Letter pubs.acs.org/JPCL
Crowding-Induced Quenching of Intrinsic Tryptophans of Serum Albumins: A Residue-Level Investigation of Different Conformations Priyanka Singh and Pramit K. Chowdhury* Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India S Supporting Information *
ABSTRACT: Macromolecular crowding has been known to influence the global conformational landscape of proteins in various ways. However, only a few studies have focused their attention on the local perturbations that can occur in the presence of crowding agents. Here we have used the sensitivity of the tryptophan (Trp) fluorescence of two homologous serum albumins (BSA and HSA) to its (Trp) surroundings to monitor the local changes in the immediate proximity of the intrinsic fluorophore. Using the tool of fluorescence quenching we have shown that the commonly used synthetic macromolecular crowders (e.g., Dextran 6, Dextran 40, Ficoll 70, and PEG 8000) can bring about dramatic conformational modulations in the two proteins. Moreover, the nature of perturbation was observed to be largely dependent on the specific crowding agent used, with Dextran 6 showing the maximum effect while PEG 8000 showed the least. Additionally the extent of local structure modulation was found to be the largest either in the native state of the proteins or under near-native conditions, signifying the important role that the surrounding amino acids play in determining the fluorescence of the Trp residues. Also, surprisingly, although BSA and HSA show ∼76% sequence homology and have almost identical structural disposition in the native state, their individual responses to the crowder-induced perturbation were found to be quite different. SECTION: Biophysical Chemistry and Biomolecules fluorescence of the tryptophan (Trp) residues of two serum albumin proteins, BSA (bovine serum albumin) and HSA (human serum albumin), to provide residue-level interrogation of the protein conformations in the presence of different crowding agents. Our results hint at significant modulation in local structure as addressed by the Trp residues, with almost all of the crowding agents bringing about quenching of the Trp fluorescence. This observed quenching occurs via a static mechanism that we hypothesize to be resulting from groundstate complex formation between Trp and the neighboring amino acid residues in response to the enhanced excluded volume effect as the crowder concentration is increased. Moreover, the different quenching profiles obtained point directly toward substantial differences in the excluded volume the crowders exert on the two proteins. The crowding agents used for this study, viz. Ficoll 70, Dextran 40, Dextran 6, and PEG 8000, are synthetic in nature and are composed of different shapes and sizes. For example, Ficoll has been shown to behave like a semirigid sphere having radius of ∼55 Å, while Dextran is cylindrical in nature.12 PEG, on the other hand, is a polyether, is highly water-soluble and has been proposed to have a mesh-like structure above the semidilute regime.14−16 BSA and HSA were chosen as model
W
ith the gradual realization that macromolecular crowding can play an important role in many cellular processes, extensive efforts are already underway to provide much needed insights into the crowding phenomenon. Both experimental and simulation studies have shown that crowding agents primarily exert their influence via the “excluded volume effect” wherein the mutual impenetrability of these crowders results in a reduced conformational space for biological macromolecules.1−4 A considerable amount of research has gone into investigating changes in the structure and dynamics of different proteins in the presence of varying concentrations of synthetic (Ficoll, Dextran, PEG, PVP) and protein-based crowding agents, both from the kinetic and thermodynamic points of view.5,6 Results obtained thus far have revealed that crowding agents not only shift the equilibrium of proteins toward the more compact native state but can also bring about functionally important changes in the structures of native enzymes, thereby enhancing their activity.7 Circular dichroism studies have shown significant gain in secondary structure for proteins in the presence of macromolecular crowders.8−10 Similar compaction effects have also been observed using FRET studies.11−13 Indeed global conformational changes in crowded media are well-documented. However conformational changes occurring locally in proteins under the influence of the crowding agents have rarely been explored and often remain masked by the larger amplitude global changes. In this study, we have used the intrinsic © 2013 American Chemical Society
Received: June 8, 2013 Accepted: July 22, 2013 Published: July 22, 2013 2610
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proteins because not only are these well-studied with regards to their folding and unfolding characteristics but also they are commercially available in very pure form. Moreover, both of these transport proteins have significant biopharmaceutical relevance as they have been shown to bind to a wide variety of drug molecules.17,18 Additionally, BSA has two tryptophan residues, while HSA has only one, the latter remaining buried in subdomain IIA.18,21 Initially we monitored the Trp fluorescence of both the serum albumins in their native state as a function of the concentration of the crowding agents. As observed, increase in the concentration of Dextran 6 leads to severe quenching of the Trp fluorescence for both BSA and HSA, with the emission intensity being the least at 200 g/L, the highest concentration of crowder used for this study (Figure 1).
