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Correlated and Anticorrelated Domain Movement of Human Serum Albumin: A Peek into the Complexity of the Crowded Milieu Saikat Biswas and Pramit Kumar Chowdhury* Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India S Supporting Information *

ABSTRACT: Protein dynamics in cells have been shown to be markedly different from that in dilute solutions because of the highly crowded cellular interior. The volume exclusion arising from the high concentration of macromolecules present can affect both equilibrium and kinetic processes involving protein conformational changes. While global changes in structure leading to modulations in the stability of the protein have been well-documented, local changes that can have a large bearing on the functional aspects of these biomolecules are rare to come across. Using the multidomain serum protein human serum albumin and a fluorescence resonance energy transfer (FRET)-based approach, with fluorescent reporters in each of its three domains, we, in this article, have provided a detailed mapping of variations in the interdomain distances (as a function of pH) in the presence of five macromolecular crowding agents, differing based on their constituent monomers and average molecular weight(s). From the observation of correlated domain movements for dextran based crowding agents to anticorrelated motion induced by Ficoll 70, and both correlated and anticorrelated action for PEG8000 (PEG8), our results reveal the inherent complexity of a crowded milieu with the serum protein serving as an able sensor for decoding such variations. Differences in the manner in which the macromolecular crowders of similar average molecular weights influence the protein conformational ensemble also provide insights into the possible variations at the molecular level that these polymeric molecules possess. Evidence is presented in support of the fact that for the large molecular weight crowding agents and PEG8, soft interactions predominate over hard sphere potentials. Finally, the nature of domain movements encountered for the serum protein are of immense significance with respect to the function of human serum albumin (HSA) as a prolific binder and transporter of small molecules.

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INTRODUCTION Proteins are empowered by their intrinsic flexibility to carry out a wide spectrum of biological functions. Nearly all large proteins are composed of multiple domains1,2 and relative movements between domains are not only important from the functional aspects (catalysis, regulation of activity, transport of metabolites, formation of protein assemblies and cellular locomotion) but also serve as the platform for large-scale conformational transitions. Most of our knowledge on the mechanism of domain movements is derived from the X- ray crystal structures of open and closed conformations of particular proteins.3,4 The intrinsic flexibility of a protein is often mirrored by the ability of different segments of the biomolecule to move relative to each other with fairly small expenditures of energy. In catalysis domain closure often excludes water from the active site and helps position the catalytic groups around the substrate. It also traps substrates and prevents the escape of reaction intermediates.5−7 Domain closure, therefore, must be fast, and the transition between open and closed forms should not involve high energy barriers. Protein interiors however often have features that can place strong constraints on the possible conformational changes: these are closely packed with main chains and side chains in preferred conformations and with buried polar groups hydro© XXXX American Chemical Society

gen bonded. In some cases, domains appear to be entirely independent of their neighbors, that is, behave as autonomous folding units, while in others domains are stabilized by interdomain interactions.8−10 Domain shuffling has resulted in the evolution of new proteins with new functionalities. However, relatively few studies exist that deal with the thermodynamic and kinetic properties of multi domain proteins.10−17 Over the recent past it has been increasingly realized that the crowded cellular interior, having a high concentration of different types of macromolecules, plays an important role in defining the features and dynamics of a host of cellular processes.18−21 These macromolecules, popularly termed as crowding agents, through their excluded volume effect, reduce the space available for the biological macromolecule of interest.22−26 Thus, decoding the internal dynamics such as domain movement, that can be critical for proper functioning of the protein, especially in physiological environments that are perennially crowded is necessary to better understand the energy landscape of multidomain proteins. Recently, we have Received: February 18, 2016 Revised: May 10, 2016

A

DOI: 10.1021/acs.jpcb.6b01671 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B shown27 that crowding agents can have appreciable influence on the movement of domains I and II in human serum albumin (HSA), in the native state of the protein and under ureainduced chemically denaturing conditions. Using the fluorescence reporter acrylodan in domain I and p-nitro-phenyl anthranilate (NPA) in domain III as the fluorescence resonance energy transfer (FRET) acceptors, we have, in this paper, studied the nature of the movements of these domains relative to domain II as a function of pH and thereby providing a comprehensive view of the overall manner in which the serum protein responds to external perturbatons. Besides the fact that HSA is one of the archetypal drug-transporting proteins known to bind a variety of small molecules and even fatty acids for transport to different locales through the bloodstream, the serum protein has also been shown to undergo different welldefined pH-dependent structural transitions.28−38 Our FRET data reveal that crowder induced domain movements of HSA under various pH conditions is not only dependent on the concentration of the crowding agent but also on the shape and size of the crowder molecule. Domain disposition in the case of the small molecular weight crowder, Dextran 6, was seen to be arising predominantly from excluded volume effect while for PEG8, electrostatics played an important role as observed from the pH dependence of the domain shifts. For Dextran 40 and Dextran 70, wherein the movements of domains I and III (with respect to that of domain II) were correlated, that is, moved in the same direction, a major part of the distance changes took place within low to moderate concentration of the crowders where excluded volume is not expected to be dominant. Ficoll 70, though having the same average molecular weight as that of Dextran 70, not only brought about anticorrelated domain motion but also showed evidence of asymmetry in its arrangement around the protein. Stern−Volmer analyses of crowder-induced domain specific quenching of the fluorophores suggest a gradual tightening of the protein structure as the pH was increased, with quenching being the maximum at the highest pH, irrespective of the crowder used. Our observations also suggest that HSA, with its multidomain nature and varying surface potentials is well-suited to sense the diversity of the interactions at the molecular level that the macromolecular crowders exhibit.

