Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 2008 Dec;17(12):2101–2110. doi: 10.1110/ps.037655.108

Proline 54 trans-cis isomerization is responsible for the kinetic partitioning at the last-step photocycle of photoactive yellow protein

Byoung-Chul Lee 1,2, Wouter D Hoff 2
PMCID: PMC2590911  PMID: 18794212

Abstract

Photoactive yellow protein (PYP), a blue-light photoreceptor for Ectothiorhodospira halophila, has provided a unique system for studying protein folding that is coupled with a photocycle. Upon receptor activation by blue light, PYP proceeds through a photocycle that includes a partially folded signaling state. The last-step photocycle is a thermal recovery reaction from the signaling state to the native state. Bi-exponential kinetics had been observed for the last-step photocycle; however, the slow phase of the bi-exponential kinetics has not been extensively studied. Here we analyzed both fast and slow phases of the last-step photocycle in PYP. From the analysis of the denaturant dependence of the fast and slow phases, we found that the last-step photocycle proceeds through parallel channels of the folding pathway. The burial of the solvent-accessible area was responsible for the transition state of the fast phase, while structural rearrangement from the compact state to the native state was responsible for the transition state of the slow phase. The photocycle of PYP was linked to the thermodynamic cycle that includes both unfolding and refolding of the fast- and slow-phase intermediates. In order to test the hypothesis of proline-limited folding for the slow phase, we constructed two proline mutants: P54A and P68A. We found that only a single phase of the last-step photocycle was observed in P54A. This suggests that there is a low energy barrier between trans to cis conformation in P54 in the light-induced state of PYP, and the resulting cis conformation of P54 generates a slow-phase kinetic trap during the photocycle-coupled folding pathway of PYP.

Keywords: photoactive yellow protein, photocycle, protein folding, slow phase, proline trans-cis isomerization, kinetic partitioning

Proteins have a funnel-shaped energy landscape for folding. The protein-folding funnel has a rugged shape with numerous intermediates (Thirumalai and Woodson 1996; Onuchic et al. 1997; Veitshans et al. 1997; Chan and Dill 1998). However, small single-domain proteins often show simple two-state unfolding/refolding transitions without folding intermediates (Jackson 1998; Fulton et al. 2005), which could hardly be reconciled with the rugged funnel view of protein folding. Among the explanations for the gap between theory and experiment in protein folding, the protein-folding kinetic partitioning has provided an ensemble nature of folding pathways with a rugged free-energy landscape (Thirumalai and Woodson 1996; Kilmov and Thirumalai 1997; Veitshans et al. 1997; Pande et al. 1998; Dinner et al. 2000). The kinetic partitioning occurs by a fast and slow folder (Thirumalai and Woodson 1996; Kilmov and Thirumalai 1997; Veitshans et al. 1997). In fast folder, there is always a direct way to reach the native state without intermediates such as the two-state folders. In slow folder, folding intermediates are involved as compact states after the initial collapse of the protein chain (Arai and Kuwajima 1996; Canet et al. 2003). Due to these properties, the rate-liming step for the fast folder involves a burial of the solvent-accessible surface area, as the slow folder is limited by the structural rearrangement from the compact state to the native state.

Protein folding often shows bi-exponential kinetics. Like the fast phase, the slow phase has been a subject of study for protein folding. The slow phase reflects the refolding from slowly interconverting unfolded states such as proline trans-cis isomerization (Creighton 1978; Kim and Baldwin 1990; Nall 1990; Schmid 1992, 2005; Ikura et al. 1997; Eyles and Gierasch 2000; Pertinhez et al. 2000; Kamen and Woody 2002a,b; Wedemeyer et al. 2002; Wu and Matthews 2002, 2003; Lowe and Itzhaki 2007; Jakob and Schmid 2008). The interconverting unfolded states confer a larger area of conformational freedom in the ensemble of the unfolded state, which diverts the pathway of protein folding into multiple folding channels (Radford et al. 1992; Wildegger and Kiefhaber 1997; Butler and Loh 2005). Because of the slow process (from tens of seconds to hours of the lifetime) and involvement of multiple prolines, the slow phase has sometimes been a hurdle for the analysis of protein folding kinetics. The slow folders are prone to encounter folding intermediates, contributing to the ruggedness of the free-energy landscape. Thus, the detailed analysis of the slow phase would provide a shape of the free-energy landscape for folding.

The kinetic partitioning predicts that the relative population of the fast and slow folder depends on various protein environments such as cosolvents, pH, temperature, etc. (Thirumalai and Woodson 1996; Kilmov and Thirumalai 1997; Veitshans et al. 1997). Investigating the effect of the protein environment on the kinetic partitioning is important for understanding how the shape of the free-energy landscape for folding is changed and why a specific folding pathway is preferred in a certain protein environment.

We chose a photoactive yellow protein (PYP) as a model system for the kinetic partitioning. PYP is a water-soluble blue-light photoreceptor in Ectothiorhodospira halophila and related eubacterial strains (Kort et al. 1996a; Meyer et al. 1987). In the absence of light, PYP displays a simple two-state folding transition (Lee et al. 2001a). However, in the presence of the blue light, apparent three-state folding transition occurs by accumulating a partially folded intermediate, pB (Lee et al. 2001a). Upon the photoactivation, the PYP proceeds through a photocycle (Meyer et al. 1987; Hoff et al. 1994b). The photocycle is coupled to protein unfolding and refolding reactions (Van Brederode et al. 1996; Lee et al. 2001a,b,c; Ohishi et al. 2001; Van der Horst et al. 2001; Hendriks et al. 2002; Imamoto et al. 2002; Sasaki et al. 2002; Zhao et al. 2006). The PYP has a p-coumaric acid chromophore (pCA) (Baca et al. 1994; Hoff et al. 1994a). The trans to cis photoisomerization of the pCA and subsequent generation of a buried negative charge inside the hydrophobic core drives the partial unfolding of PYP (Meyer et al. 1989; Kort et al. 1996b; Xie et al. 2001).

