Crowder-Induced Rigidity in a Multidomain Protein: Insights from

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Crowder Induced Rigidity in a Multidomain Protein: Insights from Solvation Saikat Biswas, Sanjib Kumar Mukherjee, and Pramit Kumar Chowdhury J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b10478 • Publication Date (Web): 28 Oct 2016 Downloaded from http://pubs.acs.org on November 25, 2016

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The Journal of Physical Chemistry

Crowder Induced Rigidity in a Multidomain Protein: Insights from Solvation Saikat Biswas, Sanjib Kumar Mukherjee, and Pramit Kumar Chowdhury* Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016

*To whom correspondence should be addressed: Email : [email protected] Tel : +911126591521

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Abstract: Global changes in conformations of proteins in presence of macromolecular crowding agents have been well documented. Here we have used solvation dynamics to monitor the changes in a specific domain of the mutlidomain protein human serum albumin (HSA) in presence of various crowders. The solvation probe BADAN (6-Bromoacetyl-2-dimethylaminonaphthalene) was sitespecifically attached to cysteine-34 of domain I of HSA. Analyses of the time resolved Stokes shift of this probe in presence of the crowding agents revealed a significant retardation of the solvent coordinate, and that too, in a crowder dependent manner. We attribute the observed slowdown primarily to increased internal protein friction in presence of these polymers, implying considerable stiffness of the protein matrix. We have discussed our findings with regards to recent reports about the cellular interior and have also made an attempt to connect to the importance of the physiological concentration of macromolecules to protein dynamics and function.


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Introduction: The cumulative excluded volume exerted by the macromolecules inside cells, known to occupy about 40% of the cellular volume1,2 has been shown to have significant effects on the structure and function of proteins.3-10 Experimental studies have reported that such congestion, commonly referred to as macromolecular crowding, favours compact states, thereby either shifting equilibrium toward the folded state,9-18 or enhancing protein-protein association,6,19 an effect that can be attributed to the hard-sphere nature of the potential that the crowders exude.4-8 Recent studies have however hinted at increased complexity of the nature of the protein-crowder interactions, with soft enthalpic type predominating in certain cases, bringing about unexpected effects.20-23 Thus to build better models of such processes, it is crucial to understand in greater detail the effect of cellular crowding on the physical determinants of protein folding and binding. In general, most of the studies of proteins in crowded environments have focused on the global structural changes associated therein, as reflected by changes in the midpoints of chemical and/or thermal denaturation along with perturbation in the secondary and tertiary structures.15-17 Not much attention has however been paid toward the unravelling of local structural modulation and dynamics in the crowded milieu leaving this area much less explored.20,21,24-26 In this report, by covalently attaching a solvation probe BADAN (6-Bromoacetyl-2-dimethylaminonaphthalene), to a specific site in domain I of the multi-domain (three domains) protein human serum albumin (HSA), we have investigated how this particular domain is influenced in presence of increasing concentrations of six different crowding agents, namely, PEG200, PEG8000 (PEG8), Dextran 6 (D6), Dextran 40 (D40), Dextran 70 (D70) and Ficoll 70 (F70). Our decision of choosing HSA as the test protein arose from its physiological significance as an avid carrier of small molecules (drugs and metabolites) and fatty acids, the binding of which induces appreciable domain 3 ACS Paragon Plus Environment


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displacements thereby reflecting on the inherent flexibility of the serum protein. Solvation data obtained from the analyses of the time resolved emission spectra (TRES) of BADAN attached to Cys-34 (free cysteine at the 34th position of the polypeptide chain) show that domain I of HSA undergoes a transition from a relatively flexible entity to an appreciably rigid moiety as the crowder concentration is increased with the nature of the transition and extent of tightening being a reflection of the manner in which the crowding agents exert their effect on this specific domain of the three-domain serum protein.

Materials and Methods Chemicals Essentially fatty-acid-free HSA, Dextran (6, 40, 70 kDa), Ficoll 70 and PEG (8000, 200) were purchased from Sigma Aldrich chemicals Pvt. Ltd. (USA) and were used as received without purification. Sodium phosphate dibasic anhydrous (Na2HPO4) and sodium phosphate monobasic anhydrous (NaH2PO4) were purchased from Merck Specialities Pvt, Ltd. (Mumbai) and used as received. BADAN was purchased from Molecular Probes Inc. (Invitrogen, USA).

