Tracking heterogenous protein aggregation at nanoscale through fluorescence correlation spectroscopy.

Various biophysical techniques have been extensively employed to study protein aggregation due to its significance. Traditionally, these methods detect aggregation at micrometer length scales and micromolar concentrations. However, unlike in vitro, protein aggregation typically occurs at nanomolar concentrations in vivo. Here, using fluorescence correlation spectroscopy (FCS), we captured bromelain aggregation at concentrations as low as ~20 nM, surpassing the detection limit of traditional methods like thioflavin T fluorescence, scattering, and fluorescence microscopy by more than one order of magnitude. Moreover, using thioflavin T fluorescence-based FCS, we have detected larger aggregates at higher bromelain concentrations, which is undetectable in FCS otherwise. Importantly, our study reveals inherent heterogeneity in bromelain aggregation, inaccessible to ensemble-averaged techniques. The presented report may provide a platform for the characterization of premature aggregates at very low protein concentrations, which are thought to be functionally significant species in protein aggregation-induced diseases.

Osmolyte induced protein stabilization: modulation of associated water dynamics might be a key factor.

The mechanism of protein stabilization by osmolytes remains one of the most important and long-standing puzzles. The traditional explanation of osmolyte-induced stability through the preferential exclusion of osmolytes from the protein surface has been seriously challenged by the observations like the concentration-dependent reversal of osmolyte-induced stabilization/destabilization. The more modern explanation of protein stabilization/destabilization by osmolytes considers an indirect effect due to osmolyte-induced distortion of the water structure. It provides a general mechanism, but there are numerous examples of protein-specific effects, i.e., a particular osmolyte might stabilize one protein, but destabilize the other, that could not be rationalized through such an explanation. Herein, we hypothesized that osmolyte-induced modulation of associated water might be a critical factor in controlling protein stability in such a medium. Taking different osmolytes and papain as a protein, we proved that our proposal could explain protein stability in osmolyte media. Stabilizing osmolytes rigidify associated water structures around the protein, whereas destabilizing osmolytes make them flexible. The strong correlation between the stability and the associated water dynamics, and the fact that such dynamics are very much protein specific, established the importance of considering the modulation of associated water structures in explaining the osmolyte-induced stabilization/destabilization of proteins. More interestingly, we took another protein, bromelain, for which a traditionally stabilizing osmolyte, sucrose, acts as a stabilizer at higher concentrations but as a destabilizer at lower concentrations. Our proposal successfully explains such observations, which is probably impossible by any known mechanisms. We believe this report will trigger much research in this area.

Understanding the intricacy of protein in hydrated deep eutectic solvent: Solvation dynamics, conformational fluctuation dynamics, and stability.

Deep eutectic solvents (DESs) are potential biocatalytic media due to their easy preparation, fine-tuneability, biocompatibility, and most importantly, due to their ability to keep protein stable and active. However, there are many unanswered questions and gaps in our knowledge about how proteins behave in these alternate media. Herein, we investigated solvation dynamics, conformational fluctuation dynamics, and stability of human serum albumin (HSA) in 0.5 Acetamide/0.3 Urea/0.2 Sorbitol (0.5Ac/0.3Ur/0.2Sor) DES of varying concentrations to understand the intricacy of protein behaviour in DES. Our result revealed a gradual decrease in the side-chain flexibility and thermal stability of HSA beyond 30 % DES. On the other hand, the associated water dynamics around domain-I of HSA decelerate only marginally with increasing DES content, although viscosity rises considerably. We propose that even though macroscopic solvent properties are altered, a protein feels only an aqueous type of environment in the presence of DES. This is probably the first experimental study to delineate the role of the associated water structure of the enzyme for maintaining its stability inside DES. Although considerable effort is necessary to generalize such claims, it might serve as the basis for understanding why proteins remain stable and active in DES.

Does Viscosity Decoupling Guarantee Dynamic Heterogeneity? A Clue through an Excitation and Emission Wavelength-Dependent Time-Resolved Fluorescence Anisotropy Study.