The change of pCA absorbance has been useful for probing the folding of PYP (Lee et al. 2001a). The native state of PYP (pG) has the absorbance maximum at 446 nm (Meyer et al. 1987). The absorbance maximum is shifted to 340 nm in the presence of denaturants for the unfolded state of PYP (pU) (Meyer et al. 1987; Lee et al. 2001a). Upon constant blue-light illumination, the blueshifted photocycle intermediate pB (λmax = 355 nm) is accumulated (Meyer et al. 1987; Hoff et al. 1994b). In the presence of moderate concentrations of denaturants, a completely unfolded state (pUcis-pCA, λmax = 340 nm) appears upon the light illumination (Lee et al. 2001a). After the light is turned off, the native state pG is thermally recovered from pB. The pB is an on-pathway folding intermediate from the pUcis-pCA (Lee et al. 2001a). Thus, protein unfolding/refolding reactions could be monitored by illuminating with blue light around 450 nm.

For years it has been observed that the last-step refolding photocycle showed bi-exponential kinetics (Meyer et al. 1987, 1989; Hoff et al. 1994b). Only the fast phase has been studied extensively. Here, we analyzed both the fast and the slow phases to understand the molecular origin of the slow phase. In order to perturb the shape of the folding free-energy landscape, we added cosolvents such as GdnHCl and sucrose. The proline trans-cis isomerization often contributes to the slow phase for folding because the energy barrier between the trans and cis isomer is low in the unfolded state. PYP has four prolines. Among the four prolines, P54 and P68 are well conserved in PYP genes (Kumauchi et al. 2008). We utilized these two proline mutants P54A and P68A to further investigate the effect of these prolines on the folding kinetics.

Results and Discussion

Kinetic partitioning observed during the last-step photocycle

We recorded and analyzed the last-step photocycle using absorbance spectroscopy. The last-step photocycle is a thermal reaction from photocycle intermediates to the native state pG. In order to obtain the last-step photocycle, we first illuminated with blue light constantly on PYP until the PYP reached a steady state. The light was then turned off to monitor the last-step photocycle (Fig. 1). With constant blue light illumination, the absorbance of the chromophore shifts >100 nm from the ground state pG (λmax = 446 nm) to the light-induced photocycle intermediates, pB and pUcis-pCA (λmax = 355 and 335 nm, respectively). This large spectral shift is advantageous in studying protein folding in PYP. We used kinetic traces at both 446 and 340 nm for the data analysis.

Figure 1.

Figure 1.

Open in a new tab

(A) The kinetic traces of the last-step photocycle at 445 nm. The blue light was constantly illuminated in order to accumulate the photoproducts (pB and pUcis). Then, the last-step photocycle was recorded after the light was turned off. The kinetic traces 1 and 2 were obtained in the presence of 1.6 M and 2.3 M GdnHCl, respectively. The fraction of the fast and slow phase is marked at each kinetic trace in the figure. (B) Standard deviation of percent of residuals relative to the change of the total amplitude for mono- (open circles) and bi-exponential (closed circles) fits at various GdnHCl concentrations.

The last-step photocycle showed a bi-phasic exponential kinetics in a range of GdnHCl concentrations (Figs. 1,2). The residuals for the mono-exponential fits had a wave-like shape with larger standard deviation than those for the bi-exponential fits. The bi-exponential fits had smaller and random residuals (Fig. 1B). This indicates that the last-step photocycle has a bi-phasic transition from the pB to pG state as it fits well with the bi-exponential kinetics.

Figure 2.

Figure 2.

Open in a new tab

Dependence of the last-step photocycle on GdnHCl. (A) Rate constants (sec−1) of the last-step photocycle in the fast (closed symbols) and slow phase (open symbols) at different GdnHCl concentrations in the presence of 0% (circles), 20% (triangles), and 40% sucrose (squares), respectively. (B) Rate constants for the fast phase with magnification of the region at low concentrations of GdnHCl in A. (C) Signal amplitudes of the fast (closed circles) and slow phase (open circles) at 445 nm in the absence of sucrose. The sum of the amplitudes of both fast and slow phase are shown as closed diamonds, indicating the accumulation of more photoproducts in the presence of higher concentrations of GdnHCl. (D) Fraction of amplitudes for the fast and slow phase. The symbols are the same as in A. (E) GdnHCl-dependent free energy difference between the fast and slow phase (= −RT ln(F_fast/F_slow), where the F_fast is the fraction of amplitude for the fast phase and F_slow is that for the slow phase. Circles, triangles, and squares for 0%, 20%, and 40% sucrose, respectively. (F) Thermodynamic cycle for the fast and slow phase that includes four states: pBfast, pBslow, pUcis- pCA fast, and pUcis- pCA slow. The equilibrium between pBfast and pBslow is responsible for regime 1 in the presence of lower GdnHCl concentrations; however, that between pUcis- pCA fast and pUcis-pCA slow contributes mainly to regime 2 in the presence of higher GdnHCl concentrations.

The amplitude of the last-step photocycle increased at higher concentrations of GdnHCl due to the accumulation of photoproducts (Fig. 2C), indicating that the photoproducts were stabilized relative to the native state in the presence of GdnHCl. The amplitude of the fast phase had a bell-shaped dependence on GdnHCl, but that of the slow phase had a sigmoidal dependence on GdnHCl (Fig. 2C). Interestingly, the fraction of amplitudes for the fast and slow phase changed abruptly above a certain concentration of GdnHCl (Fig. 1,2D). In Figure 1, the fraction of amplitude for the slow phase increased from 0.48 to 0.96 as the concentration of GdnHCl increased from 1.6 to 2.3 M. We further analyzed the change of the fraction of amplitude later in detail.

In order to characterize the transition state for the fast and slow phase, we analyzed the GdnHCl-dependent kinetics. As shown in Figure 2A, we found that both the fast and slow phases have a GdnHCl-dependent rollover. This rollover indicates that there is a kinetic folding intermediate for both the fast and slow phase. In PYP, the partially unfolded pB and fully unfolded pUcis-pCA are positioned in pre-equilibrium, resulting in this rollover (Lee et al. 2001a). We named the fast-phase intermediate pBfast and the slow-phase intermediate pBslow, and applied a three-state folding kinetic model to the GdnHCl-dependent reaction rates (see Equation 1 in Materials and Methods). The thermodynamic parameters are shown in Table 1. The fast phase has been analyzed previously (Lee et al. 2001a).

Table 1.