of this solution was taken, and acrylodan (dissolved in minimum volume of CH3CN) was added such that the molar ratio of HSA to acrylodan was 1:10.39 This mixture was stirred gently and maintained at 4 °C for 12 h in the dark. The labeled protein was then eluted through a G-25 sephadex column (molecular mass exclusion limit of 5000 for proteins) to separate it from any excess acrylodan molecules (unreacted). The concentration of the labeled protein was found by using ε278 = 8700 M−1 cm−1 and ε365 = 21 000 M−1 cm−1 for acrylodan and ε277 = 36 000 M−1 cm−1 for HSA.40 The labeling efficiency was determined to be greater than 80%. B. Chemical Modification of Tyr-411. Tyrosine-411, located in domain III, has an unusually low pKa of 8.3 and it is almost 20 times more reactive than the rest of the 19 tyrosine residues in HSA.37 Preparation of anthraniloyl-Tyr-411 -HSA was accomplished at pH 8.0 by the addition of a 10 molar excess of p-nitro-phenyl anthranilate (NPA) to HSA in 0.1 M phosphate buffer. NPA was dissolved in acetonitrile at a concentration such that the final reaction mixture contains less than 1% acetonitrile. The reaction was allowed to proceed for 7 h after which the solution was dialyzed against 0.1 M phosphate buffer at pH 7.4 to remove the p-nitrophenol produced. Spectral analysis of the dialysate and of the modified protein preparation indicated the complete removal of p-nitrophenol with labeling efficiency greater than 80%. 2.3. pH Dependent Spectroscopic Studies. Absorption measurements were performed in a double-beam Shimadzu UV−Vis Spectrophotometer (UV-2450, Japan) using 1 cm path length cuvettes. Absorbance values of the protein solutions were measured in the range of 200−600 nm and molar extinction coefficient values used are as follows: ε277 = 36 500 M−1cm−1 for HSA, ε365 = 21 000 for Ac, and 20 000 M−1cm−1 for NPA. Steady state fluorescence measurements were carried out on an Edinburgh Instruments (UK) fluorescence spectrometer (Model: FLS920). The fluorescence spectra of protein samples at different pH buffer solutions, in presence and absence of crowders were measured using fluorescence quartz cuvettes. Prior to each experiment, the concentration of every sample was measured using the UV spectrophotometer. The fluorescence spectra of the protein samples were recorded at 25 °C with the temperature being maintained by a Peltier-based controller and the protein concentration was maintained at 8 μM for all the experiments. The samples were allowed an equilibration time of 12 h (at 4 °C) before acquiring their respective spectra. Samples containing unlabeled HSA were excited at 295 nm, and emission was collected from 310 to 550 nm in 1 nm increments with an integration time of 0.5 s, using a band-pass of 4 nm in both the excitation and emission arms of the instrument. To obtain information about domains I and III Acrylodan-labeled HSA and NPA-labeled HSA samples were excited at 365 nm, with emission collected from 380 to 600 nm in 1 nm increments, using an integration time of 0.5 s, and a band-pass of 2 nm in both the excitation and emission arms of the instrument. Excited-state lifetime measurements were performed using a time-correlated single photon counting (TCSPC) spectrometer (Edinburgh FLS900). For our experiments a picosecond-pulsed diode laser having its central wavelength at 375 nm (EPL 375) was used as the source for exciting the Acrylodan (Ac) residues of Ac-HSA (labeled HSA) and NPA residues of NPA-HSA. Emission decays were subsequently collected at 420 nm (for NPA) and at 497 nm (for Acrylodan) in buffer with the crowding agents through a

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MATERIALS AND METHODS 2.1. Materials. Essentially fatty-acid-free HSA, Dextran (6, 40, and 70), Ficoll 70 and PEG8 (PEG 8000) were purchased from Sigma-Aldrich Chemicals Pvt. Ltd. (USA) and were used as received without purification. Sodium phosphate dibasic anhydrous (Na2HPO4), sodium phosphate monobasic anhydrous (NaH2PO4), potassium chloride (KCl), hydrochloric acid (HCl), acetic acid, and sodium acetate were purchased from Merck Specialties Pvt, Ltd. (Mumbai) and used as received. Acrylodan was purchased from Molecular Probes Inc. (Invitrogen, USA). NPA (p-nitro-phenyl anthranilate) was obtained from Clearsynth Laboratories (France). Buffer solutions of different pH were prepared as follows: pH 9.2 and 10.5 were prepared from 10 mM Na2CO3/ NaHCO3; for pH 7.4 10 mM sodium phosphate was used; pH 5.5 and 3.6 buffer solutions were prepared from 10 mM acetic acid/sodium acetate, and pH 2 was prepared using 10 mM KCl/HCl. 2.2. Protein Labeling and Purification. A. Chemical Modification of Cys-34. A stock solution of 50 μM HSA was prepared in 50 mM phosphate buffer (pH 7.0). A 50 μL aliquot B


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The Journal of Physical Chemistry B

Volmer quenching constant, and [Q] is the concentration of the quencher. Hence, eq 3 was applied to determine Ksv by linear regression of a plot of F0 against [Q]. A linear Stern− F Volmer plot is generally indicative of a single class of fluorophores in a protein, all equally accessible to the quencher; this also means that only one mechanism (dynamic or static) of quenching occurs. However, positive deviations from the Stern−Volmer equation are frequently observed when the extent of quenching is large. In that case, the Stern−Volmer plot exhibits an upward curvature at high [Q]. These positive deviations may arise either from a combination of both static and dynamic mechanisms or the presence of a “sphere of action” type of quenching. The latter assumes the presence of a sphere of volume, V, around a fluorophore within which a quencher will cause quenching with a probability of unity. In this situation, quenching occurs due to the quencher being adjacent to the fluorophore at the moment of excitation. These closely spaced fluorophore−quencher pairs are immediately quenched, without the need to form a ground-state complex. The modified form of the Stern−Volmer equation which describes this situation is

single monochromator with a 5 nm band-pass over a total time range (TAC) of 50 ns for all samples. Emission decays were fit with appropriate instrument response functions (IRF) collected using a scattering solution. The fwhm (full width at halfmaximum) of the IRFs collected was typically in the range of ∼250 ps. The average acrylodan lifetime was calculated using the formula ⟨τ⟩ = Σiαiτi (αi = amplitude and τi = decay time of component i). Far-UV circular dichroism (CD) experiments of HSA were carried out using an AVIV circular dichroism spectrometer (Model 420SF, Lakewood, NJ, USA). The secondary structure was monitored over the wavelength range of 200−260 nm using a 0.1 cm path length cuvette, and ellipticity was determined at 222 nm. We used 2 μM of the labeled and unlabeled HSA for the measurement of secondary structures. Each spectrum recorded is an average of 10 scans. 2.4. Fö rster Resonance Energy Transfer (FRET) Analysis. Trp-acrylodan and Trp-NPA moieties form an efficient FRET pair (Supporting Information (SI) Figure S1) and hence allows one to calculate the distance between the two domains of the protein in response to any perturbation (with Trp being in domain II and acrylodan in domain I and NPA in domain III). The efficiency of the energy transfer (E) as a function of distance (r) between two probes can be expressed as follows R6 I E = 1 − DA = 6 0 6 ID R0 + r

F0 = (1 + K sv[Q ]) exp([Q ]VN /1000) F

In this equation, V is the volume of the sphere, and N is Avogadro’s constant. To probe the contribution of dynamic quenching in the observed Stern−Volmer plots, we also performed time-resolved fluorescence studies. For a given pH, the fluorescence lifetime decays obtained at varying crowder concentrations were almost superimposable (data not shown), thereby implying that the influence of dynamic quenching for all three domains of the proteins used in this study is negligible. Macromolecular crowding agents being quite massive in size cannot penetrate into the protein interior for accessing the amino acids in specific domains. Thus, static quenching being the major factor, it can thus be envisioned that the quenching of the intrinsic Trp (unlabeled HSA), Ac attached to Cys-34 (Ac-HSA), and NPA covalently attached to Tyr 411 (NPA- HSA) is due to weak ground-state complex formation with the amino acid residues in the immediate proximity.