Traditionally, deviation from Stokes-Einstein-Debye (SED) relation in terms of viscosity dependence of medium dynamics, i.e., τx∝(ηT)p with p ≠ 1, is taken as a signature of dynamic heterogeneity. However, it does not guarantee medium heterogeneity, as the decoupling may also originate from the deviation of the basic assumption of SED. Here, we developed a method to find a stronger relation between viscosity decoupling (p ≠ 1) and dynamic heterogeneity in terms of rotational motion. Our approach exploited the fact that in heterogeneous media, a solvatochromic probe will be solvated to a different extent at different microdomains (subpopulations), and photoselection of these subpopulations can be achieved by excitation or emission wavelength-dependent measurements. We hypothesized that the dynamics of a homogeneous system might show viscosity decoupling, but the extent of decoupling at different excitations (or at different emissions) should not be different. On the other hand, in a heterogeneous medium, this extent of viscosity decoupling (p-value) should be different at different excitations (or at different emissions). As proof of concept, we investigated three versatile solvent media: squalane (viscous molecular liquid), 1-ethyle-3-methylimidazolium ethyl sulfate ionic liquid (IL), and [0.78 acetamide + 0.22 LiNO3] deep eutectic solvent (DES). We found that squalane is homogeneous, although it shows fractional viscosity dependence (p ≠ 1). Interestingly, mild heterogeneity in IL and significant heterogeneity in the DES were observed. Overall, we conclude that the difference in the p-value as a function of excitation (or emission) wavelength-dependent might be a superior way for the detection of dynamic heterogeneity.

Associated Water Dynamics Might Be a Key Factor Affecting Protein Stability in the Crowded Milieu.

Over the past 20 years, the most studied and debated aspect of macromolecular crowding is how it affects protein stability. Traditionally, it is explained by a delicate balance between the stabilizing entropic effect and the stabilizing or destabilizing enthalpic effect. However, this traditional crowding theory cannot explain experimental observations like (i) negative entropic effect and (ii) entropy-enthalpy compensation. Herein, we provide experimental evidence that associated water dynamics plays a crucial role in controlling protein stability in the crowded milieu for the first time. We have correlated the modulation of associated water dynamics with the overall stability and its individual components. We showed that rigid associated water would stabilize the protein through entropy but destabilize it through enthalpy. In contrast, flexible associated water destabilizes the protein through entropy but stabilizes through enthalpy. Consideration of entropic and enthalpic modulation through crowder-induced distortion of associated water successfully explains the negative entropic part and entropy-enthalpy compensation. Furthermore, we argued that the relationship between the associated water structure and protein stability should be better understood by individual entropic and enthalpic components instead of the overall stability. Although a huge effort is necessary to generalize the mechanism, this report provides a unique way of understanding the relationship between protein stability and associated water dynamics, which might be a generic phenomenon and should trigger much research in this area.

Site-specific Heterogeneity of Multi-domain Human Serum Albumin and its Origin: A Red Edge Excitation Shift Study†.

Conformational heterogeneity is a defining characteristic of a protein and is vital in understanding its function and folding landscape. In the present work, we interrogated the presence of conformational heterogeneity in multi-domain human serum albumin in a domain-specific manner using red edge excitation shift (REES) in its native state and also monitored its variation along the unfolding transition. We also looked into the origin of such conformational heterogeneity by varying the solution viscosity. We observed (1) even in the native state, the heterogeneity and dynamics of the side chain exhibit varied behaviors depending on which domain of the multi-domain human serum albumin (HSA) is being examined. (2) When the protein is in the unfolded state, the extent of REES is rendered unimportant since there is a greater quantity of free water present, in addition to the disruption of the protein's structure. (3) While the rigid protein matrix provides the rigidity of domain-I and domain-III, the rigidity of domain-II is provided by water molecules, which indicates that the role of water molecules in providing the rigidity is significant. Overall, our results provide direct evidence of the rigidity and alternate side chain packing arrangement of protein core that varies domain-wise in multi-domain HSA.

Macromolecular crowding: how shape and interaction affect the structure, function, conformational dynamics and relative domain movement of a multi-domain protein.

The cellular environment is crowded by macromolecules of various sizes, shapes, and charges, which modulate protein structure, function and dynamics. Herein, we contemplated the effect of three different macromolecular crowders: dextran-40, Ficoll-70 and PEG-35 on the structure, active-site conformational dynamics, function and relative domain movement of multi-domain human serum albumin (HSA). All the crowders used in this study have zero charges and similar sizes (at least in the dilute region) but different shapes and compositions. Some observations follow the traditional crowding theory. For example, all the crowders increased the α-helicity of HSA and hindered the conformational fluctuation dynamics. However, some observations are not in line with the expectations, such as an increase in the size of HSA with PEG-35 and uncorrelated domain movement of HSA with Ficoll-70 and PEG-35. The relative domain movement is correlated with the activity, suggesting that such moves are essential for protein function. The interaction between HSA and Ficoll-70 is proposed to be hydrophobic in nature. Overall, our results provide a somewhat systematic study of the shape-dependent macromolecular crowding effect on various protein properties and present a possible new insight into the mechanism of macromolecular crowding.