Figure 2. Stern−Volmer plots for (A) BSA and (B) HSA in the presence of different crowding agents. The solid lines are the fits to the respective quenching data according to eqs 1 and 2 given in the text. λem. = 341 nm. The concentration of crowders on a moles per liter scale has been shown for the different crowding agents.
F0 ′ [w] = 1 + KSV F
Figure 1. Fluorescence spectra of (A) BSA and (B) HSA in the presence of varying concentrations of Dextran 6; the spectra were acquired at 25 g/L intervals of Dextran 6.
where KSV ′ = KSV/Mc, with Mc being the average molecular weight of the crowder (6 kDa for Dextran 6, 40 kDa for Dextran 40, 70 kDa for Ficoll 70, and 8 kDa for PEG 8000), and w is the crowder concentration in grams per liter. K′SV can thus be considered to be the molar-mass-normalized form of the actual Stern−Volmer constant. The reason for reporting a modified Stern−Volmer constant will become clear if one takes a closer look at the Tables (Tables 1 and 2). For both BSA and HSA in the native state (i.e., in the absence of any chemical denaturant), Dextran 6 has a lower KSV value than Ficoll 70 and Dextran 40 for a given concentration of crowding agents, while Ficoll 70 shows the largest KSV value. However, when one considers KSV ′ , Dextran 6 has the highest value among the three. These values can be rationalized based on the fact that Ficoll 70 having the highest average molecular weight (among the aforementioned crowding agents), for a given crowder concentration (in g/L), will be having the least number of molecules. Dextran 6, being the smallest of the three crowding agents, will be having the highest number of molecules, thereby giving rise to more quenching (per g/L), resulting in a higher K′SV value. On the basis of this rationale, PEG 8000 should have had a KSV value close to that of Dextran 6 because their average molecular weights are close to each other. However, PEG for both the proteins shows a very low value of KSV and K′SV, implying that it is quite different from Dextran 6 in exerting its excluded volume effect toward the proteins. Macromolecular crowding has been shown to bring about appreciable compaction of the denatured conformations of proteins.13,20 Hence here we have also probed the quenching efficiency of the crowding agents in the unfolded conformations of the serum albumins as a function of the urea concentration, the latter being varied from 1 to 7 M (as shown in Figures 3 and 4 and Supplementary Figure 1 in the Supporting Information). Irrespective of the denaturant concentration, Dextran 6 was always observed to bring about the highest
Irrespective of the origin and mechanism of quenching, we have based our analyses on the widely used Stern−Volmer relationships as follows: F0 = 1 + KSV[Q ] F
(3)
(1)
F0 = (1 + KSV[Q ]) exp(V [Q ]) (2) F where F0 and F are, respectively, the fluorescence intensities in the absence and presence of quenchers (here the macromolecular crowders) and KSV is the quenching constant. Linear fits were modeled according to eq 1, while quenching plots showing positive (upward) curvature were analyzed based on eq 2; the latter is a modified form of the Stern−Volmer equation and is known as the sphere-of-action model, with V being the volume of the sphere surrounding the fluorophore wherein for quenching to occur no diffusion needs to take place.19 Figure 2 provides a comparison of the Stern−Volmer plots for different crowding agents and their influence on BSA and HSA. For a given concentration of crowding agent, Dextran 6 shows the maximum quenching effect, while PEG 8000 is the lowest for the proteins in the native state. Moreover, for BSA, the trends obtained in the presence of Dextran 40 and PEG 8000 show well-defined curvatures. However, for HSA, all of the crowders used give rise to linear quenching plots. Given in Table 1 are the parameters obtained from fits to the Stern− Volmer eqs 1 and 2. As can be seen, we have reported two types of quenching constants, KSV and KSV́ ; KSV carries its usual meaning as the Stern−Volmer constant and has the units of M−1 according to eq 1. Traditionally crowder concentrations are expressed in grams per liter (g/L), and hence to reflect this dependence, eq 1 was modified as follows: 2611