(1)

where R0 (Förster radius) represents the distance at which energy transfer is 50% efficient, IDA is the intensity of the donor in the presence of the acceptor, and ID is the unquenched donor intensity (that is, in the absence of the acceptor). The value of R0 (in Å) can be obtained from the equation below: R 0 = 9.78 × 103[J(λ)n−4κ 2 ΦD]1/6

(2)

where J(λ) is the overlap integral, n is the refractive index of the medium, and κ2 is the orientation factor between the donor and acceptor electronic transition dipole moments. For our study we have calculated the energy transfer efficiency from area under the curve of the Trp emission from the labeled and unlabeled proteins. The value of R0 for native protein in phosphate buffer (pH 7.4) was determined to be ∼28 Å and ∼23 Å (using donor quantum yield of 0.14, a J(λ) value of 1.18 × 10−14 nm4 M−1 cm−1 for Ac HSA and 8.18 × 10−13 nm4 M−1 cm−1 for NPA HSA and with κ2 set to 2/3 and n set to 1.34) for the Trp-Ac and Trp-NPA FRET pairs, respectively. As the donor (Trp-214 of domain II) emission maximum is dependent on the pH and polarity of the medium, to make sure that the observed changes in FRET efficiencies were not due to any abrupt modulations in the Förster distance of donor−acceptor pairs, the R0 was computed for every pH and crowder concentration, subsequent to which the efficiency of transfer was calculated. 2.5. Principles of Fluorescence Quenching. Fluorescence quenching is described by the well-known Stern−Volmer equation41

F0 = 1 + K sv[Q ] F

(4)

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RESULTS The albumin molecule is composed of three homologous, predominantly helical domains. Each domain is made up of two subdomains, which share a common helical motif. Human serum albumin undergoes several transitions as a function of pH, namely, (i) the N (native) → B (basic form) transition between pH 7.0 and 9.0, (ii) the N → F (fast moving) transition between pH 5.0 and 3.5, and (iii) the F → E (expanded) transition or acid expansion below pH 3.5. The acid-induced structural changes of human serum albumin have been studied by a wide range of methods and are characterized by alteration in the secondary as well as tertiary structures. An indication of the former is a significant decrease in helical content observed by far-UV CD measurements.42 As the pH is reduced below 3.5, further unfolding occurs until about pH 2.5, when the molecule appears to be expanded to the fullest extent as allowed by the intact disulfide bonds. The N → B transition is more subtle and more gradual in onset and has been proposed to have physiological importance in terms of fatty acid and ligand binding.4 The N → B transition arises from a

(3)

where F0 and F denote the steady-state fluorescence intensities (area) in the absence and in the presence of quencher (in this case macromolecular crowder), respectively, Ksv is the Stern− C


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Figure 1. Variation of the interdomain distance: (A) domains I and II, (B) domains II and III as a function of Dextran 6 in different pH conditions as mentioned in the legend. Below is a schematic representation of the movement of different domains of HSA based on panels (A) and (B).

structural fluctuation leading to a loss of rigidity, particularly affecting the N-terminal region and thereby impacting ligand binding too.31,33 Almost all the studies until date exploring the pH-dependent transition of HSA have discussed the secondary and tertiary structural changes in the protein. However, considering the importance of the relative movements of domains to the function of this drug transporting protein, such conformational features have been far less explored. Moreover, in a typical cell, proteins function inside an extensively crowded environment. In this study we have tried to mimic the same in vitro using several synthetic polymers. To throw light on the relative domain movements in a crowded environment during the pHinduced transitions of HSA, we have employed FRET-based analysis of covalently modified HSA. Tyr-411 of domain III has been labeled with NPA (NPA-HSA), while the free cysteine in domain I, Cys-34, was covalently attached to the dye acrylodan (Ac-HSA). Both these dyes served as acceptor moieties for the donor Trp-214 located in a hydrophobic cavity of domain II, as observed from the significant overlap between the Trp emission and the absorbance of Ac and NPA, thereby making them effective FRET pairs (SI Figure S1). To examine whether the covalent attachment of these dyes to the protein had any deleterious effect on the protein structure, we also performed far-UV CD measurements of the labeled protein. As inferred from the CD analyses (data not shown), the secondary structure for both labeled and unlabeled HSA were very similar, signifying negligible perturbation of protein structure on dye

ligation. Furthermore, it should be noted, that for any given sample, there is only one covalent label on HSA, either NPA or Ac. Interdomain distance mapping was carried out at different pH values in the presence of five macromolecular crowding agents: Dextran 6, 40, and 70, Ficoll 70 and PEG8. The pH values employed in the present study were 1.9, 4.0, 5.5, 7.4, 9.15 and 10.5, thereby spanning both the acidic and alkaline ranges. It would be of immense importance to know how the distance between the FRET pairs (and hence domains when these are intact) are affected in a cell-mimicking environment at the pH extremes. In the forthcoming sections we will discuss how HSA adjusts and adapts its geometry to reach a stable conformation at different pH environments in the presence of varying concentrations of macromolecular crowders. Crowder-Induced FRET Modulation of HSA. In buffer only, that is, in the absence of the macromolecular crowders, as one moves from the acidic pH range to a region of high pH, the interdomain separation between I−II and II−III decreases as the pH is increased, with the largest change taking place in the acidic pH range (SI Figure S2). This observation is in agreement with the F → E transition seen at low pH, wherein the HSA molecule adopts an extended conformation. Moreover, the change of interdomain distance is more prominent in the case of NPA−HSA compared to Ac−HSA, hinting at a greater flexibility of domain III−domain II interface. CD studies (SI Figure S3) indicate that the helicity of the protein decreases with decreasing pH of the medium while in the basic range the helicity remains almost unaffected, confirming previous D


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The Journal of Physical Chemistry B Table 1 (A) Δr(rx − rx−50)a in Å as a Function of Acidic pH in the Presence of the Crowdersb Dextran 6

Dextran 40

pH 1.9

I−II

II−III

A B C D ΣΔr

1.0 1.2 1.0 1.3 4.5

0.9 1.4 0.8 0.7 3.7 Dextran 6

I−II

II−III

−3.9 −2.2 −0.8 −1.6 −0.3 −0.7 −0.6 −0.8 −5.7 −5.3 Dextran 40

pH 3.6

I−II

II−III

A B C D ΣΔr

1.2 1.2 0.8 1.1 4.3

1.0 1.0 0.9 1.0 3.9

pH 5.5

I−II

II−III

A B C D ΣΔr

1.0 1.5 0.6 1.2 4.4

0.3 −4.3 0.2 −0.8 1.0 −0.3 1.0 −0.6 2.5 −6.0 (B) Δr(rx − rx−50)a in Å as a

Dextran 6

Dextran 6

Dextran 70

I−II

II−III

I−II

II−III

I−II

II−III

I−II

II−III

−2.6 −1.4 −1.6 −2.0 −1.9 −1.6 −1.4 −1.7 −7.5 −6.8 Dextran 70

−2.9 −1.5 −2.2 −3.2 −9.8

1.2 1.1 0.8 1.2 4.2

−1.1 1.8 1.7 2.3 4.6

1.0 −0.8 −1.0 −1.2 |4|

II−III

I−II

II−III

I−II

II−III

−2.9 −1.4 −2.4 −3.0 −9.8

1.7 1.1 0.7 1.2 4.8

−1.2 1.9 1.5 2.3 4.5

2.3 −1.1 −0.8 −1.3 |5.5|

I−II

I−II

I−II

II−III

I−II

II−III

−1.4 2.2 1.3 1.4 3.5

4.2 −1.5 −0.5 −1.3 |7.5|

II−III

Dextran 70

I−II

II−III

I−II

II−III

I−II

II−III

1.3 1.6 0.2 0.7 3.8

0.0 −0.6 −0.8 −1.2 −2.5 Dextran 6

−4.0 −0.8 −0.3 −0.6 −5.7

−2.1 −1.6 −0.7 −0.5 −4.8 Dextran 40

−2.9 −1.7 −1.5 −1.5 −7.6

−0.6 −1.9 −1.2 −1.9 −5.7 Dextran 70

A B C D ΣΔr

1.3 1.6 0.2 0.7 3.8

−0.1 −1.2 −1.2 −0.8 −3.4 Dextran 6

pH 10.5

I−II

A B C D ΣΔr

1.3 1.6 0.2 0.7 3.8

I−II

PEG8

II−III

I−II

PEG8c

Ficoll 70

A B C D ΣΔr

II−III

Ficoll 70

−2.6 −2.9 −0.7 −3.4 1.9 −1.8 −1.7 −2.0 −1.3 1.1 −0.7 −1.5 −1.3 −2.9 0.8 −0.4 −1.5 −1.9 −2.7 1.2 −5.5 −7.6 −5.9 −10.2 5.0 Function of Neutral and Basic pH in the Presence of the Crowdersb