Protein labelling and purification A stock solution containing 50 μM HSA was prepared in 50 mM phosphate buffer (pH = 7.0). A 50 µL aliquot of this solution was taken, and BADAN (dissolved in the minimum volume of DMSO) was added such that the molar ratio of HSA to BADAN was 1:1.28. This mixture was stirred gently and maintained at 4°C for 12 h in dark. The labelled protein was then eluted through a G-25 sephadex column (molecular mass exclusion limit of 5000 for proteins) to separate it from any excess BADAN molecules (unreacted). The concentration of the labeled


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protein was found by using ε387 = 21000 M-1 cm-1 for BADAN and ε277 = 36,000 M-1 cm-1 for HSA. The labelling efficiency was determined to be greater than 80%.

Sample preparation The phosphate buffer solution was prepared by mixing definite weighed amounts of monobasic and dibasic phosphate salts in Millipore water. The pH of the resultant buffer was measured by a pH meter (pH 510, Cyberscan, Eutech Instrument). Desired amount of crowding agents were weighed using an analytical balance (Precisa, Sweden) and dissolved in phosphate buffer solution.

Spectroscopic Measurements Absorption Absorption measurements were performed in double-beam Shimadzu UV-VIS Spectrometer (UV-2600, Japan) using 1cm path length cuvettes. Absorbance values of the protein solutions were measured in the range of 200-600 nm and molar extinction coefficient for the BADANHSA used was ε387 = 21000 M-1cm-1.

Steady state Fluorescence Steady state fluorescence measurements were carried out on an Edinburgh Instruments (UK) fluorescence spectrometer (Model: FLS920). The fluorescence spectra of protein samples at pH 7.4 phosphate 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



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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 hours (at 4 °C) before acquiring their respective spectra. BADANlabeled HSA was excited at 375 nm, with emission collected from 400 nm to 650 nm to monitor the BADAN emission alone.

Time Resolved Fluorescence 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 BADAN residues of BADAN-HSA (labeled HSA). Emission decays were subsequently collected at 410 nm to 650 nm in buffer and in presence and absence of the crowding agents through a 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 half-maximum) of the IRFs collected was typically in the range of ~220 ps. The average lifetime from the emission decays was obtained using the formula τ = ∑ α iτ i (αi = amplitude and τi = decay time i of component i).

Anisotropy Measurements of the rotational anisotropy of BADAN-HSA were performed using the abovementioned time-correlated single photon-counting (TCSPC) setup. For every measurement we


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calculated the G factor and then fitted the anisotropy decay to find the rotational time constant. The time-dependent anisotropy function, r (t ) , can be expressed as follows:

r (t ) =

I C − GI ⊥ I C + 2GI ⊥

(1)

where G is the instrument and wavelength-dependent correction factor produced by the optical system that corrects for any depolarization of the detection system. The anisotropy decays were fitted to the following equation r (t ) = ∑ r0j exp(− j =2

t

τ rj

(2)

)

where r0j are the fractional anisotropies and τ r1 and τ r 2 represent the local and global (with the protein) motions of BADAN-HSA.

Results and Discussion: The fluorescence decays of BADAN covalently bound to HSA show a significant dependence on the emission wavelength. At the blue end of the emission spectrum, only a fast decay is observed while at the red end the decay is preceded by a distinct rise at the earlier times. Such wavelength dependence is an indication of the slow solvation resolvable by the FWHM of ~220 ps of the instrument response function of our current setup. From the parameters of best fit to the emission decays and using the steady-state emission spectra, time-resolved emission spectra (TRES) of BADAN-HSA in buffer (Figure 1A) and in presence of different crowders (Figure 1B and C) have been constructed by following the usual procedure27. At first a series of decays (excited at 375 nm) were collected from 410 to 650 nm at 10 nm intervals. These decays were fit to a sum 7 ACS Paragon Plus Environment


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of exponentials, subsequent to which TRES were reconstructed from these fitted decay curves by normalizing to the respective steady state emission spectrum of BADAN-HSA (equation 3).

S (λ , t ) =

D ( λ , t ) S 0 (λ )

(3)

∫ ∞0 D(λ , t )dt

where D(λ,t) is the fluorescence decay at a particular wavelength and S0(λ) is the steady-state emission intensity at a given wavelength. The solvent correlation function was then plotted according to the following equation

C (t ) =

ν (t ) −ν (∞) ν (0) −ν (∞)

