199 in gp45W199 (KSV ∼10 (52)). (B) REES data obtained by probing W199 in T137C∗ (open symbols) and T137C∗MG (filled symbols) are plotted. REES profile of T137C∗MG in 0 M urea overlaps well with that of T137C∗ in 3.5 M urea. An average of two independent experiments is plotted with the SD. Note that REES data for both the proteins at the same concentration of urea do not overlap. (C) Given here is a BN-PAGE profile of gp45WT at three different urea concentrations and T137C∗MG at 0 M urea. T137C∗MG shows retarded mobility in 0 M urea that is similar to the retarded mobility of DMG state of gp45WT obtained in 3.5 M urea. Here, T represents trimer, M, monomer, and MG, the molten globule form. (D) Sodium dodecyl sulfate-PAGE profile of the formaldehyde-mediated, cross-linked protein samples. T137C and T137C∗ mutants show reduced cross-linking efficiency in comparison to gp45WT. The trapped MG mutant (T137C∗MG) does not stay as a trimer in solution and shows no cross-linked trimer (T) on gel. D represents the dimeric species, whereas M denotes monomeric form of the protein; CL stands for cross-linker. Molecular-weight ladder is shown on the right with two bands marked. (E) Trp fluorescence intensity of T137C∗ and T137C∗MG in four urea concentrations, i.e., 0, 3.5, 6, and 8 M. The 0 M urea spectrum for T137C∗MG shows a blue-shifted profile similar to the 3.5 M urea profile of T137C∗. (F) Given here is a FRET analysis of an IAEDANS-labeled T137C∗MG mutant with increasing urea concentration. The mutant because of being trapped in MG form shows no significant change until ∼6 M urea, when the domain unfolds. The spectra were normalized to Trp emission at varying urea concentrations (left panel). FRET data were also normalized considering emission intensity of IAEDANS at 0 M urea equal to 1 and plotted in the right panel against increasing urea concentrations. The shaded region in the right panel marks the zone of urea concentration in which T137C∗MG remains in MG form. The data shown are the average of at least two independent experiments with error bars representing the SD. (G) Given here are coplotted far-UV CD profiles of T137C∗MG in 0 M urea and gp45WT (WT) in 3.5 M urea. The WT protein spectrum was recorded only until 207 nm because of the presence of urea. Both WT and T137C∗MG proteins show the presence of secondary structure. (H) Shown here is a comparison of near-UV CD profiles of T137C∗MG with gp45WT (WT) and gp45CTD (CTD) in 0 M urea (left panel) and in 3.5 M urea (right panel); in both the panels, T137C∗MG is in 0 M urea. The CD profile of T137C∗MG in 0 M urea resembles the gp45CTD and gp45WT in 3.5 M urea (right panel). To see this figure in color, go online. Biophysical Journal 2017 113, 1738-1749DOI: (10.1016/j.bpj.2017.08.043) Copyright © 2017 Biophysical Society Terms and Conditions