pH 7.4

I−II

PEG8

−2.9 −0.8 −1.6 −2.2 −1.5 −1.6 −1.6 −1.8 −7.6 −6.3 Dextran 70

II−III

I−II

Ficoll 70

−4.6 −3.1 −0.8 −1.4 −0.3 −0.4 −0.6 −0.1 −6.3 −5.0 Dextran 40

Dextran 40

pH 9.15

PEG8c

Ficoll 70

II−III

I−II

II−III

I−II

II−III

−3.3 −1.5 −2.8 −2.6 −10.2

2.3 1.1 0.8 1.2 5.4

−1.3 1.8 1.3 1.6 3.3

5.8 −1.5 −0.5 −1.3 |9.1|

II−III

I−II

II−III 6.4 −1.2 −0.8 −1.3 |9.7|

Ficoll 70 I−II

PEG8

−3.7 −2.4 −0.8 −1.3 −0.3 −0.4 −0.6 −0.5 −5.4 −4.6 Dextran 40

−2.9 −0.7 −1.7 −2.0 −1.5 −1.4 −1.4 −1.2 −7.5 −5.3 Dextran 70

−2.7 2.4 −1.7 1.1 −2.8 0.8 −2.9 1.2 −10.0 5.5 Ficoll 70

−1.1 1.5 1.5 1.8 3.7

II−III

I−II

II−III

I−II

II−III

I−II

II−III

I−II

II−III

−0.1 −1.2 −1.3 −0.8 −3.3

−3.4 −0.8 −0.3 −0.6 −5.1

−2.8 −1.0 −0.6 −0.1 −4.5

−2.9 −1.6 −2.0 −1.0 −7.5

−0.5 −2.0 −1.6 −1.1 −5.2

−2.3 −1.6 −2.7 −2.8 −9.5

2.6 1.1 0.8 1.2 5.7

−0.7 1.4 1.4 1.9 4.0

6.7 −1.2 −0.8 −1.3 |10.0|

PEG8

a In rx − rx−50, x = 50, 100, 150, and 200 g/L. bA, B, C, and D correspond to the crowder concentration ranges, namely, 0−50 g/L, 50−100 g/L, 100−150 g/L and 150−200 g/L respectively. ΔrI−II and ΔrII−III stand for the total change in distance between domains I and II, and domains II and III, respectively (-ve signifies inward domain movement). cSince PEG8 induces movements for the same pH in both directions in a concentration dependent manner, we have reported the absolute value of ΣΔr, that is, |ΣΔr|.

literature reports. In other words, increase of pH leads to a gradual but asymmetric compaction of HSA with domain III exhibiting steeper response under those conditions. Addition of Dextran 6 leads to an increase in distance between Trp-Ac FRET pair irrespective of pH of the medium. As evident from Figure 1 and Table 1 (and SI Table S1), under acidic conditions (pH 1.9, 3.6 and 5.5) the separation (r) between domains I and II increases steadily with increasing Dextran 6 concentration (∼75−100 g/L) leading to an

appreciable decrease in the efficiency of energy transfer (SI Table S1). At neutral (pH 7.4) and basic pH ranges (pH 9.15 and pH 10.5), the increase in distance of separation showed three distinct breaks, namely, 0−25 g/L, 25−100 g/L, and 100−200 g/L. The steepest increase (Δr) was observed for the middle range, while above 100 g/L, the change was gradual, thereby further reflecting on the enhanced rigidity of the protein at basic pH. On the other hand, in the acidic pH range, while the distance between domains II and III, as monitored E


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Figure 2. Variation of the interdomain distance: (A) domains I and II and (B) domains II and III as a function of Dextran 40 in different pH conditions as mentioned in the legend. Below is a schematic representation of the movement of different domains of HSA based on panels A and B.

2A). Unlike that observed in the case of Dextran 6, the distance between domains II and III also decreases (having a slightly smaller Δr), that is, the movement between domains I−II and II−III remain correlated over the entire pH range. A closer look at the various concentration blocks (Table 1) reveal that maximum change in Δr for domains I and II occurred in between 0 and 50 g/L of Dextran 40 irrespective of the pH of the medium, whereas for domains II and III the change remains appreciable until 100 g/L, beyond which the response tails off as observed for the other FRET pair signifying an obvious but subtle difference in response. Like Dextran 40, in the presence of Dextran 70, the motions of domains I and III are correlated in that both show a sizable decrease in interdomain separation with respect to domain II (Figure 3 and Table 1). For domains I and II, the average decrease in distance is ∼7.50 Å, while the same for domains II and III is in the order of ∼6.5−5.0 Å with the change becoming smaller at higher pH values (Table 1). Moreover, it is evident from Table 1 that for domains I−II, the major change in interdomain distance occurs within 0−50 g/L while for domain III, the largest change is seen to occur between 50 and 100 g/L. Ficoll 70, having the same average molecular weight as that of Dextran 70, influences the relative domain movements in a considerably different manner. From Figure 4, with increasing Ficoll 70 concentration, domains I and III move in an opposite direction with respect to domain II, i.e., domain I approaches domain II, whereas NPA shifts away from Trp-214, irrespective of the pH of medium. Among all the sugar-based crowders used in this study, Ficoll 70 brings about

using the Trp-NPA FRET pair, also increased as a function of Dextran 6 concentration, for pH of 7.4 and above, however, these domains moved closer relative to that in buffer only, contrary to what was observed for the domain I−II separation. To provide a deeper insight into the nature of separation between the FRET pairs, we have also tabulated (Table 1) the distance changes corresponding to four distinct regions of the crowder concentrations, viz., 0−50 g/L, 50−100 g/L, 100−150 g/L, and 150−200 g/L. For neutral and basic pH values, it is evident that, with increasing Dextran 6 concentration (g/L), variation of Δr (Δr = rx − rx−50, where x = 50, 100, 150, 200 are the crowder concentrations in g/L) is quite different between domains I−II and II−III (Table 1). ΔrI−II increases with increasing Dextran 6 concentration and reaches a maximum in between 50 to 100 g/L, following which it decreases and then increases slightly between 150 and 200 g/L, whereas ΔrII−III does not follow any specific trend. In the presence of the crowder Dextran 40, there is a dramatic decrease in interdomain separation even at a very low concentration for the Trp-Ac FRET pair (domains II and I). While ΣΔr (Table 1) is in the range of ∼5−6 Å for all the pH conditions, with the negative sign indicating a decrease in the interdomain distance (inward movement toward domain II) relative to that in buffer only (i.e., in absence of the crowder, other conditions like pH and temperature remaining identical), strikingly enough, almost 90% of this distance change takes place by the time the crowder concentration is increased to 75 g/L. Beyond this, the relative distance is hardly affected (Figure F