Tracking Wormlike Micelle Formation in Solution: Unique Insight through Fluorescence Correlation Spectroscopic Study.

Although worm-like micelles were invented 35 years ago, its formation pathway remains unclear. Inspired by the fact that a single molecular level experiment could provide meaningful and additional information, especially in a heterogeneous subpopulation, herein, we present a single molecular level study on the formation of wormlike micelles by cetyltrimethylammonium bromide (CTAB) and sodium salicylate (NaSal) in water. Our results indicated a coexistence of normal spherical micelles along with a big wormlike micelle in its formation path. More interestingly, we have two unique insights into the formation mechanism, which are inaccessible in ensemble averaged experiments: (i) at extremely low concentrations of the surfactant, [CTAB]/[NaSal] ∼ 0.06, the wormlike micelle attains the highest size; and (ii) the relative concentration of wormlike micelles is highest when [CTAB]/[NaSal] ∼ 2.

Does Microsecond Active-Site Dynamics Primarily control Proteolytic Activity of Bromelain? Clues from Single Molecular Level Study with a Denaturant, a Stabilizer and a Macromolecular Crowder.

Proteins are dynamic entity with various molecular motions at different timescale and length scale. Molecular motions are crucial for the optimal function of an enzyme. It seems intuitive that these motions are crucial for optimal enzyme activity. However, it is not easy to directly correlate an enzyme's dynamics and activity due to biosystems' enormous complexity. amongst many factors, structure and dynamics are two prime aspects that combinedly control the activity. Therefore, having a direct correlation between protein dynamics and activity is not straightforward. Herein, we observed and correlated the structural, functional, and dynamical responses of an industrially crucial proteolytic enzyme, bromelain with three versatile classes of chemicals: GnHCl (protein denaturant), sucrose (protein stabilizer), and Ficoll-70 (macromolecular crowder). The only free cysteine (Cys-25 at the active-site) of bromelain has been tagged with a cysteine-specific dye to unveil the structural and dynamical changes through various spectroscopic studies both at bulk and at the single molecular level. Proteolytic activity is carried out using casein as the substrate. GnHCl and sucrose shows remarkable structure-dynamics-activity relationships. Interestingly, with Ficoll-70, structure and activity are not correlated. However, microsecond dynamics and activity are beautifully correlated in this case also. Overall, our result demonstrates that bromelain dynamics in the microsecond timescale around the active-site is probably a key factor in controlling its proteolytic activity.

Dynamic heterogeneity and viscosity decoupling: origin and analytical prediction.

The molecular-level structure and dynamics decide the functionality of solvent media. Therefore, a significant amount of effort is being dedicated continually over time in understanding their structural and dynamical features. One intriguing aspect of solvent structure and dynamics is heterogeneity. In these systems, the dynamics follow , where p is the measure of viscosity decoupling. We analytically predicted that in such cases, the Stokes-Einstein relationship is modified to due to microdomain formation, and the second term on the right-hand side leads to viscosity decoupling. We validated our prediction by estimating the p values of a few solvents, and they matched well with the literature. Overall, we believe that our approach gives a simple yet unique physical picture to help us understand the heterogeneity of solvent media.

Correlating Bromelain's activity with its structure and active-site dynamics and the medium's physical properties in a hydrated deep eutectic solvent.

Deep eutectic solvents (DESs) are emerging as new media of choice for biocatalysis due to their environmentally friendly nature, fine-tunability, and potential biocompatibility. This work deciphers the behaviour of bromelain in a ternary DES composed of acetamide, urea, and sorbitol at mole fractions of 0.5, 0.3, and 0.2, respectively (0.5Ac/0.3Ur/0.2Sor), with various degrees of hydration. Bromelain is an essential industrial proteolytic enzyme, and the chosen DES is non-ionic and liquid at room temperature. This provides us with a unique opportunity to contemplate protein behaviour in a non-ionic DES for the very first time. Our results infer that at a low DES concentration (up to 30% V/V DES), bromelain adopts a more compact structural conformation, whereas at higher DES concentrations, it becomes somewhat elongated. The microsecond conformational fluctuation time around the active site of bromelain gradually increases with increasing DES concentration, especially beyond 30% V/V. Interestingly, bromelain retains most of its enzymatic activity in the DES, and at some concentrations, the activity is even higher compared with its native state. Furthermore, we correlate the activity of bromelain with its structure, its active-site dynamics, and the physical properties of the medium. Our results demonstrate that the compact structural conformation and flexibility of the active site of bromelain favour its proteolytic activity. Similarly, a medium with increased polarity and decreased viscosity is favourable for its activity. The presented physical insights into how enzymatic activity depends on the protein structure and dynamics and the physical properties of the medium might provide useful guidelines for the rational design of DESs as biocatalytic media.