(4)

where ν(t), ν(0), and ν(∞) are the corresponding peak frequencies at times t, 0, and ∞, respectively, with ∞ ideally referring to that time at which the system has reached equilibrium. A similar approach was also used for the construction of the time-resolved emission spectra in presence of the macromolecular crowders. The normalized solvent correlation functions were then plotted (Figure 2A-H) based on equation 4. The solvation dynamics in domain I is significantly influenced in presence of the different macromolecular crowding agents. In buffer only, that is, in absence of crowders, the C(t) is well described by a biexponential fit with the average solvation time being ~6750 ps, this being incurred while responding to a time dependent Stokes shift of ~1730 cm-1. The slow component (~12 ns, Table 1) in the native protein may be assigned to the motion of the hydrogen bonded water molecules residing at the hydration layer as well as the protein matrix itself with its own share of polar and charged amino acids relaxing around the BADAN moiety28. Solvation studies using non-covalently bound probes like ANS to HSA give rise to C(t) decays that can be fit by a single exponential thereby showing that in the latter case, the solvation shell is much more 8 ACS Paragon Plus Environment

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homogeneous. The fast component (~500 ps) of solvation for the buffer may be attributed to the rapid exchange between ‘bulk-like’ (or free) water molecules and those that remain loosely associated with the protein28,29. To further confirm that the observed dynamics is indeed from the covalently bound BADAN, we have also tried to probe the solvation dynamics of BADAN only (i.e. free BADAN) in buffer and high concentrations of the crowders used. In all these systems, the BADAN fluorescence is significantly quenched resulting in very fast fluorescence decays (Supporting Information Figure S1), the latter not being resolvable by the time resolution (~220 ps) of our present TCSPC setup. This implies that the observed dynamics in our study arises solely from BADAN covalently ligated to HSA and variations in the solvation environment therein. In case of the dextran based crowding agents, at lower concentrations, that is, uptil 100 g/L, there is a decrease in the average solvation time as probed by BADAN, subsequent to which, the solvent response time increases as a function of the concentration of the crowder (Figures 2A and 3 and Supporting Information Tables S1-S6) used. The solvation times for D6 and D40 at 300 g/L were very similar at ~15.5 ns while that in presence of D70 was much faster (~ 9.2 ns) (Table 1, Figures 2B,C and 3). Thus, in general, with increasing concentration of the crowding agents, the number of crowder molecules per unit volume increases thereby restricting the structural mobility of the protein and hence the flexibility of the domain I, resulting in a retardation of solvent response for BADAN. The observed solvation times at the highest crowder concentration can be initially ascribed to the excluded volume effect that these crowders are known to exude. D6 having the smallest average molecular weight amongst the dextran based crowding agents, exhibits the maximum packing density and thereby shows the maximum excluded volume effect. Thus it is not surprising that D6 induces the lowest solvation response



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for domain I at 300 g/L. D70 on the other hand having the least excluded volume by virtue of its high molecular weight slows down the solvent coordinate the least. D40, with its molecular weight being almost midway that of D6 and D70, however induces a solvent response at 300 g/L that is very similar to D6. This observation suggests that excluded volume is not the only factor that comes into play here. This idea is further confirmed by the trend exhibited by F70. F70, having the same average molecular weight as that of D70, however induced a much different response, with the solvation time increasing from the very early concentrations of the crowder (Figure 2D and Supporting Information Table S4), and the final average solvation time () at 300 g/L being ~18.3 ns (Table 1), which is almost twice that of D70. Both the PEG based crowders, PEG8 and PEG200, gave rise to the slowest solvation times (~21 ns at 300 g/L crowder concentration) amongst the crowders used in this study; however, their manner of inducing changes in solvation were quite different. In case of PEG200 the average solvation time increases sharply from the low crowder concentration (Figure 3 and Supporting Information Table S6). On the contrary, for PEG8, initially the solvation time decreases up to 100 g/L, followed by a small increase up to 200 g/L, beyond which a sharp transition was observed (Figure 3 and Supporting Information Table S5). To ascertain that the effect of the crowders is due to their macromolecular nature, we have also carried out similar solvation experiments using glucose (monomer of the dextran based crowding agents) and sucrose (monomer of F70). As evident from Figure 2 (panels G and H) there is almost no change in solvation time even at a very high concentration of these monomeric units. Moreover, for BADAN-HSA in dilute buffer only, the emission maximum of BADAN is at 494 nm which remains typically invariant to the changing concentrations of the crowding agents for all the crowders used in this study. This observation, in combination with the fact that the emission maximum is characteristic of a