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Figure 3. Variation of the interdomain distance: (A) domains I and II, (B) domains II and III as a function of Dextran 70 in different pH conditions as mentioned in the legend. Below is a schematic representation of the movement of different domains of HSA based on panels A and B.

g/L was independent of pH (SI Table S5) during which the interdomain distance decreased by ∼3 Å. To have a glimpse into the global conformational changes in HSA in the presence of the crowders, CD experiments were performed (SI Figure S3) as a function of pH at the fixed crowder concentration of 200 g/L. Monitoring the helical signal at 222 nm, there was increase in the secondary structural content with increase in pH for all the solvent conditions, as was expected. Among the crowding agents, PEG8 and Dextran 6 brought about an appreciable increase in ellipticity as compared to that in dilute buffer, while all the high molecular weight crowders (Dextran 40, Dextran 70 and Ficoll 70) caused a noticeable decrease in the helical content, especially under basic conditions. These results thus further reinforce the enhanced excluded volume effect that the small molecular weight crowders can exert. Crowder-Induced Domain Specific Quenching. Recently it has been shown that macromolecular crowding agents can significantly quench the fluorescence of the intrinsic Trp residue of HSA, thereby providing residue-level information on protein structure perturbation.43 The macromolecular crowders being massive in size cannot directly access the Trp residues and hence the only way these can influence the fluorophore is by inducing protein structural changes in the immediate proximity of the Trp fluorophore. It has been hypothesized that crowder-induced static quenching of Trp-214 residue is a result of ground state complex formation between Trp and the neighboring amino acid residues in response to the enhanced excluded volume effect as the crowder concentration is

the maximum decrease in distance for domains I and II. On the other hand, the separation between domains II and III increases by ∼5 Å, that is, about half of the absolute movement shown by domain II (Table 1). A closer examination of distance modulation reveals that for domains I−II, the major change occurs at 0−50 g/L and 150−200 g/L Ficoll 70, a feature that was not observed for the dextran-based crowders mentioned before. PEG8 also brings about an interesting case of anticorrelated domain movement. With increasing PEG8 concentration, interdomain compaction (I and II) is observed up to 75 g/L beyond which the Trp-Ac FRET pair moved farther from one another. From 0 to 75 g/L PEG8 concentrations there was a ∼ 2 Å decrease in interdomain separation while beyond 75 g/L the Trp-Ac FRET pair distance increased by ∼3−4 Å (Figure 5 and SI Table S5). This observed movement of domain I with respect to II was quite similar over the entire pH range. However, an entirely different phenomenon was observed in the case of domains II−III. A closer look at Figure 5B confirms that this domain movement is dependent on the pH of the medium although disposition of domain III with respect to domain II follows a similar kind of pattern at all pH. The increment in interdomain separation (Δr) between domains II and III in low to moderate concentration range (0−75 g/L) of PEG8 increases with increasing pH of the medium as follows: ∼1.5 Å for pH 1.9, ∼3 Å for pH 3.6, ∼5 Å for pH 5.5, ∼6 Å for pH 7.4, and ∼7−7.5 Å for pH 9.15 and 10.5. On the other hand, the Trp-NPA FRET pair distance modulation beyond 75 G


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Figure 4. Variation of the interdomain distance: (A) domains I and II and (B) domains II and III as a function of Ficoll 70 in different pH conditions as mentioned in the legend. Below is a schematic representation of the movement of different domains of HSA based on panels A and B.

Figure 5. Variation of the interdomain distance: (A) domains I and II and (B) domains II and III as a function of PEG8 in different pH conditions as mentioned in the legend.

Table 2. Stern−Volmer Constant, K′sv (g−1L), Ksv (M−1), and Volume of Sphere, V (M−1) of Domain I of HSA (Based on Acrylodan Fluorescence) with Increasing pH in the Presence of the Crowders Dextran 6

Dextran 40

Dextran 70

Ficoll 70

PEG8

pH

K′sv *10−3

Ksv

V

K′sv *10−3

Ksv

K′sv *10−3

Ksv

K′sv *10−3

Ksv

K′sv *10−3

Ksv

V

1.9 3.6 5.5 7.4 9.2 10.5

6.4 8.0 11.4 14.8 19.8 23.6

38.2 48.1 68.4 88.8 118.7 141.4

10.8 4.2

4.7 5.8 6.4 7.9 8.7 9.4

189.2 231.6 257.2 314.4 347.2 377.2

2.8 3.6 4.3 5.0 5.9 6.3

200.9 250.6 298.9 353.5 410.9 442.4

4.0 4.6 5.5 6.1 6.6 7.1

280.7 321.3 382.9 424.2 462.0 494.9

1.49 1.8 2.9 3.9 4.4 4.8

11.9 14.4 22.9 31.8 35.4 38.8

5.84 3.2

increased.43 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. Thus, using the respective signals from the three fluorophores in the individual domains of HSA, namely, Ac in domain I, Trp-214 in domain II and NPA H


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Table 3. Stern−Volmer Constant, K′sv, Ksv (M−1), and Volume of Sphere, V (M−1) of Domain II of HSA (Based on Trp-214 Fluorescence) with Increasing pH in the Presence of the Crowders Dextran 6 pH 1.9 3.6 5.5 7.4 9.2 10.5

−3

K′sv *10 4.2 5.7 7.2 9.2 17.6 22.6

Dextran 40

Ksv

V

25.4 34.2 43.0 55.0 105.7 135.5

14.6 5.6

−3

K′sv *10

Dextran 70 −3

K′sv *10

Ksv

8.5 8.8 9.3 9.8 11.1 12.4

338.8 351.6 370.8 393.6 443.6 496.4

Ficoll 70 K′sv *10

Ksv

4.39 5.29 6.43 7.71 8.29 8.94

307.3 370.3 450.1 539.7 580.3 625.8

−3

PEG8 K′sv *10

Ksv

6.29 6.79 7.53 8.02 9.1 9.7

440.3 475.3 527.1 561.4 637.0 679.0

−3

0.51 0.92 1.3 1.85 2.17 2.97

Ksv 4.08 7.36 10.4 14.8 17.36 23.76

Table 4. Stern−Volmer Constant, K′sv, Ksv (M−1) and Volume of Sphere, V (M−1) of Domain III of HSA (Based on NPA Fluorescence) with Increasing pH in the Presence of the Crowders Dextran 6 pH 1.9 3.6 5.5 7.4 9.2 10.5