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1.4 1.2 0.3 52.7 370.5 0.4 2.1 1.0 0.9
18.7 20.3 20.1
20.9
16.5 21.3 20.4
23.2
degree of quenching for both BSA and HSA. To have a better perception of the observed quenching of the intrinsic Trp fluorescence of the two proteins belonging to the serum albumin family, we have also compared the Stern−Volmer quenching plots for the same crowding agent but at different urea concentrations (Supplementary Figures 2−5 in the Supporting Information). Linear quenching profiles were observed for both of the proteins in the presence of Dextran 6 for 0 M and 4−6 M urea solutions (Supplementary Figure 2 in the Supporting Information). In the intermediate urea concentration range of 1−3 M, the plots exhibit distinct curvatures, showing that the proteins exhibit a different conformational response in these denaturant concentrations. Moreover, for BSA, the maximum quenching occurs at the urea concentration of 2 M, followed closely by 1, 0, and 4 M, the latter three being quite similar. For traditional quenchers like acrylamide or iodide, it is known that, in general, with increase in denaturant concentration and hence an increase in exposure of the intrinsic fluorophores, the extent of accessibility increases, thereby giving rise to enhanced quenching. However in the present study, no such trend is observed. Additionally, quenching is observed to be the highest for the lower urea concentration(s), wherein the change in structure is very small, that is, where the protein is still native-like, with the Trp residues remaining largely unchanged in their degree of exposure to the surrounding solvent. To probe the contribution of dynamic quenching in the observed Stern−Volmer plots, we also performed time-resolved fluorescence studies. For a given denaturant concentration, the fluorescence lifetime decays obtained at varying crowder concentrations were almost superimposable (Supplementary Figure 6 in the Supporting Information), thereby implying that the influence of dynamic quenching for both the proteins used in this study is negligible. This observation can be explained based on the fact that the macromolecular crowding agents are quite massive in size and hence their penetration into the protein interior for accessing the intrinsic Trp residues is hindered. Having established that static quenching mechanism is the dominant one, it can thus be envisioned that the quenching of the intrinsic Trp residues of the serum albumins is due to weak ground-state complex formation with the amino acid residues in the immediate proximity. This therefore explains why the observed quenching is at its maximum in the native- or near-native-like (low denaturant concentration) conformations, wherein the proteins are compact in size and the surrounding amino acids remain in close proximity to the intrinsic fluorophore. Subsequently, upon increase in concentration of Dextran 6, the proteins experience increasing excluded volume effect, thereby leading to an even closer approach of the neighboring amino acids and hence enhanced quenching of Trp. Consistent with these observations, for the rest of the crowding agents as well (except the effect of PEG 8000 on HSA), the extent of the quenching induced is maximum in the absence of urea or at low concentrations of the denaturant. On the basis of the aforesaid argument, the quenching is therefore expected to decrease as the protein unfolds in response to increase in the concentration of the chemical denaturant urea. Indeed, according to our expectations, in general, it is observed that at higher urea concentrations the Trp emission is less quenched because the surrounding amino acids have moved further apart (as the protein swells on denaturation), leading to reduced complex formation.
r is the radius calculated on the basis of volume of a sphere (V). bLehrer’s plot (L.P.), wherever indicated, has been tabulated separately in Supplementary Table 1 in the Supporting Information.
318.3 2.0 1.2 1.4
142.8 84.6 98.1 L.P. L.P. 97.2 81.5 26.8 40.5 168.2 2.3
93.1 L.P.b L.P. L.P. 15.7 83.1 40.9 37.3 7.5 4.8 4.7 3.5 8.1 4.4 5.9 3.7 44.7 28.7 28.3 21.0 48.5 26.6 35.4 21.6 0 1 2 3 4 5 6 7
Letter
a
50.1
r V
108.7 0.3 2.5 L.P. L.P. L.P. L.P. L.P. L.P.