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medium having a dielectric constant of ~35 for the microenvironment of BADAN, suggest that the changes in solvation pattern solely arise from the combined retardation of the protein matrix and hydration environment in the proximity of the solvation probe. Taken together these data not only reveal the enhanced rigidity in and around domain I of HSA as observed by the slowdown of the solvent coordinate, but also bring to light the intrinsic differences in the manner that these crowding agents influence the solvation of BADAN. A closer analysis of Figure 3 with regards to the trends in the average solvation time as a function of the concentration of the crowders reveals some interesting features. For the three dextran-based crowding agents and PEG8, the profiles can be divided into multiple and distinct concentration regimes. For example, for D40 and D70, that is, the higher molecular weight polymers, the following are the different phases observed: (i) an initial part corresponding to 0– 100 g/L of the crowders wherein the solvent relaxation time is seen to decrease, that is solvation becomes faster as compared to that in buffer only, (ii) a middle phase spanning 100–200 g/L where there is a sharp increase in the solvation time, and (iii) the final phase beyond 200 g/L, characterized by the near saturation of the observed solvation, that is, accompanied by small changes in the relaxation time. For the smaller macromolecular crowders of D6 and PEG8, in addition to the aforementioned regimes, one observes a fourth phase, with the last one being characterized by a second steep increase in the solvation time. In other words, for these crowders, the solvation change stalls midway en route to the final concentration of 300 g/L. Schreiber and coworkers have proposed that the intrinsic packing of these polymeric crowders may vary in a concentration dependent manner, starting from dilute conditions wherein the individual polymers seldom overlap/interact with each other until a ‘cross-over’ concentration is reached, the latter signifying the onset of such overlap.23 Additionally, at higher concentrations, 11 ACS Paragon Plus Environment


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extensive overlap takes place leading to entanglement of the polymer chains. Our results can also be correlated to a certain extent with the presence of such different polymer regimes. We hypothesize that the transition from the first phase to the second one, corresponds to the onset of the ‘cross-over’ concentration. For D6 and PEG8, the presence of the middle plateau, that is, the third phase can be associated with the second polymer regime that signifies a changeover from semi-dilute to highly concentrated. Based on these arguments, the absence of the fourth phase for D40 and D70 can be rationalized if one considers the number density of the different crowders involved. D6 and PEG8 being the lower molecular weight polymers, have the highest number of molecules per unit weight amongst the macromolecular crowders used here. Hence, the transition to the third phase occurs much earlier, that is, at much lower crowder concentrations, as compared to that of D40 and D70. This implies that for the latter (D40 and D70), the presence of only three phases arises from the fact that even at 300 g/L, the number density is not high enough so as to get a signature of the next level of entanglement, unlike that of D6 and PEG8. Moreover, the absence of a plateau, that is, saturation of the solvation time, at the highest concentration of these lower molecular weight crowders (D6 and PEG8), further suggests the possibility of more extensive entanglement at concentrations beyond 300 g/L. The next logical question one would like to pose is the significance of the presence of these different solvation phases with regards to the serum protein, HSA. BADAN is covalently ligated to the cysteine residue in domain I of the multidomain protein. Hence our observations are limited mostly to the changes in the manner in which the protein matrix and/or solvent molecules surrounding the BADAN chromophore respond to the variation in crowder concentration. A recent study based on the changes in interdomain separation between domains I and II of this protein showed that the major percentage of such distance modulation happened in the 0–100 g/L


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crowder concentration range for the dextran based crowding agents.21 Interestingly enough, this is also the region where the solvation becomes faster with respect to that in buffer only (i.e. in absence of the crowding agents). Thus under these conditions, it is evident that existence of interdomain motion entails the presence of a relatively loose protein matrix, that is, a state characterized by relatively low internal friction, thereby leading to faster solvation. Beyond 100 g/L, the distance changes ware minimized to a large extent. In other words, below 100 g/L of crowder(s), HSA responded to the external conditions by allowing the domains to undergo relative movement amongst each other. However, increase of the crowder concentration leads to a significant decrease in conformational subspace thereby limiting the scope of large-scale changes that would take place in domain separation. Hence, at least with respect to domain I, with its motion stalled due to lack of space, the protein matrix this time responded by becoming increasingly rigid leading to a slowdown in the relaxation around the BADAN chromophore, that is, an increase in the average solvation time. For PEG8, the argument however is slightly different since our previous study shows that at the lower crowder concentration, the separation between domains I and II was the least affected.21 This arises from the intrinsic difference in the manner in which PEG8 influences the serum protein. Finally, the remaining two crowding agents, F70 and PEG200 induced a completely different response with an initial rapid increase followed by a near stagnation in the observed solvent relaxation at high crowder concentrations. The observed trend of the average solvation time in case of PEG200 is as expected. This being a small molecule crowding agent, even at very low concentrations, its number density is quite high, leading to a sharp increase in the solvation time. Beyond 150 g/L, the effect of this crowder saturates, but not before it has induced the slowest solvation time of ~21.0 ns amongst the crowders used in this study. This effect is in agreement with a recent article wherein it was