K′sv *10 5.8 7.2 9.9 17.3 21.4 27.0

−3

Dextran 40

Ksv

V

35.2 43.3 59.7 103.6 128.6 162.1

11.0 3.8 6.9 8.3 9.1 9.3

−3

K′sv *10 5.2 6.0

Dextran 70 Ksv

−3

K′sv *10

209.2 239.6 274.8 331.6 362.8 373.6

2.5 3.3 3.9 4.5 5.1 5.9

Ficoll 70 Ksv

177.1 232.4 277.9 314.3 357.0 410.2

K′sv *10

−3

4.9 5.8 6.6 7.9 8.6 9.1

PEG8

Ksv

V

K′sv *10−3

Ksv

340.9 406.0 462.7 558.6 599.9 640.5

56.7 14.0

0.43 0.83 0.93 1.2 1.7 1.9

3.4 6.6 7.4 9.8 13.3 15.8

Figure 6. Stern−Volmer plots for different domains of HSA: (A) domain I in terms of Acrylodan; (B) domain II in terms of Trp-214 and (C) domain III in terms of NPA in the presence of dextran 6 at various pH conditions as mentioned in the legend.

grams per liter (g/L), and hence, to reflect this dependence, eq 3 was modified as follows:43

in domain III, we have investigated how each of the component structural units of the serum protein is being affected by the crowding agents. It should be noted that the Trp quenching analyses were carried out with unlabeled HSA, that is, wherein neither Ac nor NPA was present, such that no complication from FRET could arise. Given in Tables 2−4 are the parameters obtained from fits to the Stern−Volmer eqs 3 and 4. As can be seen, we have reported two types of quenching constants, KSV and K′SV. KSV carries its usual meaning as the Stern−Volmer constant and has the units of M−1 according to eq 3. Traditionally crowder concentrations are expressed in

F0 = 1 + K ′sv [w] F

(5)

where the molar mass normalized Stern Volmer constant has been expressed as K ′sv =

K sv 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 8), and w is the crowder concentration in g/L. I


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Ficoll 70, where domains III and I were moving in opposite directions. However, both Trp-214 (domain II) and NPA (domain III) were almost equally quenched, suggesting that some other factors might be in operation here. In this regard the data for PEG8 also suggest that the type of influence observed depends significantly on the nature of the crowder in vogue.

Linear quenching profiles were observed for all the three domains at neutral and basic pH medium in the presence of Dextran 6 (Figure 6 and Tables 2−4). At pH 1.9 and 3.6 the plots exhibit a slight upward curvature showing that the domains exhibit a different conformational response under these conditions. With increasing pH of the medium enhancement of quenching occurred for all the domains as observed from the final F0/F values at 200 g/L of the crowder. From Tables 2−4 it is evident that domains I and III experienced a similar kind of quenching for the various pH solutions. At pH 9.15 the emission maximum of Trp-214 showed a significant red shift of ∼12 nm (from 340 to 352 nm) with increasing Dextran 6 concentration from 100 to 150 g/L. This coincided with a sudden increase in the Stern−Volmer quenching constant observed for this residue, with the quenching being further augmented at pH 10.5 (Table 3). This signifies that exposure of Trp 214 leads to enhanced quenching, mostly solvent induced, resulting from progressive increase in interaction with the surrounding water molecules of the intrinsic tryptophan moiety. On the other hand for the Ac and NPA moieties, the spectral reporters of domains I and III, respectively, no such spectral shift in neutral and basic pH range occurred but in acidic medium a small blue shift (∼2 nm) was observed. Like Dextran 6, for all other crowders too, the quenching constant (K′sv) increased with increasing pH of the medium (SI Figures S4−S7). This is in agreement with the fact that at higher pH (basic) the secondary structure is almost intact, while at lower pH (acidic range) there is a significant loss of helicity. Thus, in the latter, because of the swelling of the protein (as observed from FRET) resulting from disruption of the secondary and tertiary structure-stabilizing forces, the surrounding amino acids are no longer in close proximity to the fluorophore, thereby bringing about a decrease in the extent of quenching. The reverse scenario is seen to occur for the basic pH values, suggesting progressive tightening of the individual domains as compared to that in the physiological pH (7.4). Closer analyses of the quenching plots provide us with some more interesting insights. The extent of quenching is highest for Dextran 6 and decreases as the average molecular weight of the crowders increase. This is a direct fallout of the excluded volume effect variation arising from the change in packing density, the latter increasing as the average molecular weight of the crowder becomes smaller. In this regard PEG8 is an exception as it gives rise to the least F0/F values among all indicating a perceptible change in the way it influences the serum protein. This aspect is in good quantitative agreement with the previous report from our group wherein a similar trend was observed.43 Moreover in the presence of Dextran 6, the extent of quenching is almost similar for all domains, suggesting a uniform disposition of amino acids around the fluorophore in the respective domains, in the presence of this crowder. However, the same does not hold true for the other crowding agents. For example, in the case of Dextran 40 and Dextran 70, the F0/F values at different pH conditions are the maximum for Trp-214 in domain II, while in the presence of Ficoll 70, Trp214 and NPA experience very similar extent of quenching. In PEG8, the acrylodan moiety in domain I is seen to have its fluorescence quenched the most. In the case of Dextran 40 and Dextran 70, we hypothesize that since both domains I and III move toward domain II (Figure 6), there is an excess of compressive force exerted on the second domain leading to enhanced quenching of Trp-214. Based on this, one would have expected the quenching to be relieved for this Trp residue in

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DISCUSSION There is a striking disparity between the heart-shaped structure of human serum albumin observed in single crystals and the elongated ellipsoid model used for decades to interpret the protein solution hydrodynamics at neutral pH.42,49 Moreover, recent reports have suggested that the solution structure of HSA was more spherical than previously assumed,50 thereby reflecting on the intrinsic flexible nature of this serum protein. Such a feature of HSA can be significant from the physiological point of view, as the protein is known to be an active transporter of small molecules. Adding to this disparity is the fact that the physiological interior is highly crowded, thereby further influencing the structural disposition of HSA in its functional form. Recently we have shown that the influence of macromolecular crowders on domain movement of HSA can be attributed to both entropic as well as enthalpic depletion forces.43−48 We hypothesized the existence of a distinct crossover in the nature of depletion forces operational for the polymeric crowding agents, with effects of Dextran 6 being mainly entropic in nature, Dextran 70 showing evidence of both entropic and appreciable enthalpic contributions, while Dextran 40 has characteristics of both Dextran 70 and Dextran 6, it being midway with regards to the average molecular weight of the other two crowders.27 However, in the previous report of ours we could obtain information about only a part of the HSA molecule, with the rest of the protein remaining silent with respect to the FRET changes. Hence here we have strived to expand our knowledge by providing insights about the simultaneous movements of the domains of the serum protein in native HSA as a function of pH induced denaturation in the presence of macromolecular crowding agents. From its inception, the general approach of macromolecular crowding has been based on the excluded volume approach arising from the hard sphere potential exhibited by the crowder molecules. Dextran 6, being the smallest crowder used in this study, has the highest number of molecules at any given concentration (g/L), thereby presenting a high packing density to HSA. Thus, if one assumes the inert-like environment of the crowders, then Dextran 6 should have had provided the maximum excluded volume thereby minimizing the Δr values for the domains. The fact that Dextran 6 brings about the least changes in the domain distances (ΣΔr values from Table 1) is further proof of the fact that its effect is primarily entropic. One would thus be surprised to observe an increase in the interdomain distance for domains (I and II) and also partly for domains II and III (in the acidic pH regimes). This increase in distance is however very much in accordance with our previous results where we had attributed such changes to the intrinsic compressibility of HSA.27 Based on the same argument, the larger crowders providing lesser excluded volume gives rise to greater interdomain separations with Ficoll 70 inducing the largest change (Δr) for domains I and II (Trp-Ac FRET pair) this being followed by Dextran 70 and Dextran 40. For domains II and III (Trp-NPA FRET pair) a similar picture also emerges, with Dextran 70 showing a slight more of an J