′ × 10−3 KSV V KSV
′ × 10 KSV
V
r
a
KSV
′ × 10 KSV
−3 −3
V
r
KSV
′ × 10 KSV
−3
Ficoll 70 Dextran 40 Dextran 6
conc. of urea (M)
Table 1. KSV (M
−1
), V (M
−1
), and r (Å) values for BSA for Different Crowders in the Presence and Absence of Denaturant
r
KSV
PEG 8000
35.0
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−1
), V (M
−1
), and r (Å) values for HSA for Different Crowders in the Presence and Absence of Denaturant
Dextran 6 conc. of urea (M)
KSV
′ × 10−3 KSV
0 1 2 3 4 5 6 7
55.8 53.4 18.0 27.0 48.9 43.3 24.9 28.9
9.3 8.9 3.0 4.5 8.1 7.2 4.1 4.8
a
Letter
Dextran 40
V
25.9 71.2
14.4
r
21.7 30.4
17.9
KSV
′ × 10−3 KSV
127.7 82.3 21.7 9.0 4.2 52.8 21.8 35.8
3.1 2.1 0.54 0.22 0.10 1.3 0.54
PEG 8000a
Ficoll 70
V
r
86.5 253.0 341.1 460.3
32.5 46.4 51.3 56.7
KSV
′ × 10−3 KSV
192.9 133.2 105.6 76.9 25.2 12.2 9.2 7.8
2.7 1.8 1.4 1.0 0.36 0.17 0.13 0.11
V
570.3 573.7 598.1 622.4
r
KSV
′ × 10−3 KSV
8.1
1.0
V
r
60.9 61.0 61.9 62.7
For PEG 8000, from 1 M urea, HSA shows an enhancement in Trp fluorescence; hence no quenching parameter could be obtained.
Figure 4. Stern−Volmer plots for HSA in the presence of different crowding agents at different concentrations of urea. The solid lines (except those for the plots showing negative curvature, wherein these lines should be considered as just visual aids) are the fits to the respective quenching data according to eqs 1 and 2 given in the text. λem. = 341 nm. The plots showing enhancement in tryptophan fluorescence in the case of PEG 8000 have been fitted using a negative slope. Error bars have also been shown in the Figure. (The error is in the range of 3 to 4%.)
Figure 3. Stern−Volmer plots for BSA in the presence of different crowding agents at different concentrations of urea. The solid lines (except those for the plots showing negative curvature wherein these lines should be considered as just visual aids) are the fits to the respective quenching data according to eqs 1 and 2 given in the text. λem. = 341 nm. Error bars have also been shown in the Figure. (The error is in the range of 3 to 4%.)
To provide a better comparison of the quenching profiles, we have also plotted the F0/F values obtained at different urea concentrations for 50, 100, 150 g/L, and 200 g/L of crowder concentrations (Supplementary Figures 7−10 in the Supporting Information). For Dextran 6, the overall variations in pattern for both the proteins are quite similar, signifying that the serum albumins are influenced in the same manner by this crowding agent. For Dextran 40 and Ficoll 70, the obtained data reveal a marked difference in the manner in which the excluded volume effect is experienced by BSA and HSA; for example, HSA in both of these crowding agents shows a steady decrease in the F0/F values as the urea concentration is increased, while for BSA, the extent of quenching goes through a maximum at the intermediate urea concentrations of 2−4 M. These data thus suggest that in the aforesaid urea concentration range, BSA (unlike HSA) undergoes a sudden conformational collapse around the Trp residue(s), giving rise to enhanced quenching. The quenching profiles obtained in the presence of varying concentrations of PEG 8000 are also quite interesting to compare (Supplementary Figures 5 and 10 in the Supporting
Information). For HSA, except for buffer, under all urea concentrations, PEG shows an enhancement in fluorescence unlike any of the other macromolecular crowders used, with the maximum enhancement occurring at 7 M urea. Moreover, only at 7 M urea and at the highest concentration of PEG used does one observe an enhancement in fluorescence in BSA. Taken together, our experimental data clearly reveal the complexity of the unfolding process involving the myriad of conformations in the presence of the different crowding agents and also bring out the differences between the two proteins having similar structure and nearly 76% sequence homology.21 Over the entire denaturant range employed here, the quenching plots show distinct curvatures, both positive and negative, for the different crowders used in this study. While plots with upward curvature have been analyzed using the sphere-of-action quenching, those with negative deviation were analyzed based on Lehrer’s plot (Supplementary Figure 11 in the Supporting Information), wherein the fractional accessi2613
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bility of fluorophores was taken into account. The plots showing downward curvature were obtained for BSA only, thus further confirming the differential accessibilities of the two tryptophan residues to the quenchers. The fractional accessibility (fa) values obtained for the different crowding agents have been provided in Supplementary Table 1 in the Supporting Information. Dextran 40 in the presence of 2 M urea has the maximum fa value of 0.55, while PEG 8000 has the lowest. The tabulated data reveal that there is not much change in the fractional accessibility of intrinsic tryptophan for Ficoll 70 and PEG 8000 as the denaturant condition is varied. However, with Dextran 40, with increase in urea concentration from 1 to 2 M there is an increase in fractional accessibility of the Trp residues, while the same decreases as one goes from 2 to 3 M, implying that the tryptophan residues are more accessible to surrounding environment at the lower urea concentration range. Moreover, the much higher fa values obtained for Dextran 40 in comparison with the other two crowding agents also suggest that in general the Trp residues will undergo more