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reported that smaller the crowding agent, better it is at exerting excluded volume, provided that the effect of the crowder is exclusively steric in nature.30 The most striking effect of the intrinsic architecture of the crowder and its subsequent nature of entanglement is the difference observed between F70 and D70, in spite of these having the same average molecular weight. Evidence of this was also available in our previous report on domain separation wherein the difference was attributed to enhanced soft interactions between F70 and HSA.21 This observation, coupled with the fact that solvent correlation functions are also being affected at concentrations less than 50 g/L where excluded volume effect should be at its lowest, is another evidence of the prevalent enthalpic interactions that these polymeric entities exhibit with the serum protein. To further check that the increase in solvation times is not due to a sudden jump in the magnitude of time resolved Stokes shift, we have also plotted the time taken (in ps) per cm-1 (=

τs ), that Δυ

is, the time taken per unit Stokes shift, for the surroundings (protein matrix and bound water molecules if present) to relax around the non-equilibrium charge distribution being brought about by the sudden change in dipole moment of BADAN immediately after excitation. The plot (Supporting Information Figure S2) is almost identical to Figure 3, signifying that our observed changes are indeed a result of the progressive slowing down of the time taken for the protein as a unit, that is, along with the surrounding hydration layer, to respond to the changes in solvation at increased crowder concentrations. Subtle changes are however noticed. At 300 g/L crowder concentration (Table 1), the time taken for unit Stokes shift is almost the same for D6, D40, F70 and PEG8, implying that under these conditions the response of the protein matrix is quite similar. The highest value (Table 1) is obtained for PEG200 at ~13 (ps/cm-1) with that observed


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in buffer being the lowest at ~4 ps/cm-1, the latter being in good agreement with a recent report by Sen et al.31 To put our results in perspective of the solvation times in proteins and organised assemblies as reported in literature, we have also provided a brief summary of the same below. When the probe DCM is incorporated in the micellar environment (TX-100, SDS), the time taken for unit Stokes shift is ~2-3 ps/cm-1 32 while it reduces to ~1 ps/cm-1 when the same probe is covalently attached to HSA28. For the coumarin based dye CPM covalently attached to the HSA, the observed time per unit Stokes shift was ~1.6 ps33 which is quite similar to that obtained for the Aladan labelled streptococcal protein G (~1-1.2 ps/cm-1)34. Hochstrasser and co-workers investigated the solvation of Coumarin 343 bound to the protein Calmodulin and reported a solvation time of ~4 ps/cm-1 35, very similar to that of Coumarin 480 in AOT reverse micelles36. A low temperature (278 K) study of ANS bound to BSA revealed a moderately higher time of ~5-6 ps required per unit Stokes shift37. For acrylodan labeled HSA in AOT reverse micelles a relatively higher solvation time of about 12 ps/cm-1 was incurred for the surroundings to relax in response to the sudden perturbation in equilibrium.38 For systems like 4-aminophthalimide in β-cyclodextrin and Coumarin 343 in propane at near critical temperature, solvation times as high as ~16 ps/cm-1 have been reported39,40; interestingly, for some of the crowders used in this study, the values were quite similar. Based on the penetration depth, the solvation time in lipid bilayers can vary from ~0.5 ps/cm-1to ~1-2 ps/cm-1, with the solvation becoming slower as the probe embeds deeper into the bilayer41-49. A newly synthesised fluorophore MUC7 in AOT reverse micelle (w0 = 2) at room temperature shows a higher solvation time of ~3 ps per unit Stokes shift50. Slow protein dynamics at the tunnel mouth position of enzymes has been proposed to be the cause for the slow solvation of a coumarin dye (~6-7 ps/cm-1).51 Thus, in presence of crowders (300 g/L),



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the time required for solvating BADAN is quite similar or even slower than that observed in some of the organized assemblies as mentioned above, thereby providing us a glimpse into the extent of rigidity being induced on this specific domain (domain I) of HSA. For analysing segmental and global motions time resolved fluorescence anisotropy experiments of BADAN covalently bound to the serum protein were carried out. The anisotropy decay of BADAN was found to be biexponential with components of 300 ps and 16.4 ns (Supporting Information Table S7(A-E)). The initial fast component that can be attributed to the local wobbling motion of the probe only52, does not show significant dependence on the crowder concentration for any of macromolecular crowding agents used here. The slower component, which is due to unresolved segmental motions and the tumbling of the protein as a whole unit, shows some variation depending on the specific crowder in question. For the dextran based crowding agents, the longer component stays nearly the same, while changes are observed for F70 and the PEG based crowders. Considering the fact that the global tumbling component provides a read-out of the viscosity of the medium, the fact that there are appreciable changes in the average solvation times for D6, D40 and D70, suggests that the observed retardation of dynamic solvation arises from the intrinsic freezing out of the solvent coordinate as dictated by the protein matrix and associated water molecules in the immediate vicinity of BADAN, and hence to a large extent is insensitive to the viscous drag of the surrounding bulk solvent. A comparative plot (Supporting Information Figure S4) of the ratios of the average solvation times for a specific crowder and ratios of the longer rotational time constant, τr2, provides a detailed picture of the extent to which the viscosity of the solutions might affect the solvation slowdown. Thus, except PEG200, for no other crowding agent did we observe a strong correlation between the two ratios thereby implying that for all the higher molecular weight crowders the observed