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III to move farther from domain II. This should also be regarded as a compaction but an asymmetrical one wherein the crowding agent (Dextran 6), induces such changes through excluded volume only. On moving to the neutral and basic pH, where the HSA is native (pH = 7.4) and native-like (pH = 9.15 and 10.5) respectively, the protein as a whole becomes more compact. Hence, because of the lesser space available, the pressure brought about by the Dextran 6 molecules, under these conditions, causes the inward movement of domain III, the latter thereby also helping in pushing domain I farther from domain II. The stress exerted by the Dextran 6 on HSA was further evident from the significant red-shift in the Trp-214 emission maxima (∼12 nm shift), this feature only being observed at the basic pH and also exclusively for this crowding agent only. However, the same argument does not hold true for Ficoll 70 wherein the anticorrelated movement was present in all pH conditions, since packing density is far less than that of Dextran 6. Moreover, the similarity in Δr values throughout, that is, for all pH solutions, suggest that the interactions between Ficoll 70 and HSA are not driven by electrostatics, but rather by hydrophobic forces between the protein side chains and Ficoll 70 carbon backbone. To extend this hypothesis, the trend induced by Ficoll 70 thus implies a difference in the overall hydrophobicity around domains I and III. To end this part of our discussion, while our data suggest that Dextran 6 exerts its action mostly through excluded volume, we do acknowledge that the presence of soft interactions cannot be ruled out completely. Based on the recent report about the existence of different concentration regimes in a polymer solution,54 we further divided our crowder concentration ranges into four distinct regimes as follows: 0−50 g/L, 50−100 g/L, 100−150 g/L, and 150−200 g/L. Focusing initially on the domain I−II distance as monitored by the Trp-Ac FRET pair, in the presence of Dextran 6 the distance change is quite uniform throughout all the concentration intervals except at higher pH, wherein, for the higher crowder concentrations, the rigidity of the protein inhibits large-scale domain distance modulation. Moving on to the other dextran based crowders, both Dextran 40 and Dextran 70 induce the maximum distance change by 50 g/L. Moreover, in the case of Dextran 40, beyond 50 g/L the change is very nominal. This is in agreement with our previous observation where we had hypothesized that by ∼75 g/L, the primary layer of Dextran 40 around HSA is already well-formed and hence subsequent increase of concentration of the crowding agent has no further impact on the domain separation.27 On the contrary, for Dextran 70, moderate changes take place over the entire concentration range, a feature that we attribute to progressive increase in the number of crowder molecules in the vicinity of HSA, thereby causing a gradual decrease in FRET distance.27 For Ficoll 70, except the 50−100 g/L range, distance changes for all other regions are quite appreciable and similar to each other. Thus, to analyze the overall picture obtained thus far, changes at the lower crowder concentrations (less than 50 g/L) must arise from the enthalpic or soft interactions that these exhibit with the protein, since the effect of excluded volume under these conditions would be minimized. Furthermore, for all the high molecular weight crowding agents, viz., Dextran 40, Dextran 70, and Ficoll 70, the modulation in the interdomain distance is independent of the pH of the medium suggesting that the aforementioned soft interaction is not necessarily governed by electrostatics but is rather dominated by hydrophobic interactions between the nonpolar amino acid

effect on the distance change, as compared to the other crowding agents. Moreover, the disparity in terms of the direction of domain movement is also quite striking. For Ficoll 70, for all pH values, the movement of domains I and III with respect to domain II are anticorrelated (Scheme in Figure 4). On the other hand, for Dextran 40 and Dextran 70, both the domains move inward, i.e., show correlated motion (Schemes in Figures 2 and 3 respectively). However, for Dextran 6, at pH 7.4 and beyond, the direction gets reversed for domains II and III (Scheme in Figure 1). Taken together, the aforementioned summary of the FRET related structural changes brings to the fore not only the differences between various crowders at the molecular level but also the heterogeneity in terms of the nature of spatial distribution and/or interaction, for the same macromolecular crowding agent. Additionally, these data reveal that excluded volume is not the only factor that needs to be considered while addressing such crowded milieu. Had this been the only contributor influencing the protein structure, one would have observed similar changes occurring with respect to the directions of domain motion without any bias whatsoever. In the presence of Dextran 40 and Dextran 70, both domains I and III move inward, i.e., toward domain II, to nearly equal extent, with domain I showing a slightly more movement. This suggests that in the presence of these crowding agents HSA undergoes a near uniform compaction in size as compared to that of buffer only. In this context, the anticorrelated movement observed for Ficoll 70 is surprising, given the fact that it has the same average molecular weight as that of Dextran 70. This anticorrelated movement is indicative of an asymmetric distribution of Ficoll 70 around the serum protein. Ficoll 70 is a highly branched copolymer of sucrose and epichlorohydrin and reportedly behaves much like a rigid sphere with a radius of ∼55 Å.21,51 In contrast, Dextran 70 is a flexible, long chain polymer of D-glucose with sparse, short branches, and is better modeled as a rod-like particle.21,52,53 Putting this in context with a recent finding wherein HSA was reported to assume a more spherical disposition in solution,50 our data suggest that this might not be the case, since then the Ficoll 70 molecules (also being spherical) would have given rise to similar effects throughout arising from a uniform distribution around HSA. On the other hand, it is also possible that HSA through its extensive surface area, being a large protein, induces changes in the Ficoll molecules through soft interactions (enthalpic) resulting in an asymmetric distribution of the crowder molecules around the protein. A similar proposition is also applicable in case of Dextran 6, albeit in a pH dependent manner, since a shift from acidic to basic pH induces the correlated movement of domains to become anti correlated in disposition. For Dextran 6, it can be argued that since the protein retains its native structure to a significant extent in the basic medium, hence in the presence of the dense packing environment of Dextran 6, the protein strikes a thermodynamic balance between the extent of compression it can endure and disruption of structure stabilizing forces, thus forcing the domains to move in an opposite sense. To explain in a different manner, the rigidity of the structure ensures that the inward movement of domain III forces domain I to move out, thereby minimizing pressure-induced destabilization from the surrounding crowder molecules. Thus, in acidic pH, wherein the protein is unfolded to a certain extent as observed from the changes in secondary structure (using CD), the volume expansion of the protein allows preferential compression in a lateral direction, thereby forcing both domains I and K


Article


Figure 7. Relative changes in Stern−Volmer quenching constant [Ksv/Ksv(1.9)] as a function of pH in for the three domains of HSA (as mentioned in legend) in the presence of the macromolecular crowders: (A) Dextran 6, (B) Dextran 40, (C) Dextran 70, (D) Ficoll 70, and (E) PEG8.