Denaturant dependence of the last-step photocycle at different sucrose concentrations

graphic file with name 2101tbl1.jpg

Open in a new tab

Protein folding reaction is often complex, and it has been difficult to discriminate a sequential from a parallel mechanism (Kiefhaber 1995; Wallace and Matthews 2002). From the GdnHCl dependences of reaction rates and their relative amplitudes of the fast and slow phases, we believe that the two intermediates (pBfast and pBslow) are positioned in parallel folding pathways. If the folding pathway is sequential with two intermediates, there should be one energy barrier above the rollover where the unfolded state pUcis-pCA is stabilized. The fast and slow phases should merge together with one phase of reaction kinetics after the rollover. However, if the folding pathway is parallel, there should be a partitioning of reaction rates in any conditions, which is the case of our PYP (Fig. 2A). As evidence for the parallel pathway shown later in this paper, we found that only a single phase of the last-step photocycle was observed in the P54A mutant, suggesting the slow phase is induced by the cis isomer of P54 (see Fig. 6).

Figure 6.

Figure 6.

Open in a new tab

Origin of the slow phase in the last-step photocycle. (A) Last-step photocycles for P54A and P68 mutants in the absence of denaturant. The kinetic traces were fitted by mono- or bi-exponential curve for P68A or P54A, respectively. The percent of residuals from both mono- (colored in black) and bi-exponential (colored in red) fits for P54A and P68A are shown, respectively, in the lower two panels. The standard deviations of the percent of residuals for P54A are 0.597 and 0.322 for mono- and bi-exponential fits, respectively. The standard deviations of the percent of residuals for P68A are 5.248 and 0.507 for mono- and bi-exponential fits, respectively. In P68A, the fast and slow phase contributes 80% and 20% of the kinetic trace, respectively. (B) Rate constants for the last-step photocycle of P54A at different concentrations of GdnHCl (closed triangles). The rate constants for the wild type presented in Figure 2A are also shown.

Interestingly, the rate constants of the slow phase had no dependence on the GdnHCl until the rollover at 2.0 M of GdnHCl, in contrast to that of the fast phase (Fig. 2A; Table 1). This indicates that the folded states of the two intermediates pBfast and pBslow are different. The kinetic m-value, which is a denaturant dependence of the logarithm of reaction rates, describes the change of the solvent-accessible area between an initial state and transition state (Myers et al. 1995). Thus, pBfast and pBslow have different exposure to the solvent-accessible area at the transition state. pBfast is partially unfolded, but pBslow is compacted in terms of the solvent-accessible area. From these data, it is concluded that the folding from pBslow to pG involves a structural rearrangement, as the folding from pBfast to pG involves a burial of the solvent-accessible surface area at the transition state. Therefore, we hypothesize that the parallel channel of folding during the photocycle of PYP results from the kinetic partitioning (Kilmov and Thirumalai 1997; Veitshans et al. 1997; Thirumalai and Woodson 1996).

The fast and slow folders were equally populated in the native condition (Fig. 2C–F). The relative amplitude of the fast phase increased in the presence of moderate concentrations of GdnHCl, which is believed to have stabilized the fast phase (Fig. 2D,E). Above certain concentrations of GdnHCl, for example, 2.2 M of GdnHCl in the absence of sucrose (Fig. 2D,E), the slow phase was preferred as a major channel of folding. This cosolvent-dependent change of the major folding channel also supports the kinetic partitioning in protein folding, which predicts that the dominant folding pathway is dependent on the solvent environment (Thirumalai and Woodson 1996; Kilmov and Thirumalai 1997). A thermodynamic cycle could be derived from these data to show the unfolding and refolding of the fast and slow phases (Fig. 2F). Regime 1 involves the partitioning of pBfast and pBslow in the presence of moderate concentrations of GdnHCl. The equilibrium of the unfolded species pUcis-pCA fast (unfolded state of pBfast) and pUcis-pCA slow (unfolded state of pBslow) contributes to regime 2 above a certain concentration of GdnHCl because the photocycle intermediate pB is unfolded in these conditions. We could obtain the thermodynamic parameters from the equilibrium of the fast and slow phases in two different regimes (Table 1). The free energy difference in pBfast and pBslow was almost zero in the native condition (Fig 2E; Table 1). However, the free energy difference between pUcis-pCA fast and pUcis-pCA slow was larger than that between pBfast and pBslow (Table 1). In addition, based on the m-values (Table 1), the difference in the solvent-accessible area between pBfast and pBslow was less than that between pUcis-pCA fast and pUcis-pCA slow.

Effect of sucrose on the parallel channel of protein folding

As described above, either the fast or slow phase was preferred, depending on the concentration of the destabilizing cosolvent GdnHCl. In order to see the effect of a cosolvent on the parallel channel of protein folding in more detail, we used a stabilizing cosolvent such as sucrose for the last-step photocycle of PYP. First, we titrated the PYP with GdnHCl in the presence of sucrose. Second, we titrated PYP with sucrose in the presence of GdnHCl.

From the data of the GdnHCl-dependent last-step photocycle in the presence of 20% or 40% sucrose, we found that the folding rate increased more for the fast phase in the presence of a higher percentage of sucrose; however, the slow phase had similar folding rates until the rollover (Fig. 2A). The thermodynamic parameters in the presence of sucrose are shown in Table 1. This data supports the idea that the transition state of the fast phase involves the burial of the solvent-accessible surface area susceptible to solvent environments, but the structural rearrangement is responsible for the transition state of the slow phase with no dependence on the cosolvents.

Unexpectedly, additional log-linear slopes for the fast phase were found below 0.4 M and 1.2 M GdnHCl in the presence of 20% and 40% sucrose, respectively (Fig. 2B). This additional log-linear slope was more pronounced, but became less steep as more sucrose was added from 20% to 40%. This suggests that there might be another intermediate involved in the last-step photocycle of PYP and is hidden in the absence of sucrose. It remains to be resolved what causes this leveling off of the GdnHCl-dependent folding rates in the presence of low concentrations of GdnHCl.

The chevron analysis is often carried out for protein folding to capture the nature of the transition state along the reaction coordinate of the solvent-accessible area (Fersht 1999; Oliveberg 2001). In general, mutations and protein environments such as temperature, pH, and cosolvents change the shape of the chevron plot. Especially, the effect of the mutation of amino acid side chains into alanine has been framed into phi-analysis, Tanford β value, etc., providing valuable insight for the transition state of protein folding (Fersht 1999). In addition, by looking at the change of the chevron plot under different protein environments, a useful insight could be obtained for the effect of the protein environment on the folding. For example, the chevron plot is shifted vertically if viscosity plays a major role (Fig. 3A). The viscosity affects the reaction kinetics without changing the energy surface of protein folding. The chevron plot is shifted horizontally when there is a change in protein stability (Fig. 3A). In most cases, both horizontal and vertical shifts occur. In order to discriminate the effect of viscosity from that of protein stability, the horizontal chevron-shift analysis, called the isostability technique, is often carried out (Plaxco and Baker 1998; Bhattacharyya and Sosnick 1999).