Fluorescence correlation spectroscopy as a tool to investigate the directionality of proteolysis.

Enzymatic proteolysis or protein digestion is the fragmentation of protein into smaller peptide units under the action of peptidase enzymes. In this contribution, the directionality of proteolysis has been studied using fluorescence correlation spectroscopy (FCS), taking human serum albumin (HSA) as the model protein and papain, chymotrypsin and trypsin as the model enzymes. Domain-I of HSA has been tagged with tetramethylrhodamine-5-maleimide (TMR) and domain-III with p-nitrophenylcoumarin ester (NPCE) separately and subjected to proteolysis. Following the change in hydrodynamic radius, as monitored by FCS, it has been confirmed that under similar experimental conditions the order of efficiency of digestion is papain > trypsin > chymotrypsin. More interestingly, a faster decrease of hydrodynamic radius was observed when the fluorescence from domain-I was monitored in FCS, compared to that of domain-III. This observation clearly indicates that all these enzymes prefer to start cleaving HSA from domain-I. We assign this preference to the hydrophilic natures of the enzyme active site and domain-I surface. The dependence of the proteolysis on temperature and enzyme concentration has also been studied for papain using the same approach. Reverse-phase HPLC results are found to be in line with the FCS results and validates the applicability of our proposed method.

Partial Viscosity Decoupling of Solute Solvation, Rotation, and Translation Dynamics in Lauric Acid/Menthol Deep Eutectic Solvent: Modulation of Dynamic Heterogeneity with Length Scale.

Deep eutectic solvents (DESs) are new-generation media that can be fine-tuned to have desired properties circumventing economic and environmental issues. Typically, these are ionic, and only recently, nonionic DESs, having interesting properties, are being explored. In this report, we examined the structure and dynamics of a nonionic lauric acid/menthol (LA/Men) DES through steady-state emission, solvation dynamics, time-resolved fluorescence anisotropy, and translational diffusion dynamics. The zero shift in the emission spectra of coumarin 153 (a solvatochromic dye) as a function of the excitation wavelength suggests that LA/Men DES is spatially homogenous. Decoupling (p = 0.63) of the average solvation time, ⟨τs⟩, from medium viscosity suggests the presence of mild dynamic heterogeneity in the system. Rotational time, ⟨τr⟩, which reflects the nature of the first solvation shell, shows little decoupling (p = 0.81), suggesting it to be fairly dynamically homogeneous at a shorter length scale. An Arrhenius-type analysis also proves that rotation is mainly controlled by medium viscosity. Translational diffusion time, ⟨τD⟩, which provides information at a larger length scale, is strongly decoupled from medium viscosity (p = 0.29). This indicates that at a larger length scale, the DES is quite dynamically heterogeneous. The slow component of solvation time, which is believed to originate at a larger length scale, correlates well with the translational diffusion timescale having similar activation energies. This suggests that their origin is same. Expectedly, for the long component of solvation time, the decoupling is quite strong (p = 0.30). Overall, our result demonstrates the structure and dynamics of the nonionic LA/Men DES, and the existence of length scale-dependent heterogeneity has been proposed.

Shape-Dependent Macromolecular Crowding on the Thermodynamics and Microsecond Conformational Dynamics of Protein Unfolding Revealed at the Single-Molecule Level.