would be worthwhile to revisit the FRET changes for the domains as induced by Ficoll 70. The anticorrelated movement so observed for Ficoll can thus be considered to be related to the aforesaid difference in the nature of the interacting surfaces, with the inward movement (decrease in FRET distance) for domains I−II implying the enhanced hydrophobic interactions between the serum albumin and the crowder molecules. Subsequently, an asymmetry in the distribution around HSA is introduced wherein for the domain II−III surface, not only is the extent of interaction at a reduced level, but also the same arises from a decrease in the number density of Ficoll molecules, thereby allowing the domain with the NPA moiety to move further apart from Trp-214 (domain I). The fact that Dextran 40 and Dextran 70 induce uniform correlated domain motions (inward) for both domains I and III reinforce the molecular level differences between these dextran-based crowders and Ficoll 70. Another aspect that can influence the conformational modulations of proteins is the change in the size of the macromolecular crowders with increase in their concentrations, arising from the self-crowding phenomenon.58−61 These polymers have been reported to undergo a decrease in size at larger polymer concentrations, a feature that can also affect the extent of domain movement over the different crowder intervals. Supporting residue-level quenching studies from the independent reporters of the three individual domains of HSA add to the inherent complexity of the crowded milieu. From the Stern−Volmer constants (Tables 2−4), as expected, Dextran 6 has the maximum K′sv among all the crowders arising from its large excluded volume effect, while the quenching constant is minimum for PEG8, where soft interactions predominate over excluded volume. On the other hand, if

side chains and the extended backbones of the polymeric crowders.55 Surprisingly, except for Dextran 40 wherein the maximum change occurs by 100 g/L of the crowder, the domain II−III distance trends do not show any of the above preferences, again confirming our conjecture of different average interaction potentials of the two faces of the serum protein. This feature is further amplified when one probes the influence of PEG8 on the serum protein. Unlike the other crowders, the PEG8 induced effects exhibit significant pH dependence for the Trp− NPA distance at low-to-moderate concentrations (until 75 g/L) but becomes pH independent at high concentrations of the crowder. At the low concentration regime, PEG8 is monomeric in nature and known to exhibit hydrophilic soft interactions with protein backbone, 56,57 thereby enhancing domain separation as the pH is increased. With increase in concentration, the monomeric nature of the PEG molecules is replaced by an entangled polymeric network, wherein excluded volume effect predominates and causes a decrease in the interdomain distance. On the other hand, no such pH dependence is seen to be in operation for the domain I−II separation, though the crossover region from soft interactions to that of excluded volume is also clearly visible, and that too exhibiting a trend opposite to that of the separation between the Trp-NPA FRET pair. These results additionally imply that the interaction surface presented to the crowding agents by domains II and III is predominantly hydrophilic while that of domains I and II is mostly hydrophobic. Thus, for the hydrophilic surface, the PEG8 polymers interact through the polar −OH groups until the crossover concentration is reached while for the hydrophobic surface of HSA the −CH2−O− CH2− moieties of the polymer predominate. In this context, it L


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The Journal of Physical Chemistry B one were to decide by the traditional KSV values, then the overall quenching picture undergoes a major transformation with Ficoll 70 turning out to be the one having the maximum KSV. This is a direct fallout of the different molecular masses of the crowding agents, and with K′sv being the mass-weighted form of Ksv, the normalization factor is high for the larger crowding agents. To further aid in our understanding of the crowder-induced domain specific quenching, we have plotted the KSV values relative to that observed at pH 1.9 for the different crowders in Figure 7. The increase in quenching with increasing pH is an obvious signature of the gradual enhancement of the rigidity of the domains and hence of the overall protein. Moreover, it is also evident that for the low molecular weight crowders (Dextran 6 and PEG8), Trp always experiences the highest quenching, suggesting enhanced conformational pressure on domain II. Although the bimolecular quenching constant is lowest in the case of PEG8, however, the change in Ksv is as high as that of Dextran 6 confirming the fact that the serum protein is also modulated to a significant extent by PEG8, an aspect that we have encountered during the FRET based variations in the interdomain distances. On the other hand, the increased quenching for Ac in domain I and NPA in III with respect to that of Trp-214 (domain II) is a further proof of the difference in the manner in which these crowders (Dextran 40, Dextran 6 and Ficoll 70) interact with the serum protein as opposed to that of the Dextran 6 and PEG8. Recently Poolman and co-workers have used a genetically encoded FRET sensor to provide a quantitative assessment of the extent of crowding both in vitro and in vivo, adding to the PEG based sensor developed by Ebbinghaus’ group.62,63 Their results emphasized upon the fact that the sensor was only amenable to changes in excluded volume and was oblivious to any other modifications in solution conditions like salt and/or pH or the type of crowder used in vitro. In this regard, the intrinsic ability of HSA to respond to the varied interaction potentials that the complex medium has to offer, based on its domain movements (from FRET) and domain specific response (through quenching), also shows the possibility of it being as a sensor for the crowded environment. Moreover, the fact that this serum protein is able to reveal the asymmetry in the spatial disposition of the crowder molecules around its domains provides an extra handle based on which the characteristic features of the crowded medium can be better addressed. Finally keeping in mind the functional aspects of human serum albumin that involve fatty acid and anesthetic binding, leading to significant domain movement and pocket expansion,64,65 and the different levels of crowding that the physiological interior possesses, understanding the dynamic response of HSA to the changing external conditions would go a long way in deciphering the underlying details of how the serum protein maintains its status as a prolific transporter of small molecules.

such crowded milieu. The influence of Dextran 6 was found to be predominantly via excluded volume, while PEG8, having a similar molecular weight, exhibited a distinct crossover from enthalpy-dominated interactions to entropy-based potentials. pH dependence of the PEG8-induced interdomain distance changes confirm the presence of a hydrophilic surface for the domain I−III side of HSA, while that for domains I and II is more hydrophobic. For the larger molecular weight crowders, the majority of the FRET distance modulation was seen to be taking place within 50 g/L crowder concentration implying significant soft interactions with HSA. Residue-level fluorescence quenching studies for the three domains coupled to the FRET results provide us further insights into the manner in which the serum protein responds to and adapts itself to varying pressure(s) of the crowded medium. Such insights into local protein motion bears tremendous significance with respect to the various functions that the protein is destined to perform in the physiological interior.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b01671. Variation of interdomain distances between domain I−II (Ac-HSA) and II−III (NPA-HSA), efficiency of energy transfer (E), R0 with increasing Dextran 6, Dextran 40, Dextran 70, Ficoll 70, and PEG 8 in different pH buffer was shown in SI Tables S1 to S5. Figure S1 shows the overlap between the FRET pairs. Interdomain distance variation between domains I and II (Ac-HSA) and domains II and III (NPA-HSA) with increasing pH of the buffer solution has been plotted in Figure S2. Figure S3 shows the effect of pH on the far-UV CD at 222 nm of HSA with increasing pH in buffer and different crowder (200 g/L) solutions. Figures S4−S7 show the quenching profiles of the three domains of HSA in the presence of the different crowders. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; Tel: +911126591521. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS S.B. thanks CSIR for his fellowship, and P.K.C. 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. We also thank Vaisnavi Vishi and Samadrita Biswas for their help with regards to some of the experiments.

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CONCLUSION In this paper we have attempted to decode the environment around the multidomain protein HSA in crowded media by mapping the changes in interdomain distances as a function of pH. The observation of correlated and anticorrelated domain movements specific to the macromolecule crowding agent being used is an apt reflection of the molecular level differences among the crowders, their underlying interactions with the serum protein and hence the heterogeneity associated with

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