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retardation of dynamic solvation arises primarily from the intrinsic freezing out of the solvent coordinate as dictated by the protein matrix and associated water molecules in the immediate vicinity of BADAN. On the other hand for PEG200, the sheer number density gives rise to a rather intricate network, the same being reflected in the increased tumbling time for HSA (Supporting Information Table 8F) and also contributing to the solvation slowdown. Hydration dynamics of water associated with biomolecule surfaces/interfaces has attracted researchers for long. Starting with the idea of protein conformational motions being slaved by solvent fluctuations53,54, an extensive amount of work has gone into investigating the various timescales associated with the aforementioned dynamics of solvent molecules55-62. With water as the physiological solvent considered far from being a silent spectator63, indeed recent reports have brought to the forefront the varied manner and mechanisms by which hydration dynamics can affect protein structure and function64-67. With the finding of the existence of a hydration layer extending over an appreciable distance around proteins68,69, the concept of protein-coupledsolvent motion, that is, solvent molecules making their presence felt on diffusive or functionally relevant protein structural transitions has been further reinforced69. The cellular interior presents a crowded environment arising from a dense packing of several macromolecules. Under these conditions the water properties have been shown to be different than that of bulk water, as characterised by lower polarity, higher viscosity, and slower solvation response.70-73 Thus it is not surprising that protein dynamics also stands to be modulated when placed in the crowded milieu. However, studies addressing this aspect are rare to come across. A very recent article has reported the observation of a ‘jamming-like’ transition in presence of both synthetic and protein based crowders, for the protein lysozyme, occurring in the picosecond timescale.74 Such a phenomenon featured a sudden slow-down of the protein motion beyond a critical crowder



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concentration, which the authors have attributed to an ‘independent-to-collective’ hydration induced by the crowding agents. Our data on the solvation of a specific domain (domain I) of HSA also show a similar phenomenon, albeit in a crowder dependent manner and also on the time-scale of nanoseconds, which is much longer than that observed in the aforementioned study. We propose that the observed increase in solvation times for BADAN as a function of the crowder concentration is a reflection of how the internal viscosity of the protein, in this case domain I, is enhanced, thereby rendering these so-called fast protein modes/fluctuations less likely to happen. Support of this proposition comes from recent articles wherein the retardation in solvation times arises from the slowness in the protein dynamic/conformational response as opposed to the water properties at the protein surface.75,76 Further proof of this phenomenon is obtained from the observation that solvent exerts its frictional effect only for some of the very slow protein modes, these being involved in major changes pertaining to shapes of surfaces and hence water accessibility, thereby needing to invoke extensive interactions with the surrounding solvent molecules.77 However, we also acknowledge that though we hypothesise the protein matrix to be the primary cause of the observed increase of the average solvation times, the coupling and influence of hydration water, that is water bound to the protein surface, on the observed solvation dynamics cannot be ruled out. Considering the fact that conformational motions in proteins spans several decades of time (ranging from picoseconds to seconds),78 our observation of the slow-down of the solvent coordinate signifies an alternate mechanism by which cells can modulate functions by changing the intracellular hydration levels thereby affecting the concentrations of macromolecules79. Moreover the apparent rigidification of protein motion is very similar to that observed in glasses, the latter being characterised by a protein dynamic transition beyond a certain temperature. Indeed recent evidence suggests a glassy


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behaviour of the cytoplasm with occasional fluidity being introduced through metabolic activity.80 It is worth noting that protein internal friction arises from the manner in which individual segments of the polypeptide chain move/slide with respect to one another81; hence the enhanced restriction of such movements seen in this study hints at greater interdigitization of the amino acid residues surrounding BADAN in domain I of HSA, thereby giving rise to a relatively ‘locked’ conformation of the protein domain. Finally the cytoplasm has of late been regarded as a poroelastic medium, with cellular rheology being dynamically controlled based on the pore size and entanglement length.82 Hence to a first assumption, the entangled nature of the synthetic polymers, and the possibility of tuning the same as a function of the concentration of the polymer24 based on the nature of the specific macromolecular crowding agent, provides one with ample opportunity to throw further light into the possible dependence of protein dynamics on the varying ‘poroelastic’ behaviour of the polymers.