Figure 3.

Figure 3.

Open in a new tab

Chevron-shift analysis of the last-step photocycle. (A) Chevron shift. The horizontal shift of the chevron plot under certain environment indicates the change of protein-folding stability. The change of viscosity induces the vertical shift of the chevron plot only affecting the folding kinetics. (B) The data in Figure 2A were used for the chevron-shift analysis to see the effect of sucrose on the last-step photocycle. The x-axis in the presence of 20% and 40% sucrose was shifted by subtracting 0.6 M and 1.0 M GdnHCl, respectively.

We performed the chevron-shift analysis using the refolding arm in order to see the effect of sucrose on PYP folding. We found that denaturant-dependent refolding rates in the presence of 0%, 20%, and 40% sucrose overlapped in both the fast and slow phases when they were horizontally shifted (Fig. 3B), but no match occurred when they were vertically shifted. Based on the exact match of horizontally shifted profile of the folding rates (Fig. 3B), we conclude that sucrose stabilizes PYP during the photocycle. As shown in Table 1, both fast and slow phase intermediates, pBfast and pBslow, were stabilized relative to the unfolded state in the presence of sucrose.

The slow phase was dominant above 2.2 M GdnHCl in the absence of sucrose (Fig. 2C,D). In order to reverse the dominant folding channel from the slow to fast phase, sucrose was added in the presence of 2.3 M of GdnHCl (Fig. 4). The total amplitude of the last-step photocycle decreased as sucrose was added (Fig. 4B), indicating that the native state was stabilized relative to the photoproducts by sucrose. The amplitude of the fast phase had a bell-shaped dependence on sucrose, but that of the slow phase had a sigmoidal dependence on sucrose (Fig. 4B). As shown in Figure 4C, the fast phase was preferred at higher sucrose concentrations. We found that there was also a rollover for sucrose-dependent folding rates with a kink at 1.2 and 0.5 M for the fast and slow phase, respectively (Fig. 4A). The rate constants for the slow phase had no dependence on the sucrose above the rollover, which is in line with our conclusion that structural rearrangement is the rate-limiting step of the folding of pBslow. Sucrose increased the stability of both pBfast and pBslow intermediates relative to their unfolded states (Table 2).

Figure 4.

Figure 4.

Open in a new tab

Dependence of the last-step photocycle on sucrose in the presence of 2.3 M GdnHCl. (A) The rate constants of the last-step photocycle in the fast (closed circles) and slow phase (open circles) at different sucrose concentrations in the presence of 2.3 M GdnHCl. (B) Signal amplitudes of the fast (closed circles) and slow phase (open circles) at 445 nm at various concentrations of sucrose. The sum of the amplitudes of both fast and slow phase is shown as closed diamonds. (C) The fraction of amplitudes for the fast and slow phase. The symbols are the same as in A.

Table 2.

Sucrose dependence of last-step photocycle at 2.3 M GdnHCl

graphic file with name 2101tbl2.jpg

Open in a new tab

Origin of the slow phase in the last-step photocycle

In order to see if there is a time-dependent interconversion between the light-induced states of PYP, we varied the time-of-light exposure from 0.5 sec to 400 sec and measured the last-step photocycle in the presence of 2.0 M GdnHCl. This is analogous to the double-jump refolding assay using a stopped-flow device (Kiefhaber 1995; Wallace and Matthews 2002). If there is a slow interconversion between the states like peptidyl proline trans-cis isomerization, the amplitude of the slow phase is going to increase as the time-of-light exposure increases.

The kinetic traces of the last-step photocycle at various delays in time were analyzed by bi-exponential kinetics. The kinetic trace in Figure 5A shows an accumulation of the photoproduct during the blue-light illumination. The signal of the photo steady-state was normalized to 1. The fraction of amplitudes and folding rates for the fast and slow phases were obtained and shown in Figure 5A and B, respectively. The fraction of the slow phase increased mono-exponentially with an observed rate constant of 0.056 ± 0.015 sec−1 as the time-of-light exposure increased (Fig. 5A). Even after a 0.5-sec illumination of the blue light, the slow phase was already evolved. The kinetic partitioning to both fast and slow phases occurred instantaneously. We believe that the rate constant for the increase of the slow phase we observed here (0.056 ± 0.015 sec−1) reflects the interconversion between the unfolded states pUcis-pCA slow and pUcis-pCA fast (see Fig. 8) because the pBfast and pBslow are unfolded in the presence of 2.0 M GdnHCl.

Figure 5.

Figure 5.

Open in a new tab

Accumulation of the slow phase in the last-step photocycle. (A) Fraction of amplitudes of the fast (closed circles) and slow (open circles) phase in the presence of 2.0 M GdnHCl at various time-of-light exposures. This is analogous to the double-jump refolding assay. The kinetic trace shows the time-dependent accumulation of the photoproducts with a normalization of 1. (B) The observed rate constants for the fast and slow phase at various time-of-light exposures.

Figure 8.

Figure 8.

Open in a new tab

Schematic diagram of kinetic partitioning during the photocycle. Lifetimes and transition midpoints are indicated using Table 1 and Figure 5A.

In order to investigate the molecular origin of the slow phase, we utilized two proline mutants near the pCA chromophore of PYP, P54A, and P68A. P54 and P68 are well conserved in PYP genes (Kumauchi et al. 2008). In general, proline trans-cis isomerization is often responsible for the slow phase of protein folding and considered to be a rate-limiting step for the structural rearrangement at the transition state (Creighton 1978; Kim and Baldwin 1990; Nall 1990; Schmid 1992, 2005; Ikura et al. 1997; Eyles and Gierasch 2000; Pertinhez et al. 2000; Kamen and Woody 2002a,b; Wedemeyer et al. 2002; Wu and Matthews 2002, 2003; Lowe and Itzhaki 2007; Jakob and Schmid 2008). The rate of the slow phase that we observed here for the last-step photocycle (∼0.06 sec−1) is similar to that of refolding from the cis isomer of proline in proteins (Eyles and Gierasch 2000; Kamen and Woody 2002b; Wu and Matthews 2002; Lowe and Itzhaki 2007).