quenching in presence of Dextran 40 as compared with Ficoll and PEG, thus further validating our prior observations involving the Stern−Volmer plots. Comparison of the volume of sphere (V) obtained from fitting the quenching profiles showing upward curvature using the sphere of action model also provides some useful insights. In the presence of Dextran 6, V increases from 16.5 (at 1 M urea) to 23.2 M−1 (at 7 M urea) for BSA, while in the case of Dextran 40, V increases to 370.5 M−1 at 4 M urea (Table 1). For HSA, the quenching sphere volume also increases in the case of Dextran 40 and Ficoll 70 as the concentration of the chemical denaturant is increased (Table 2). These observations thus further confirm our previous hypothesis that the extent of ground-state complexation leading to static quenching decreases with increase in denaturant concentration because the latter also brings about a simultaneous increase in the sphere-of-action volume. The fact that the type of crowder can influence the sphere-of-action volume is evident (Tables 1 and 2) wherein V is the smallest for Dextran 6 while it is the largest for Ficoll 70; hence, as has been observed, under the given conditions Dextran 6 exhibits the maximum quenching effect. This we propose is a direct measure of the extent to which the respective crowder based on its size and shape exerts its excluded volume effect on the two serum albumin proteins. The sphere-of-action quenching, as alluded to before, is based on the premise that a group of quenchers remains very close (i.e., adjacent) to the fluorophores and hence gives rise to instantaneous quenching without any diffusion needing to take place. Because the effect of dynamic quenching has already been shown to be insignificant in this study, the presence of two types of static quenching mechanisms, the linear variation and the sphere-of-action, implies a distribution of amino acids around the Trp residue(s). In other words, the relatively low association constants (KSV) obtained from the linear portion of the plot arise from weak complex formation of Trp with some of the surrounding amino acids; that is, it represents the combined effect of the rate constants, kon and koff (KSV = kon/ koff). The latter thus further signifies the dynamic nature of the association governed by the local, low-amplitude breathing motions of the protein giving rise to complex formation in the ground state of the fluorophore. The exponential factor can be attributed to those neighboring residues that exhibit negligible relative motion with respect to the Trp moiety and hence are static in the real sense. Taken together, these data thus provide
insight into the complexity of the protein interior in the immediate vicinity of the intrinsic fluorophore, wherein a section of the protein shows conformational flexibility while another part of the protein remains relatively rigid. We have also tabulated the radius of the active volume (assuming the volume to be spherical), which reveals that in the presence of Dextran 6 the radius is the smallest (∼18 Å for HSA), while Ficoll gives rise to the largest (∼62 Å for HSA). The obtained radii are larger than the maximum possible sum of the van der Waals radii (estimated to be ∼10 Å) involving Trp and any other amino acid; this suggests that although the amino acids responsible for the sphere-of-action quenching are not in physical contact, their side chains can diffuse over an appreciable length scale to exhibit instantaneous quenching.22 This latter process is further amplified by the protein matrix in combination with the high concentration of the crowding agents which, together, hold the amino acids in close proximity (without allowing them to meander away from the active volume), thereby increasing the residence time near Trp. Global Conformational Changes. Large changes in Trp fluorescence (as previously seen) are generally accompanied by distinct changes in the secondary and tertiary structures of proteins. To confirm these observations, we investigated the modulation in the secondary structure of the predominantly helical albumins, BSA and HSA, by monitoring the change in the CD (circular dichroism) signal at 222 nm in the presence of urea and the crowding agents; the respective percentage changes have been tabulated in Table 3 for the crowder concentration of 200 g/L. Significant changes in the helical signal can be seen in the presence of the crowders, especially at higher concentrations of urea. Dextran 6 showed maximum increase in helicity (∼88%) for HSA at 7 M urea when compared with the same in the absence of the crowding agent, Table 3. Percentage Change in Ellipticity of BSA and HSA for a Crowder Concentration of 200 g/L in the Presence of Varying Concentrations of Ureaa concentration of urea (M)
Dextran 6
Dextran 40
Ficoll 70
PEG 8000
1.6 −13.9 −15.5 5.0 8.9 17.4 39.8 40.2
−8.1 18.4 −6.8 −8.1 13.9 11.0 22.4 23.0
−1.2 −16.7 −9.4 −23.0 −3.0 21.6 −5.1 −5.6
−27.9 −35.2 −5.7 −2.2 −13.5 3.4 23.9 25.5
−12.6 −3.2 3.0 −6.7 11.0 39.3 19.7 36.2
−8.1 −6.5 −20.3 −8.2 −2.3 8.6 50.3 76.8
BSA 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7
−13.5 12.4 17.5 13.6 13.2 19.0 29.2 28.7 HSA 22.3 1.4 2.8 18.4 15.0 52.3 60.0 88.2
a Percentage change = ((Ec − E0)/E0) × 100, where E0 is the ellipticity in buffer only, that is, in the absence of crowding agent, and Ec is the ellipticity at the reported crowder concentration. The ellipticity changes were monitored at 222 nm with negative values signifying a decrease in the helical signal at that wavelength.