Conclusion: The interaction between proteins and water is crucial for protein folding and stability. Water molecules confined in various biological and organized assemblies strongly influence the structure, function, and dynamics of biological systems. The dynamics of water near protein surfaces has been studied using several theoretical and experimental approaches64. MD simulations predict that the dynamics of water slow down near protein surfaces, with the amount of retardation observed strongly depending on the protein surface topology.65,83-85 NMR studies have also revealed a similar slowdown effect in water of hydration and protein segmental motions86-90 and while time-resolved fluorescence91,92 studies find a wide distribution of water reorientation times in the protein hydration layer, with both techniques requiring the embedding



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of specific probes in the protein (or protein encapsulation93). In this report we have shown that the internal dynamics of protein matrix is highly dependent on the crowder concentration and the nature of entanglement of the polymer. Modulation in solvation dynamics at lower crowder concentrations is indicative of the presence of soft enthalpic type interactions between the crowding agents and the serum protein, in agreement with earlier studies. Self-crowding at increased crowder concentrations not only has been shown to have substantial effects on the hydrodynamic radii of the polymers but is also associated with changing interaction surfaces and surface areas, the differences in the same being exhibited in the profiles of the average solvation times so observed.94,95 Given the fact that the degree of macromolecular crowding in the physiological interior is different for the various organelles, our data suggest that the protein dynamics are influenced accordingly, thereby spanning a range of frequencies/timescales relevant to the function of that specific polypeptide chain. For example, in the blood plasma, the concentration of macromolecules is in the range of ~80-100 g/L96,97. Under these conditions, the protein matrix of HSA is quite flexible, as observed from our study. Being a major carrier serum protein, HSA shows a dramatic conformational flexibility upon fatty acid binding involving domain I, the latter being the domain under investigation in the present study98-102. Crystal structure of fatty acid (FA) bound HSA shows that binding of the FA at the interface between subdomains IA and IIA appears to stabilise the rotation of domain I relative to domain II100. Moreover, upon binding of FAs, Y150 from subdomain IB moves to interact with the carboxylate moiety of the lipid bound to the site that straddles domains I and II. Rotation of the side chains of Tyr-138 and Tyr-161 by about 90˚ opens up a specific hydrophobic channel to allow FA binding which thus confirms the enhanced flexibility and functional importance of domain I in HSA102. Additionally, upon binding with drugs like azapropazone, indomethacin and


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warfarin in subdomain IB, domain I of HSA also undergoes extensive hinge binding motion99-101. In other words, the level of congestion in the serum seems to be optimal with respect to HSA being able to perform its functions involving domain movement in a satisfactory manner.

Supporting Information: Tables of the average salvation time for the crowders and tables on the rotational anisotropy decays of BADAN-HSA with increasing concentration (g/L) of the crowding agents have been included in Table S1-S7. Figure S1 consists of the plot of time resolved decays of BADAN and BADAN-HAS in pH 7.4 buffer. The dynamic Stokes shift per unit time (ps/cm-1) as a function of the crowders’ concentrations is given in Figure S2 while Figure S3 shows some representative anisotropy decay curves of BADAN covalently attached to HSA. Figure S4 includes comparative plots between ratios of solvation times and the longer rotational times for each individual crowder.

Acknowledgment SB thanks CSIR and SKM thanks UGC for their fellowships. PKC thanks Council of Scientific and Industrial Research (CSIR), India, Grant No. 01(2827)/15/EMR-II, for financial support and IIT Delhi for startup funding.



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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1: Time-resolved Stokes shift and solvation response time (τs) of domain I (BADANHSA) in phosphate buffer (pH 7.4) only and in presence of 300 g/L of the various macromolecular crowding agents.

Sample

a1

τs1 (ps)

a2

τs2 (ps)

(ps)

Δν (cm-1)

/ Δν (ps/cm-1)