We found that P54A showed mono-exponential kinetics for the last-step photocycle, although the bi-exponential kinetics still remained in P68A (Fig. 6A). Similarly in other proline-limited folding, proline-to-alanine mutants eliminated some of the slow phases in protein refolding (Eyles and Gierasch 2000; Kamen and Woody 2002b; Wu and Matthews 2002; Lowe and Itzhaki 2007). We conclude from these mutant studies that the slow phase of the last-step photocycle in wild type originated from the cis isomer of proline 54 in the light-induced state of PYP. We measured the last-step photocycle of P54A at different concentrations of GdnHCl, and obtained various thermodynamic parameters (Fig. 6B; Table 3). Although the kinetics from the pB to pG in the absence of GdnHCl decreased fivefold in P54A, the stability of pB to pUcis-pCA and m- value increased by 4 kJ/mol and 1.2 M−1, respectively, in comparison with wild type (Table 3). This result indicates that the solvent-accessible area for pUcis-pCA in P54A is greater than in wild type. As shown in Figure 7, the P54 is located in the fourth helix (residues 53–57) from the N terminus (Borgstahl et al. 1995). It suggests that the P54A mutant destabilizes this particular helix in the light-induced state, yielding a larger difference in the solvent-accessible area between the native and light-induced unfolded state than wild type. The benefit of proline 54 in the wild type of PYP might be the faster kinetics even though it generates the slow phase of protein folding.

Table 3.

Denaturant dependence of the last-step photocycle of P54A in comparison with wild type

graphic file with name 2101tbl3.jpg

Open in a new tab

Figure 7.

Figure 7.

Open in a new tab

Cartoon of PYP structure with pCA (yellow), P54, and P68. The PDB code is 2PHY (Borgstahl et al. 1995).

PYP provides a unique system in which the thermodynamic cycle of the proline trans-cis isomerization is coupled to the photocycle. Crystal structures of PYP in native pG, pR, and pB states were solved previously using time-resolved X-ray crystallography (Borgstahl et al. 1995; Genick et al. 1997; Genick et al. 1998; Perman et al. 1998; Ren et al. 2001; Ihee et al. 2005); however, all PYP structures indicate the trans conformation for all four prolines. In our experiments, the trans and cis conformations of proline 54 were equally populated in the light-induced intermediate pB state (Fig. 2D,E). It might not be obvious to detect the cis conformation of proline 54 in time-resolved X-ray crystallography. However, it is necessary to revisit the time-resolved X-ray data, in order to investigate the long-range molecular coupling between the cis form of pCA and P54. P54 is located in a well-defined helical structure of PYP (Fig. 7; Borgstahl et al. 1995). We expect that the chromophore pCA and proline 54 are coherent in molecular motion. It is likely that the cis of pCA drives the trans to cis isomerization at P54 and subsequently, destabilizes the helix (residues 53–57).

Conclusions

The slow phase of PYP folding upon photoactivation was analyzed in detail using cosolvents GdnHCl and sucrose, and two mutants: P54A and P68A. The thermodynamic cycle for the parallel channel of both fast and slow phases was coupled to the photocycle of PYP (Fig. 8). In contrast to the fast phase, which involved the burial of surface area at the transition state, the structural rearrangement from the compact intermediate to native state, which is the cis-trans isomerization of proline 54, was responsible for the rate-limiting step of the slow phase. The cis conformation of proline 54 was evolved during the photocycle and yielded a nonproductive kinetic trap with a compact folding intermediate. The relative amplitudes of the fast and slow phases depended on the protein environment. These findings were in line with kinetic partitioning. It can be envisioned that the fast folder occurs at the smooth surface of the folding funnel, but the slow folder bumps around the rugged energy surface for the structural rearrangement. We believe that detailed analysis of the slow phase in protein folding would bridge the gap between the theory and experiment in protein folding.

Materials and Methods

Absorbance spectroscopy

Wild-type, P54A, and P68A PYP were expressed in Escherichia coli and purified as described previously (Xie et al. 1996). We used various concentrations of PYP ranging from 6 μM to 16 μM in the presence of 20 mM potassium phosphate buffer (pH 7.3). We illuminated these PYP samples with the highest available intensity of light using a Cuda I-150 light source and a broadband filter around 450 nm. In order to measure the last-step photocycle, we first illuminated the PYP with this blue light until it reached the photo steady-state, then turned off the light and recorded the last-step photocycle using a Cary 300 UV-Vis spectrophotometer at 446 nm and 340 nm. The kinetic traces for the last-step photocycle of the wild-type and P68A mutants were fitted by a bi-exponential curve and those for the P54A mutant were fitted by a mono-exponential curve.

Data analysis for the last-step photocycle

GdnHCl- or sucrose-dependent folding rates (k obs) of the last-step photocycle were averaged from both wavelengths 446 nm and 340 nm, and analyzed using Equation 1 for both the fast and slow phases (Lee et al. 2001a).

graphic file with name 2101equ1.jpg

where k pB-‡ 0 is the rate constant of the pB-to-pG transition extrapolated to the absence of the denaturant. The mpB-‡ and mpB↔pUcis describe denaturant dependences of the kinetic transition from the pB to pG and the thermodynamic equilibrium between pB and pUcis, respectively. Inline graphic is the standard state free energy difference between the pB and pUcis state in the absence of GdnHCl. [D] is the concentration of GdnHCl. Sucrose-dependent folding rates of the last-step photocycle in the presence of 2.3 M GdnHCl were also fitted by Equation 1.

The GdnHCl-dependent free energy differences between the fast and slow phases [−RT ln(F_fast/F_slow)], where F_fast and F_slow are the fraction of amplitude for the fast and slow phase of the last-step photocycle, were analyzed by Equation 2.

graphic file with name 2101equ2.jpg

where ΔG fast↔slow ([D]) is the free energy difference at each GdnHCl concentration, and Inline graphic is the standard state free energy difference between the fast and slow phase in the absence of GdnHCl. mfast↔slow describes denaturant dependence of the thermodynamic transition between the fast and slow phase. Both regimes 1 (transition between pBfast and pBslow) and 2 (transition between pUcis-pCA fast and pUcis-pCA slow) were analyzed by Equation 2.

Acknowledgments

We thank Dr. Andrew Philip for constructing the two PYP proline mutants P68A and P54A. We also thank Michael Connolly and Tammy Chu for the critical reading of this manuscript. B.-C.L. is currently supported by the Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract DE-AC02-05CH11231. W.D.H. gratefully acknowledges support from NIH Grant GM063805 and OCAST Grant HR07-135S.