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while for BSA the increase was only ∼29%. Similarly, PEG 8000 showed a small helicity decrease for BSA at 7 M urea, while for HSA the helicity increased by ∼76%. For HSA, while the Trp fluorescence was drastically quenched in the presence of Dextran 6, when PEG 8000 was used as the crowding agent, the Trp fluorescence was considerably enhanced (Supplementary Figure 5 in the Supporting Information) despite the fact that both the crowding agents brought about substantial increase in helicity (for HSA) at 7 M urea, as previously mentioned. These observations thus suggest that the local and global changes seen in the presence of the crowding agents might not always be correlated. As a further proof of the same, we have also attempted to correlate the changes in fluorescence (as denoted by F0/F shown in Supplementary Figures 7−10 in the Supporting Information) to the change in ellipticity (θ0/θ) (Supplementary Figures 12−15 in the Supporting Information), where θ0 is the observed ellipticity at 222 nm in the absence of any crowding agents (at a given urea concentration), while θ is that in the presence of varying concentrations of the crowding agents at the same urea concentration. If the ellipticity ratio is greater than 1, then it implies that the protein undergoes a decrease in the helical signal at 222 nm as compared with its urea-only counterpart, while the reverse (increase) occurs if the ratio is less than 1. In general, the maximum stabilization (minimum θ0/θ) is observed at 200 g/L crowder when the concentration of the denaturant is 7 M; this is expected because the proteins would be most extended in the unfolded state and hence would experience the highest excluded volume effect of the crowding agents. The extent of stabilization in the presence of the crowders is much more for HSA when compared with that of BSA at the higher urea concentrations, with Dextran 40 being the only exception wherein the trend is reversed. Moreover, at higher urea concentrations, PEG 8000 was found to be significantly destabilizing for BSA, in particular, at 50 and 100 g/L concentrations of the crowding agent. These data therefore reflect the discernible differences in the inherent plasticity of the two proteins, with HSA showing greater conformational flexibility/adaptability, as has also been suggested by a very recent study.21 Summary. Dramatic modulations in the local structure of BSA and HSA as monitored through intrinsic tryptophan fluorescence have been observed in this study in the presence of four macromolecular crowding agents. In light of the data previously reported, we have presented a summary of our most important observations so as to further enforce and elaborate on the significance of our findings as follows: (i) Dextran 6 exhibited the maximum quenching for both of the proteins under all concentrations of urea. PEG 8000 brings about the minimum quenching for BSA, while for HSA it brings about an increase in the Trp fluorescence, the latter occurring to the greatest extent at 7 M urea (Figure 4 and Supplementary Figures 1, 5, and 10 in the Supporting Information). This implies that the conformational response for BSA is in stark contrast with that of HSA. Enhancement in the single Trp fluorescence in HSA symbolizes that the increase in the concentration of the crowding agent induces a conformational change wherein the Trp moiety moves into a more hydrophobic environment and thus the fluorescence quantum yield increases. This was further supported by the Trp emission maximum shifting to the blue by ∼8 nm (from 343 nm in 7 M urea only to 335 nm in the presence of 200 g/L PEG 8000 and 7 M urea; data not shown) and the concomitant dramatic
increase in helical signal at 222 nm seen for HSA (at 7 M urea), thus revealing that in presence of PEG 8000 the domain surrounding the Trp gains a significant amount of helical structure. Under identical conditions, the Trp fluorescence of BSA shows a slight reduction in helicity. Taken together, these therefore clearly point out the remarkable differences in the manner in which the serum albumin proteins emanate their respective conformational adaptabilities toward PEG 8000. (ii) The aforesaid correlation of the extent of Trp fluorescence enhancement with that of gain in structural content cannot, however, be generalized for the other three crowding agents used in this study. As evident from Table 3, despite appreciable increase in the helical signal at 222 nm, the Trp fluorescence gets quenched by Dextran 6, Dextran 40, and Ficoll 70 under identical denaturing conditions. A similar picture also emerges from comparison of the F0/F plots (Supplementary Figures 7−10 in the Supporting Information) and the fractional change in ellipticity (θ0/θ) plots (Supplementary Figures 12−15 in the Supporting Information). Specifically, in the presence of Dextran 6 and Ficoll 70, at 7 M urea, the Trp emission maximum of HSA undergoes a ∼5 nm shift to the blue but unlike PEG 