0 g/L

0.46

476.5

0.54

12090.5

6757.5

1728.3

3.9

Dextran 6

0.23

659.7

0.77

19421.0

15129.5

1455.5

10.4

Dextran 40

0.18

1000.4

0.82

18785.0

15580.7

1656.8

9.4

Dextran 70

0.21

799.7

0.79

11418.2

9193.8

1546.4

5.9

Ficoll 70

0.07

491.3

0.93

19742.2

18351.6

1819.3

10.1

PEG200

0.02

516.9

0.98

21800.0

21374.3

1637.4

13.0

PEG8000

0.15

649.4

0.85

23938.3

20445.0

1782.8

11.5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 40

Figure Captions: Figure 1: Representative normalized time-resolved emission spectra (TRES) of BADAN labelled HSA in (A) Buffer, (B) Dextran 6 (300 g/L) and (C) Ficoll 70 (300 g/L) (as mentioned in the legend). A few corresponding time resolved decays as a function of the emission wavelengths have been provided below the respective panels. λexc=375 nm. Figure 2: Comparison of the decays of the solvent response function C(t) of domain I (BADANHSA) as a function of the concentrations of various crowders: (A) Dextran 6, (B) Dextran 40, (C) Dextran 70, (D) Ficoll 70, (E) PEG 8000, (F) PEG 200, (G) Glucose and (F) Sucrose. Figure 3: Plot of the average solvation time, (ps) as a function of the concentration of the different crowding agents (as mentioned in the legend) used in this study.


Page 35 of 40


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



Figure 1:

A

0.4 0.2

0.8 0.6

1.0

B

0.4 0.2 0.0

0.0 16000 18000 20000 22000 24000 -1 Wavenumber (cm )

Normalised Counts

0.1

5

10 15 Time (ns)

0.4

20

IRF 410 nm 450 nm 650 nm

0.1

0

Ficoll 70 300 g/L

C

0.2

16000 18000 20000 22000 24000 -1 Wavenumber (cm ) 1 IRF 410 nm 450 nm 650 nm

0.1

0.01

0.01

0.01

0.6

0 ps 500 ps 1000 ps 2000 ps 3000 ps 6000 ps 8000 ps 14000 ps

0.0

1 IRF 410 nm 450 nm 650 nm

0.8

16000 18000 20000 22000 24000 -1 Wavenumber (cm )

1

0

Dextran 6 300 g/L


Normalised Intensity

0.6

1.0

pH 7.4 0 g/L

Normalised Counts

0.8




1.0

Normalised Counts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 36 of 40

5

10 Time (ns)

15

20

0

5

10 15 Time (ns)

20


Page 37 of 40

Dextran 6 A

1.0

0.8

0 g/L 50 g/L 100 g/L 150 g/L 200 g/L 300 g/L

0.8

0.6 0.4

C(t)

1.0

0.2

0.0

0.0 6000

12000 18000 24000 30000 Time (ps)

0

1.0

Dextran 70 C

1.0

0.8

0 g/L 50 g/L 100 g/L 150 g/L 200 g/L 300 g/L

0.8

0.6 0.4 0.2

0 g/L 50 g/L 100 g/L 150 g/L 200 g/L 300 g/L

0.4

0.2

0

Dextran 40 B

0.6

C(t)

C(t)

Figure 2:

C(t)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


6000

Ficoll 70 D 0 g/L 50 g/L 100 g/L 150 g/L 200 g/L 300 g/L

0.6 0.4 0.2

0.0

12000 18000 24000 30000 Time (ps)

0.0 0

6000

12000 18000 24000 30000 Time (ps)

0

6000

12000 18000 24000 30000 Time (ps)



1.0

0 g/L 50 g/L 100 g/L 150 g/L 200 g/L 300 g/L

0.6 0.4 0.2

PEG 200 F

0.8 C(t)

C(t)

1.0

PEG 8000 E

0.8

0 g/L 50 g/L 100 g/L 150 g/L 200 g/L 300 g/L

0.6 0.4 0.2

0.0

0.0 0

1.0

6000

12000 18000 24000 30000 Time (ps)

0

1.0

Glucose G

6000

12000 18000 24000 30000 Time (ps)

Sucrose H

0.8

0.6

0 g/L 100 g/L 200 g/L 300 g/L

0.4 0.2 0.0

C(t)

0.8 C(t)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 40

0.6

0 g/L 100 g/L 200 g/L 300 g/L

0.4 0.2 0.0

0

6000 12000 18000 24000 30000 Time (ps)

0

6000 12000 18000 24000 30000 Time (ps)


Page 39 of 40

Figure 3:

D40 D70 D6 PEG8 PEG200 F70

20000 15000

ps

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


10000 5000 0

50

100 150 200 Crowder (g/L)

250

300



TOC Figure:

large domain motion

Average Solvation Time (ps)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 40

domain motion stalls

Possible evidence of high protein friction

slow solvation

semi dilute

20000

PEG 200

dilute PEG 8

F 70

15000

D6

D 40

10000

highest number density – extensive entanglement

D 70

5000

concentrated

0

50

100 150 200 Crowder (g/L)

250

300


Crowder-Induced Rigidity in a Multidomain Protein: Insights from

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