One of the main differences in the intercellular environment compared to the laboratory condition is the presence of macromolecular crowders of various compositions, sizes, and shapes. In this article, we have contemplated a systematic shape dependency of macromolecular crowders on the thermodynamics and microsecond conformational fluctuation dynamics of protein unfolding by taking human serum albumin (HSA) as the model protein and similar-sized crowders, namely, dextran-40, ficoll-70, and PEG-35 as macromolecular crowders of different shapes, to mimic the cell environment. We observed that dextran-40 and ficoll-70 counteract the thermal denaturation and PEG-35 assists it. A complete thermodynamic analysis suggests that the stabilization by dextran-40 and ficoll-70 occurs mainly through stabilizing entropic effect, which is somewhat counteracted by the destabilizing enthalpic effect, in line with what is expected from the traditional interpretation of excluded volume and soft interaction. Surprisingly, the destabilizing effect of PEG-35 is not through unfavorable interaction but through a destabilizing entropic effect, which is opposite to the excluded volume prediction. Our speculation is that the modulation of the associated water structure due to crowder-induced distortion plays a crucial role in modulating the entropic component. Moreover, while a two-state model can approximate the overall thermal denaturation of HSA in the absence and presence of various crowders, the thermal denaturation profile of domain III of HSA involves a distinct intermediate state. The active-site dynamics of HSA are altered significantly in the presence of all the three differently shaped crowders used in the study. Rod-shaped dextran-40 and spherical ficoll-70 hinder the microsecond conformational dynamics of domain III of HSA in all states except the intermediate state. Mesh-like PEG-35 in addition to hindering the microsecond conformational dynamics shifts the intermediate state from 40 to 30 °C. Overall, our results provide new insight into deciphering the mechanism of crowder-induced changes in protein. Through our interpretation, we not only explain the unfavorable entropic contribution but also provide a physical basis to explain the entropy-enthalpy compensation.

Reversible Ultra-Slow Crystal Growth of Mixed Lead Bismuth Perovskite Nanocrystals: The Presence of Dynamic Capping.

An ultra-slow crystal growth over a period of 24 h of a newly synthesized CH3 NH3 Pb1/2 Bi1/3 I3 perovskite (MPBI) nanocrystal in non-polar toluene medium is reported here. From several spectroscopic techniques as well as from TEM analysis we found that the size of nanocrystals changes continuously with time, in spite of being capped by the ligands. Using a single molecular spectroscopic technique, we also found that this size change is not due to the stacking of nanocrystals but due to crystal growth. The notable temperature dependence and reversible nature of the nanocrystals growth is explained by the dynamic nature of the capping. The observed temperature-dependent ultra-slow growth is believed to be a pragmatic step towards controlling the size of perovskite NC in a systematic manner.

Size-dependent macromolecular crowding effect on the thermodynamics of protein unfolding revealed at the single molecular level.

The real biological environment involves a high degree of complexity and the macromolecular crowder is the best candidate to somewhat mimic this. In this contribution, we have used two different sized dextrans as model crowders and human serum albumin (HSA) as a model protein to decipher how the thermal stability of protein is modulated inside the crowded milieu and also to understand the effect of the size of the crowders. In our previous report (Biochemistry 2018, 57, 6078-6089) we have proposed the presence of some interaction between dextran-6 and HSA, which are probably not present between the larger dextrans and HSA. Complete thermodynamic analysis of thermal denaturation profile of HSA suggests that small crowders increase protein stability mainly via the enthalpy of denaturation while larger crowders increase stability primarily through entropy. Further, the active site dynamics is altered significantly in the presence of larger dextran-40, but not by smaller dextran-6. Surprisingly, the dynamics of the more compact intermediate state does not get modified by the crowders. Overall, our result indicates that biomacromolecules of similar chemical composition and shape may exert their effect not only by different extent but also by a different mechanism, owing to their different sizes.

ß-carboline-based turn-on fluorescence chemosensor for quantitative detection of fluoride at PPB level.

A novel ß-carboline-based chemosensor, having an acidic NH proton that leads to fluoride-induced deprotonation involving a vivid color change from colorless to yellow is described. The absorption spectrum of the chemosensor in acetonitrile has a peak at 375 nm, which changes to 428 nm with the gradual addition of only fluoride in the solution with a clear isosbestic points at 357 nm and 392 nm. More interestingly, the chemosensor gives a turn-on type of fluorescence at 554 nm in the presence of fluoride. Further it was found that the sensor is highly selective towards fluoride over other anions including chloride, bromide, iodide, nitrate, borate, perchlorate and can quantitatively detect fluoride at ppb level with a limit of detection of 0.02 mg/ L or 20 ppb. The chemosensor was successfully demonstrated to assess the fluoride concentration in the tap water.

Spectroscopic Insight on Ethanol-Induced Aggregation of Papain.

