Figure 1.
Structural domain arrangements of wild-type Sis1 and mutants.
(A) Sis1 contains the J-domain and the G/F region located in the N-terminal region (121 amino acids residues – red box). In the central portion, between the G/F domain and the C-terminus, Sis1 has a G/M-rich (Glycine/Methionine-rich – green box) domain and Ydj1 has a zinc-finger-like region (ZFLR; yellow box). The C-terminal domains (CTD) I and II follow (orange and blue boxes, respectively). In Sis1_Δ124–174 the G/M region is absent and in Sis1_Δ121–257 both the G/M region and the CTDII are deleted. The chimera SYS is formed by the J-domain and the CTDII from Sis1 and the middle region from Ydj1. Ydj1 is shown for comparison. PDB accession numbers: 2O37 (red, residues 1–81), 1C3G (yellow, residues 180–255; blue: 256–349) [16], 1NLT (yellow, residues 110–255 and blue 256–337) [13]. (B) Amino acid sequence alignment of Sis1, SYS and Ydj1. Red box, J-domain; Green, G/M-rich region; red, CTDI; blue, CDTII; yellow, permuted region between Ydj1 and Sis1; gray, dimerization region of Sis1.
Figure 2.
Functional tests of wild-type Sis1 and mutants and Ydj1.
The ability of yeast DnaJ and mutants in binding to Hsp70 Ssa1 (A) and in cooperating with Hsp70/Ssa1 to refold heat-denatured firefly luciferase (B) was monitored. Sis1 activity was set as a standard (100%).
Figure 3.
Urea- and thermal-induced unfolding measurements followed by CD.
The recombinant proteins (0.5 mg/mL) were submitted to thermal-unfolding followed by CD at 222 nm. (A) Urea-induced unfolding experiments were followed by CD at 222 nm, using a 1 mm pathlength cell in buffer A, at 20°C after 90–120 min of equilibration. Data are shown as fraction of unfolded protein and represent the mean of three independent experiments. The three-state unfolding transition model (Eq. 1) was used to fit the fraction of unfolding data and the fitting is shown by the line (see text for details). The unfolding experiments (in triplicate) were measured from 20°C to 90°C with a scan rate of 1.0°C/min. Data is presented as [θ] (B) and in delta of signal (C), which was fitted using a sigmoidal function (full line) yielding the TmCD.
Figure 4.
Urea disturbs the dimer structure of Sis1_Δ121–257 and SYS at lower concentrations than for Sis1 and Sis1_Δ122–174.
(A) SV-AUC experiments were done for all proteins (250–750 µg/mL) in the presence of urea at concentrations ranging from 1–5 M in buffer A. The curves of s020,w versus urea concentrations were fitted by sigmoidal functions (continuous line) leading us to obtain the CmAUC (Table 1). Data at 0 M urea were obtained from [22] and [21]. (B) SEC-MALLS experiments were performed with all proteins in concentrations from 50 µM to 75 µM and in the presence of urea at concentrations ranging from 1–5 M in buffer A. The curves of MM versus urea concentration were fitted by sigmoidal functions (lines) resulting in the CmSEC-MALLS, which represents the urea concentration of the midpoint of the MM transition (Table 1). The molecular masses for wild-type Sis1, SYS, Sis1_Δ124–174 and Sis1_Δ121–257 were approximately 78, 75, 68 and 48 kDa in the absence of denaturant, and approximately 39, 42, 35 and 24 kDa in 4 M urea, respectively. Taken together, the results suggest that Sis1 and Sis1_Δ124–174 are dimers at higher urea concentrations than SYS and Sis1_Δ121–257.
Table 1.
Summary of the thermodynamic data obtained from unfolding experiments.
Figure 5.
Thermal-induced unfolding measurements followed by DSC.