8000 is accompanied by a decrease in fluorescence. This latter observation suggests that the local structure or distribution of amino acids around the single Trp residue for HSA is quite different for these crowders despite comparable gains in the helical content (especially for Dextran 6 and PEG 8000). (iii) Maximum quenching of the intrinsic Trp fluorescence quenching is seen to occur under native-like conditions for the proteins, that is, either in the absence of urea or in the presence of low concentrations of the chemical denaturant. In general, variation of intrinsic Trp fluorescence among different proteins has been attributed to the influence of the surrounding charged amino acids and the peptide bond with the quenching occurring through excited-state electron or proton-transfer mechanisms.23,24 Such processes are invariably accompanied by a decrease in the fluorescence lifetime of the involved Trp residue(s). However, as previously mentioned, the Trp lifetime remains nearly unchanged in our study irrespective of the crowder concentration used. This suggests that the quenching primarily arises from ground-state interactions between Trp and the amino acids (possibly hydrophobic interactions with the nonpolar side chains) in the immediate proximity of the intrinsic fluorophore (Trp). As a further support of our hypothesis, the quenching is observed to decrease at higher denaturant concentrations; this we propose is as a result of volume expansion of the protein on denaturation, whereby the surrounding residues move farther apart, giving rise to weaker ground-state complex formation. Such an observation thus provides excellent insight into the similarities and differences in the local conformational modulation under different perturbing conditions. (iv) The heterogeneity of the conformational landscape in the presence of the crowding agents is further borne out by the differential quenching profiles (linear, sphere-of-action, Lehrer’s) induced by the crowders in the presence and absence of the denaturing agent urea. In general, the plots with upward curvatures are observed mostly when BSA and HSA are in their native or native-like states (low urea concentrations); referring to our previous discussion on the implications of the sphere-ofaction model, such an observation seems logical because the proximal amino acids are close enough to bring about instantaneous quenching of Trp. The only exception in this 2615
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The Journal of Physical Chemistry Letters
Letter
Conclusions. In conclusion, via the quenching of the Trp emission, we have shown how different the macromolecular crowding agents are in exerting their respective excluded volume effects in the immediate proximity of the intrinsic fluorophore. Whereas we have also monitored global changes of BSA and HSA at the secondary structure level using CD, this in combination with the significant local modulation observed (based on Trp fluorescence) provides a more detailed and complete description of the manner in which different parts of proteins respond to the congestion imposed by the crowders. The advantage of this method is that unlike FRET studies, where in most cases one has to label the protein with at least one external fluorophore (except for proteins having an intrinsic donor−acceptor pair like Trp-heme in myoglobin or cytochrome c), here the fluorescence modulation of an intrinsic fluorophore can be made use of for the much needed information on local perturbation of protein structure. Also, the macromolecular crowding agents used here are quite massive in size; the only way these are able to influence the Trp environment is not by direct collision but indirectly by inducing structural changes in the proximity of the Trp fluorophore. This approach thus provides an excellent means for correlating between changes observed at the secondary or tertiary structure level with those occurring at or near a specific site in a protein based on the location of the Trp moiety. Indeed crowding agents have been shown to affect the shapes of proteins, this being a cumulative effect of how different parts of the protein molecule respond to the volume excluded. In this regard, a thorough understanding of the manner in which the various independent parts of a multidomain protein are individually influenced in a crowded environment becomes essential. Additionally, in as much as it is difficult to rationalize all of our observations to the minutest of details, a significant finding of this study is that two proteins showing high-sequence homology still show substantial differences with respect to their conformational changes (both local and global) in response to the macromolecular crowders. This aspect along with the fact the quenching mechanisms for the same crowding agents can differ considerably (linear, sphere of action, or Lehrer’s plot) based on the solvent conditions thereby further allude to the complexity of the energy landscape of proteins.