In this contribution, the structural and dynamic changes occurring to papain in ethanol-water binary solvent mixtures have been investigated and compared with its denatured state. Steady-state fluorescence, solvation dynamics, time-resolved rotational anisotropy, circular dichroism (CD), and single molecular-level fluorescence correlation spectroscopic (FCS) studies were performed for this purpose. In ethanol-water mixtures with XEtOH = 0.6, N-(7-dimethylamino-4-methylcoumarin-3-yl)iodoacetamide (DACIA)-tagged papain was found to undergo a blue shift of 12 nm, while in the presence of 5 M GnHCl, a red shift of 5 nm was observed. Solvation dynamics of the system was also found to be different in the presence of these external agents. In ethanol-water mixtures, the average solvation time was found to increase almost 2-fold as compared to that in water, while in the presence of GnHCl, only a marginal increase could be observed. These changes of DACIA-tagged papain in ethanol-water mixtures are attributed to the aggregation of the protein in the presence of ethanol. The residual anisotropy was found to increase 14-fold, and the rotational time component corresponding to the rotation of the probe molecule was found to increase by 4-fold in the ethanol-water mixture which also gives a notion of the papain aggregation. Atomic force microscopy (AFM) confirms this aggregate formation, which is also quantified by the FCS study. The hydrodynamic radius of the protein aggregates in ethanol-water mixtures was calculated to be ∼155 Å as compared to the corresponding value of 18.4 Å in the case of native monomer papain. Also, it confirmed that the aggregate formation takes place even in the nanomolar concentration of papain. Analysis of circular dichroism spectra of papain showed that an increase in the ß-sheet content of papain at the expense of α-helix and the random coil with an increase of the ethanol mole fraction may be responsible for this aggregation process.

Domain-Specific Stabilization of Structural and Dynamic Responses of Human Serum Albumin by Sucrose.

BACKGROUND: Human Serum Albumin (HSA) is the most abundant protein present in human blood plasma. It is a large multi-domain protein with 585 amino acid residues. Due to its importance in human body, studies on the interaction of HSA with different external agent is of vital interest. The denaturation and renaturation of HSA in presence of external agents are of particular interest as they affect the biological activity of the protein. OBJECTIVE: The objective of this work is to study the domain-specific and overall structural and dynamical changes occurring to HSA in the presence of a denaturing agent, urea and a renaturing agent, sucrose. METHODS: In order to carry out the domain-specific studies, HSA has been tagged using N-(7- dimethylamino-4-methylcoumarin-3-yl) iodoacetamide (DACIA) at Cys-34 of domain-I and pnitrophenyl coumarin ester (NPCE) at Tyr-411 site in domain-III, separately. Steady-state absorption, emission and solvation dynamic measurements have been carried out in order to monitor the domain-specific alteration of HSA caused by the external agents. The overall structural change of HSA have been monitored using circular dichroism spectroscopy. RESULTS: The α-helicity of HSA was found to decrease from 65% to 11% in presence of urea and was found to further increase to 25% when sucrose is added, manifesting the denaturing and renaturing effects of urea and sucrose, respectively. The steady state studies show that domain-III is more labile towards denaturation as compared to domain-I. The presence of an intermediate state is observed during the denaturation process. The stabilization of this intermediate state in presence of sucrose is attributed as the reason for the stabilization of HSA by sucrose. From solvation dynamics studies, it could be seen that the solvation time of DACIA inside domain-I of HSA decreases and increases regularly with increasing concentrations of urea and sucrose, respectively, while in the case of NPCE-tagged domain-III, the effect of sucrose on solvation time is evident only at high concentrations of urea. CONCLUSION: The denaturing and renaturing effects of urea and sucrose could be clearly seen from the steady state studies and circular dichroism spectroscopy measurements. A regular change in solvation time could only be observed in the case of domain-I and not in domain-III. The results indicate that the renaturing effect of sucrose on domain-III is not very evident when protein is in its native state, but is evident in when the protein is denatured.

Structural, Functional, and Dynamical Responses of a Protein in a Restricted Environment Imposed by Macromolecular Crowding.

The intercellular environment is known to be very different from the environment where most of the elementary biological processes are studied in the laboratory. As a result, there was a considerable effort on cell mimicking either by confinement or by introducing macromolecular crowding. In the present study, dextran of varying sizes has been used to crowd the environment of a protein, human serum albumin (HSA), and its structure, dynamics, and activity were studied as a function of crowder concentration. By employing bulk and single molecular level spectroscopic measurements, we elucidate the overall structure and local microsecond dynamics of HSA. Further, we have attempted to correlate these structural changes with its activity.