(A) Sis1; (B) Sis1_Δ124–174; (C) Sis1_Δ121–257 and (D) SYS. The figures represent the first and second scans after baseline treatment. Sis1 and Sis1_Δ124–174 showed two overlapping transitions with similar Tms of approximately 59 and 67°C (Table 1). Sis1_Δ121–257 and SYS showed two well separated unfolding transitions where the second Tm was within 64–66°C (Table 1). For all proteins, the second unfolding transition was more than 95% reversible. The first unfolding transition of Sis1, Sis1_Δ124–174 and Sis1_Δ121–257 was approximately 90% reversible when the proteins were heated to 90°C. SYS (Fig. D) presented a reversibility of approximately 60% when heated to 90°C. However, when SYS was heated to 50°C (Fig. D, inset) it was more than 95% reversible. Similar behaviors were observed with other proteins. E–F) Kirchoff plots showing the dependence of ΔHcal with Tm for Sis1 and its mutants. Urea concentrations from 0.25 M to 2.0 M were used in order to disturb both ΔHCal and Tm that were monitored by DSC. The figure shows the Kirchoff plot for the first (E) and second (F) thermal-induced unfolding transition. The curves were fitted using Eq. 3 in order to obtain the ΔCp for each unfolding transition.
Figure 6.
Fluorescence studies of Sis1 and Sis1_Δ124–174.
The lone tryptophan residue in Sis1 and Sis1_Δ124–174 (5 µM protein concentration) was used as a probe to investigate changes in the local protein structure and in hydrodynamic parameters, such as rotational diffusion, caused by the deletion of 50 amino acids in Sis1. The (A) fluorescence emission spectra, (B) tryptophan anisotropy and (C) tryptophan quenching results are shown. All experiments were performed in triplicate.
Table 2.
Summary of fluorescence experiment data. Errors are shown inside parentheses.
Figure 7.
NMR experiments for Sis1 and Sis1_Δ124–174.
(A) Superimposed TROSY NMR spectra of Sis1 (black signals) and Sis1_Δ124–174 (red signals) showing that Sis1_Δ124–174 presents less signals, all of them in the central region of the TROSY spectrum with chemical shifts around 8.2. (B) Zoom of the 2D 1H 15N chemical shifts of Sis1 showing the crosspeaks (marked in green) which are present in the wild-type Sis1 spectra but absent in the Sis1_Δ124–174 spectra. The NMR experiments suggest that the deleted region of Sis1 is intrinsically disordered.
Figure 8.
A) Model of the Sis1 binding and delivery.
A model that combines Sis1 observations showing that the disordered G/M region participates in the binding and delivery of client proteins to Hsp70. A schematic Sis1 structure is represented as a dimer showing its main domains and regions. Light blue, Sis1 CTD (PDB accession number 1C3G); red, Sis1 J-domain (PDB accession number 2O37); dark blue, Hsp70 NBD (PDB accession number 1HJO); green, DnaK SBD (PDB accession number 1DKX). The unstructured G/F and G/M regions of Sis1 are presented in black and red lines, respectively. The schematic substrate is in orange. B) Model of the Sis1 unfolding pathway. We propose a model for the Sis1 unfolding pathway which involves at least 2 events based on the results obtained in this study (see text for details).
Table 3.
Estimation of ΔCpresidue (the ratio of the ΔCp per amino acid residue of the predicted domain) values for the first and second transitions observed in DSC experiments.
Figure 9.
Interface contacts between CTDI and CTDII of Sis1, SYS and Ydj1.
The contacts between CTDI and CTDII of the crystallographic model of Sis1 (A and B) and Ydj1 (G and H) were scouted and compared to the chimerical model of the CTD of SYS (D and E) generated by molecular homology modeling (data not shown). Panels C), F) and I) present the amino acids at the interface that make hydrophobic contacts (full line) and electrostatic contacts (dashed lines). Some of the later contacts involve atoms of the peptide bond of the indicated amino acids. Amino acids not indicated are on the surface or turned inwards towards the core of the CTD subdomains.
Table 4.
Number of amino acids in the subdomain interface that are in contact.