Footnotes

Reprint requests to: Byoung-Chul Lee, Biological Nanostructures Facility, The Molecular Foundry, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA; e-mail: bclee@lbl.gov; fax: (510) 495-2376.

References

  1. Arai M., Kuwajima K. Rapid formation of a molten globule intermediate in refolding of α-lactalbumin. Fold. Des. 1996;1:275–287. doi: 10.1016/s1359-0278(96)00041-7. [DOI] [PubMed] [Google Scholar]
  2. Baca M., Borgstahl G.E.O., Boissinot M., Burke P.M., Williams D.R., Slater K.A., Getzoff E.D. Complete chemical structure of photoactive yellow protein: Novel thioester-linked 4-hydroxycinnamyl chromophore and photocycle chemistry. Biochemistry. 1994;33:14369–14377. doi: 10.1021/bi00252a001. [DOI] [PubMed] [Google Scholar]
  3. Bhattacharyya R.P., Sosnick T.R. Viscosity dependence of the folding kinetics of a dimeric and monomeric coiled coil. Biochemistry. 1999;38:2601–2609. doi: 10.1021/bi982209j. [DOI] [PubMed] [Google Scholar]
  4. Borgstahl G.E.O., Williams D.R., Getzoff E.D. 1.4 Å structure of photoactive yellow protein, a cytosolic photoreceptor: Unusual fold, active site, and chromophore. Biochemistry. 1995;34:6278–6287. doi: 10.1021/bi00019a004. [DOI] [PubMed] [Google Scholar]
  5. Butler J.S., Loh S. Kinetic Partitioning during folding of the p53 DNA binding domain. J. Mol. Biol. 2005;350:906–918. doi: 10.1016/j.jmb.2005.05.060. [DOI] [PubMed] [Google Scholar]
  6. Canet D., Lyon C.E., Scheek R.M., Robillard G.T., Dobson C.M., Hore P.J., van Nuland N.A. Rapid formation of non-native contacts during the folding of HPr revealed by real-time photo-CIDNP NMR and stopped-flow fluorescence experiments. J. Mol. Biol. 2003;330:397–407. doi: 10.1016/s0022-2836(03)00507-2. [DOI] [PubMed] [Google Scholar]
  7. Chan H.S., Dill K.A. Protein folding in the landscape perspective: Chevron plots and non-Arrhenius kinetics. Proteins. 1998;30:2–33. doi: 10.1002/(sici)1097-0134(19980101)30:1<2::aid-prot2>3.0.co;2-r. [DOI] [PubMed] [Google Scholar]
  8. Creighton T.E. Possible implications of many proline residues for the kinetics of protein unfolding and refolding. J. Mol. Biol. 1978;125:401–406. doi: 10.1016/0022-2836(78)90411-4. [DOI] [PubMed] [Google Scholar]
  9. Dinner A.R., Sali A., Smith L.J., Dobson C.M., Karplus M. Understanding protein folding via free-energy surfaces from theory and experiment. Trends Biochem. Sci. 2000;25:331–339. doi: 10.1016/s0968-0004(00)01610-8. [DOI] [PubMed] [Google Scholar]
  10. Eyles S.J., Gierasch L.M. Multiple roles of prolyl residues in structure and folding. J. Mol. Biol. 2000;301:737–747. doi: 10.1006/jmbi.2000.4002. [DOI] [PubMed] [Google Scholar]
  11. Fersht A. Structure and mechanism in protein science. W.H. Freeman and Co; New York: 1999. [Google Scholar]
  12. Fulton K.F., Devlin G.L., Jodun R.A., Silvestri L., Bottomley S.P., Fersht A.R., Buckle A.M. PFD: A database for the investigation of protein folding kinetics and stability. Nucleic Acids Res. 2005;33:D279–D283. doi: 10.1093/nar/gki016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Genick U.K., Borgstahl G.E., Ng K., Ren Z., Pradervand C., Burke P.M., Srajer V., Teng T.Y., Schildkamp W., McRee D.E., et al. Structure of a protein photocycle intermediate by millisecond time-resolved crystallography. Science. 1997;275:1471–1475. doi: 10.1126/science.275.5305.1471. [DOI] [PubMed] [Google Scholar]
  14. Genick U.K., Soltis S.M., Kuhn P., Canestrelli I.L., Getzoff E.D. Structure at 0.85 Å resolution of an early protein photocycle intermediate. Nature. 1998;392:206–209. doi: 10.1038/32462. [DOI] [PubMed] [Google Scholar]
  15. Hendriks J., Gensch T., Hviid L., van Der Horst M.A., Hellingwerf K.J., Van Thor J.J. Transient exposure of hydrophobic surface in the photoactive yellow protein monitored with Nile Red. Biophys. J. 2002;82:1632–1643. doi: 10.1016/S0006-3495(02)75514-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hoff W.D., Düx P., Hård K., Devreese B., Nugteren-Roodzant I.M., Crielaard W., Boelens R., Van Beeumen J., Hellingwerf K.J. Thiol ester-linked p-coumaric acid as a new photoactive prosthetic group in a protein with rhodopsin-like photochemistry. Biochemistry. 1994a;33:13959–13962. doi: 10.1021/bi00251a001. [DOI] [PubMed] [Google Scholar]
  17. Hoff W.D., Van Stokkum I.H.M., Van Ramesdonk H.J., Van Brederode M.E., Brouwer A.M., Fitch J.C., Meyer T.E., Van Grondelle R., Hellingwerf K.J. Measurement and global analysis of the absorbance changes in the photocycle of the photoactive yellow protein from Ectothiorhodospira halophila . Biophys. J. 1994b;67:1691–1705. doi: 10.1016/S0006-3495(94)80643-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ihee H., Rajagopal S., Srajer V., Pahl R., Anderson S., Schmidt M., Schotte F., Anfinrud P.A., Wulff M., Moffat K. Visualizing reaction pathways in photoactive yellow protein from nanoseconds to seconds. Proc. Natl. Acad. Sci. 2005;102:7145–7150. doi: 10.1073/pnas.0409035102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ikura T., Tsurupa G.P., Kuwajima K. Kinetic folding and cis/trans prolyl isomerization of staphylococcal nuclease. A study by stopped-flow absorption, stopped-flow circular dichroism, and molecular dynamics simulations. Biochemistry. 1997;36:6529–6538. doi: 10.1021/bi963174v. [DOI] [PubMed] [Google Scholar]