regard is Ficoll 70 for HSA, where the sphere-of-action model had to be invoked at the higher urea concentrations (4−7 M), the latter probably arising because of its difference in shape as compared with the other crowding agents used here (see below), thus leading to quenching being dominated by those amino acids in the active volume that do not have to exhibit extensive diffusive motion. The Lehrer’s plot applied for quenching profiles showing downward curvature arises from the differential exposure of multiple tryptophans and hence was observed only for BSA. PEG 8000 induced the maximum number of such profiles, suggesting that its excluded volume interaction results in distinct partitioning of the two tryptophan residues into buried and exposed regions of the protein under almost all conditions (except the native state, i.e., 0 M urea, where an upward curvature is seen). Finally, it should be kept in mind that BSA has two tryptophans in two different domains while HSA has only one; hence the differences in the quenching profiles between the two proteins can also be partially attributed to the fact that the two domains in BSA might not be influenced uniformly by the respective crowding agent. (v) The structure and shape of the crowding agents also play an important role here besides the general conformational adaptability of the two proteins. Dextran-based crowding agents assume a rodlike shape, whereas Ficoll 70 has a spherical geometry. Hence the excluded volume so presented by these crowders to the different protein structural ensembles will be quite different.12 Additionally, the number density of the crowding agents is also an important parameter in determining the packing efficiency; for example, in this study, Dextran 6 has the lowest average molecular weight and hence will be having the largest number of molecules (for any given concentration in g/L); thus it will give rise to the maximum packing and hence the highest excluded volume, leading to enhanced quenching (Figure 2 and Supplementary Figures 2−5 in the Supporting Information). For example, at the highest crowder concentration, that is, 200 g/L, the concentration for different crowding agents is 0.033 M for Dextran 6, 0.0050 M for Dextran 40, 0.0028 M for Ficoll 70, and 0.025 M for PEG 8000. In agreement with this hypothesis, Dextran 40 follows Dextran 6 with regards to the quenching efficiency, while Ficoll 70, which has the highest average molecular weight, shows the least effect on Trp fluorescence. PEG 8000-induced conformational changes have been dramatically different from the other crowding agents, especially for HSA. Indeed it has been suggested that PEG-based crowding agents can exhibit nonspecific attractive interactions with proteins.4,14 Further proof of the same is the difference in the manner in which HSA and BSA respond to PEG 8000, which implies that the interaction of the crowding agents with the two proteins showing high sequence homology (and structural similarity) is very dissimilar. A recent report has suggested that HSA differs from BSA not only in being structurally more flexible, this being attributed to the penetration of the water deeper into the interdomain crevices, but also in exposure to the solvent (water) based on differential clustering of amino acids. We hypothesize that this observation along with the fact that PEGbased crowding agents are not particularly inert but have the propensity to show nonspecific interactions with proteins, leads to the dramatic disparity in response between the two proteins, as observed in our study, at both the local (Trp fluorescence) and the global (secondary structure) level.
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ASSOCIATED CONTENT
S Supporting Information *
Details of the materials and methods and Lehrer’s plot analysis have been included. Additional Stern−Volmer plots for the same crowder but different concentrations of urea. Tables showing fractional accessibility values for different crowders at a fixed urea concentration and fluorescence lifetimes of BSA and HSA in the presence of Dextran 40 in the absence and presence of denaturant. Lifetime plots and the F0/F and θ0/θ plots as a function of increasing concentration of urea in the presence of different crowding agents. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel: +911126591521. Notes
The authors declare no competing financial interest. 2616
dx.doi.org/10.1021/jz401179z | J. Phys. Chem. Lett. 2013, 4, 2610−2617
The Journal of Physical Chemistry Letters
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ACKNOWLEDGMENTS Priyanka Singh thanks the Indian Institute of Technology (IIT) Delhi for her institute fellowship. Pramit K. Chowdhury thanks the Department of Science and Technology (DST), New Delhi, India, for financial support under the Fast Track Scheme for Young Scientists (SR/FT/CS-007/2010) and IIT Delhi for startup funding.
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