  20. Imamoto Y., Kamikubo H., Harigai M., Shimizu N., Kataoka M. Light-induced global conformational change of photoactive yellow protein in solution. Biochemistry. 2002;41:13595–13601. doi: 10.1021/bi0264768. [DOI] [PubMed] [Google Scholar]
  21. Jackson S.E. How do small single-domain proteins fold? Fold. Des. 1998;3:R81–R91. doi: 10.1016/S1359-0278(98)00033-9. [DOI] [PubMed] [Google Scholar]
  22. Jakob R.P., Schmid F.X. Energetic coupling between native-state prolyl isomerization and conformational protein folding. J. Mol. Biol. 2008;377:1560–1575. doi: 10.1016/j.jmb.2008.02.010. [DOI] [PubMed] [Google Scholar]
  23. Kamen D.E., Woody R.W. Folding kinetics of the protein pectate lyase c reveal fast-forming intermediates and slow proline isomerization. Biochemistry. 2002a;41:4713–4723. doi: 10.1021/bi0115129. [DOI] [PubMed] [Google Scholar]
  24. Kamen D.E., Woody R.W. Identification of proline residues responsible for the slow folding kinetics in pectate lyase C by mutagenesis. Biochemistry. 2002b;41:4724–4732. doi: 10.1021/bi0115131. [DOI] [PubMed] [Google Scholar]
  25. Kiefhaber T. Kinetic traps in lysozyme folding. Proc. Natl. Acad. Sci. 1995;92:9029–9033. doi: 10.1073/pnas.92.20.9029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kilmov D.K., Thirumalai D. Viscosity dependence of the folding rates of proteins. Phys. Rev. Lett. 1997;79:317–320. [Google Scholar]
  27. Kim P.S., Baldwin R.L. Intermediates in the folding reactions of small proteins. Annu. Rev. Biochem. 1990;59:631–660. doi: 10.1146/annurev.bi.59.070190.003215. [DOI] [PubMed] [Google Scholar]
  28. Kort R., Hoff W.D., Van Ist M., Kroon A.R., Hoffer S., Vlieg K., Crielaard W., Hellingwerf K.J. The Xanthopsins: A new family of eubacterial blue-light photoreceptors. EMBO J. 1996a;15:3209–3218. [PMC free article] [PubMed] [Google Scholar]
  29. Kort R., Vonk H., Xu X., Hoff W.D., Crielaard W., Hellingwerf K.J. Evidence for the trans-cis isomerization of the p-coumaric acid chromophore as the photochemical basis of the photocycle of photoactive yellow protein. FEBS Lett. 1996b;382:73–78. doi: 10.1016/0014-5793(96)00149-4. [DOI] [PubMed] [Google Scholar]
  30. Kumauchi M., Hara M.T., Stalcup P., Hoff W.D. Identification of six new photoactive yellow proteins–diversity and structure–function relationships in a bacterial blue light photoreceptor. Photochem. Photobiol. 2008;84:956–969. doi: 10.1111/j.1751-1097.2008.00335.x. [DOI] [PubMed] [Google Scholar]
  31. Lee B.-C., Pandit A., Croonquist P.A., Hoff W.D. Folding and signaling share the same pathway in a photoreceptor. Proc. Natl. Acad. Sci. 2001a;98:9062–9067. doi: 10.1073/pnas.111153598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lee B.-C., Croonquist P.A., Sosnick T.R., Hoff W.D. PAS domain receptor photoactive yellow protein is converted to a molten globule state upon activation. J. Biol. Chem. 2001b;276:20821–20823. doi: 10.1074/jbc.C100106200. [DOI] [PubMed] [Google Scholar]
  33. Lee B.-C., Croonquist P.A., Hoff W.D. Mimic of photocycle by a protein folding reaction in photoactive yellow protein. J. Biol. Chem. 2001c;276:44481–44487. doi: 10.1074/jbc.M104362200. [DOI] [PubMed] [Google Scholar]
  34. Lowe A.R., Itzhaki L.S. Biophysical characterization of the small ankrin repeat protein myotrophhin. J. Mol. Biol. 2007;365:1245–1255. doi: 10.1016/j.jmb.2006.10.060. [DOI] [PubMed] [Google Scholar]
  35. Meyer T.E., Yakali E., Cusanovich M.A., Tollin G. Properties of a water soluble, yellow protein isolated from a halophilic phototrophic bacterium that has photochemical activity analogous to sensory rhodopsin. Biochemistry. 1987;26:418–423. doi: 10.1021/bi00376a012. [DOI] [PubMed] [Google Scholar]
  36. Meyer T.E., Tollin G., Hazzard J.H., Cusanovich M.A. Photoactive yellow protein from the purple phototrophic bacterium, Ectothiorhodospira halophila. Quantum yield of photobleaching and effects of temperature, alcohols, glycerol, and sucrose on kinetics of photobleaching and recovery. Biophys. J. 1989;56:559–564. doi: 10.1016/S0006-3495(89)82703-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Myers J.K., Pace C.N., Scholtz J.M. Denaturant m-values and heat capacity changes: Relation to changes in accessible surface areas of protein unfolding. Protein Sci. 1995;4:2138–2148. doi: 10.1002/pro.5560041020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Nall B.T. Proline isomerization and folding of yeast cytochrome c . In: Gierasch L.M., King J., editors. Protein folding, deciphering the second half of the genetic code. American Association for the Advancement of Science; Washington, DC: 1990. pp. 198–207. [Google Scholar]
  39. Ohishi S., Shimizu N., Mihara K., Imamoto Y., Kataoka M. Light induces destabilization of photoactive yellow protein. Biochemistry. 2001;40:2854–2859. doi: 10.1021/bi001846i. [DOI] [PubMed] [Google Scholar]
  40. Oliveberg M. Characterization of the transition states for protein folding: Towards a new level of mechanistic detail in protein engineering analysis. Curr. Opin. Struct. Biol. 2001;11:94–100. doi: 10.1016/s0959-440x(00)00171-8. [DOI] [PubMed] [Google Scholar]
  41. Onuchic J.N., Luthey-Schulten Z., Wolynes P.G. Theory of protein folding: The energy landscape perspective. Annu. Rev. Phys. Chem. 1997;48:545–600. doi: 10.1146/annurev.physchem.48.1.545. [DOI] [PubMed] [Google Scholar]
  42. Pande V.S., Grosberg A.Yu., Tanaka T., Rokhsar D.S. Pathways for protein folding: Is a new view needed? Curr. Opin. Struct. Biol. 1998;8:68–79. doi: 10.1016/s0959-440x(98)80012-2. [DOI] [PubMed] [Google Scholar]
  43. Perman B., Srajer V., Ren Z., Teng T., Pradervand C., Ursby T., Bourgeois D., Schotte F., Wulff M., Kort R., et al. Energy transduction on the nanosecond timescale: Early structural events in a xanthopsin photocycle. Science. 1998;279:1946–1950. doi: 10.1126/science.279.5358.1946. [DOI] [PubMed] [Google Scholar]
  44. Pertinhez T.A., Hamada D., Smith L.J., Chiti F., Taddei N., Stefni M., Dobson C.M. Initial denaturing conditions influence the slow folding phase of acylphosphatase associated with proline isomerization. Protein Sci. 2000;9:1466–1473. doi: 10.1110/ps.9.8.1466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Plaxco K.W., Baker D. Limited internal friction in the rate-limiting step of a two-state protein folding reaction. Proc. Natl. Acad. Sci. 1998;95:13591–13596. doi: 10.1073/pnas.95.23.13591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Radford S.E., Dobson C.M., Evans P.A. The folding pathway of hen lysozyme involves partially structured intermediates and multiple pathways. Nature. 1992;358:302–307. doi: 10.1038/358302a0. [DOI] [PubMed] [Google Scholar]
  47. Ren Z., Perman B., Srajer V., Teng T.Y., Pradervand C., Bourgeois D., Schotte F., Ursby T., Kort R., Wulff M., et al. A molecular movie at 1.8 Å resolution displays the photocycle of photoactive yellow protein, a eubacterial blue-light receptor, from nanoseconds to seconds. Biochemistry. 2001;40:13788–13801. doi: 10.1021/bi0107142. [DOI] [PubMed] [Google Scholar]
  48. Sasaki J., Kumauchi M., Hamada N., Oka T., Tokunaga F. Light-induced unfolding of photoactive yellow protein mutant M100L. Biochemistry. 2002;41:1915–1922. doi: 10.1021/bi011721t. [DOI] [PubMed] [Google Scholar]
  49. Schmid F.X. Protein isomerization as a rate-limiting step. In: Creighton T.E., editor. Protein folding. W.H. Freeman; New York: 1992. pp. 197–241. [Google Scholar]
  50. Schmid F.X. Prolyl isomerization in protein folding. In: Buchner J.K.T., editor. Protein folding handbook. Wiley-VCH; Weinheim, Germany: 2005. pp. 916–945. [Google Scholar]
  51. Thirumalai D., Woodson S.A. Kinetics of folding of proteins and RNA. Acc. Chem. Res. 1996;29:433–439. [Google Scholar]
  52. Van Brederode M.E., Hoff W.D., Van Stokkum I.H., Groot M.L., Hellingwerf K.J. Protein folding thermodynamics applied to the photocycle of the photoactive yellow protein. Biophys. J. 1996;71:365–380. doi: 10.1016/S0006-3495(96)79234-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Van der Horst M.A., Van Stokkum I.H., Crielaard W., Hellingwerf K.J. The role of the N-terminal domain of photoactive yellow protein in the transient partial unfolding during signalling state formation. FEBS Lett. 2001;497:26–30. doi: 10.1016/s0014-5793(01)02427-9. [DOI] [PubMed] [Google Scholar]
  54. Veitshans T., Kilmov D.K., Thirumalai D. Protein folding kinetics: Timescales, pathways and energy landscapes in terms of sequence-dependent properties. Fold. Des. 1997;2:1–22. doi: 10.1016/S1359-0278(97)00002-3. [DOI] [PubMed] [Google Scholar]
  55. Wallace L.A., Matthews C.R. Sequential vs. parallel protein-folding mechanisms: Experimental tests for complex folding reactions. Biophys. Chem. 2002;101-102:113–131. doi: 10.1016/s0301-4622(02)00155-2. [DOI] [PubMed] [Google Scholar]
  56. Wedemeyer W.J., Welker E., Scheraga H.A. Proline cis-trans isomerization and protein folding. Biochemistry. 2002;41:14637–14644. doi: 10.1021/bi020574b. [DOI] [PubMed] [Google Scholar]
  57. Wildegger G., Kiefhaber T. Three-state model for lysozyme folding: Triangular folding mechanism with an energetically trapped intermediate. J. Mol. Biol. 1997;270:294–304. doi: 10.1006/jmbi.1997.1030. [DOI] [PubMed] [Google Scholar]
  58. Wu Y., Matthews C.R. Parallel channels and rate-limiting steps in complex protein folding reactions: Prolyl isomerization and the α subunit of Trp synthase, a Tim barrel protein. J. Mol. Biol. 2002;323:309–325. doi: 10.1016/s0022-2836(02)00922-1. [DOI] [PubMed] [Google Scholar]
  59. Wu Y., Matthews C.R. Proline replacements and the simplification of the complex, parallel channel folding mechanism for the α subunit of Trp synthase, a Tim barrel protein. J. Mol. Biol. 2003;330:1131–1144. doi: 10.1016/s0022-2836(03)00723-x. [DOI] [PubMed] [Google Scholar]
  60. Xie A., Hoff W.D., Kroon A.R., Hellingwerf K.J. Glu46 donates a proton to the 4-hydroxycinnamate anion chromophore during the photocycle of photoactive yellow protein. Biochemistry. 1996;35:14671–14678. doi: 10.1021/bi9623035. [DOI] [PubMed] [Google Scholar]
  61. Xie A., Kelemen L., Hendriks J., White B.J., Hellingwerf K.J., Hoff W.D. Formation of a new buried charge drives a large-amplitude protein quake in photoreceptor activation. Biochemistry. 2001;40:1510–1517. doi: 10.1021/bi002449a. [DOI] [PubMed] [Google Scholar]
  62. Zhao J.M., Lee H., Nome R.A., Majid S., Scherer N.F., Hoff W.D. Single-molecule detection of structural changes during PAS domain activation. Proc. Natl. Acad. Sci. 2006;103:11561–11566. doi: 10.1073/pnas.0601567103. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES