- 07524805558
- Biophysical Chemistry, Fluorescence Correlation Spectroscopy, Macromolecular crowding, Protein Folding, Protein Aggregation, Deep Eutectic Solvents, and 8 moreSolvation Dynamics, Protein Structure and Dynamics, Perovskites, Heterogeneity, Protein Dynamics, Protein folding/unfolding, Sains Malaysiana, and Fluorescence Microscopyedit
- I am a PhD student in the Chemistry department, IIT Kanpur. My primary research interest includes unraveling and corr... moreI am a PhD student in the Chemistry department, IIT Kanpur. My primary research interest includes unraveling and correlating various protein properties like structure, dynamics, activity and stability of a protein in the presence of macromolecular crowder.edit
Research Interests:
Many current methods for detecting molecular-level heterogeneity are complex and require stringent data analysis, limiting their widespread use. Herein, we propose a novel edge effect, i.e., the shift of excitation spectra at the blue... more
Many current methods for detecting molecular-level heterogeneity are complex and require stringent data analysis, limiting their widespread use. Herein, we propose a novel edge effect, i.e., the shift of excitation spectra
at the blue edge of emission (which we termed as Blue Edge Emission Shift, BEEmS), to perceive the structural heterogeneity. This method is simple and can be easily implemented with commonly available fluorimeter. Red
edge excitation shift (REES), a related technique, is already in use, but like most other known techniques, its usefulness is constrained by its dependence on environmental rigidity. Our method does not suffer from this
drawback significantly. We showed the generality of the proposed method taking various chemically and biologically heterogenous systems including molecular liquid, deep eutectic solvents, organic cavitand, micelle and
protein. BEEmS certainly comes out as a more effective sensor of heterogeneity than REES in certain cases (like denatured protein, hydrophobic deep eutectic solvent and SDS micelle) where solvation time is not sufficiently slow to be detected by REES, but can be measured though BEEmS. Furthermore, unlike most existing techniques, domain-specific heterogeneity of a model multi-domain protein is successfully measured.
at the blue edge of emission (which we termed as Blue Edge Emission Shift, BEEmS), to perceive the structural heterogeneity. This method is simple and can be easily implemented with commonly available fluorimeter. Red
edge excitation shift (REES), a related technique, is already in use, but like most other known techniques, its usefulness is constrained by its dependence on environmental rigidity. Our method does not suffer from this
drawback significantly. We showed the generality of the proposed method taking various chemically and biologically heterogenous systems including molecular liquid, deep eutectic solvents, organic cavitand, micelle and
protein. BEEmS certainly comes out as a more effective sensor of heterogeneity than REES in certain cases (like denatured protein, hydrophobic deep eutectic solvent and SDS micelle) where solvation time is not sufficiently slow to be detected by REES, but can be measured though BEEmS. Furthermore, unlike most existing techniques, domain-specific heterogeneity of a model multi-domain protein is successfully measured.
Research Interests:
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... more
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.
Research Interests:
Deep eutectic solvents (DESs) are new-generation solvents with exquisite and tuneable properties. Molecular-level heterogeneity has been identified as an intriguing feature of such solvents. Herein, we examined the spatio-temporal... more
Deep eutectic solvents (DESs) are new-generation solvents with exquisite and tuneable properties. Molecular-level heterogeneity has been identified as an intriguing feature of such solvents. Herein, we examined the spatio-temporal heterogeneity of a potential non-ionic biocatalytic DES, acetamide/urea/sorbitol (0.5Ac/0.3Ur/0.2Sor), and compared the result with corresponding binary acetamide/urea (0.6Ac/0.4Ur) DES, and another related non-ionic ternary DES (0.55Ac/0.36Ur/0.09PEG). The effect of the addition of a third component on the spatio-temporal heterogeneity of a DES was investigated. The excitation wavelength-dependent emission measurement suggests an induction of spatial heterogeneity in acetamide/urea/sorbitol compared to spatially homogenous acetamide/urea and acetamide/urea/PEG. The dynamic heterogeneity measurements in terms of solvation dynamics, dielectric relaxation, and rotational/translational diffusion indicate a length and timescale dependency. Overall, acetamide/urea/sorbitol is found to be dynamically more heterogenous than the other two related DESs.
Research Interests:
Research Interests:
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... more
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.
Research Interests:
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... more
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.
Research Interests:
Research Interests:
Generally, dynamic heterogeneity in a media is identified by recognizing the viscosity decoupling of the dynamics (i.e., p ≠ 1 in the equation). While dynamically heterogeneous media will show a breakdown from the Stokes-Einstein (SE)... more
Generally, dynamic heterogeneity in a media is identified by recognizing the viscosity decoupling of the dynamics (i.e., p ≠ 1 in the equation). While dynamically heterogeneous media will show a breakdown from the Stokes-Einstein (SE) relationship (p ≠ 1), the vice-versa is not automatically true. One should be cautious in relating viscosity decoupling to dynamic heterogeneity. Herein, we developed a novel analysis for identifying dynamic heterogeneity in a better way. The analysis is based on probing different dynamics in a heterogeneous system from a single measurement and observing their temperature evolution. To prove our concept, we studied temperature dependent translational diffusion of a fluorophore using fluorescence correlation spectroscopy. We used two molecular liquids and two complex deep eutectic solvents in our study. All four solvents show viscosity decoupling, albeit to a different extent. Following the previous knowledge, this could be explained by dynamic heterogeneity in all these solvents. However, our analysis suggests that two molecular liquids, DMF and glycerol, are dynamically homogenous and the two DESs, acetamide/urea and lauricacid/menthol, are dynamically heterogeneous. We believe that the newly developed approach will be a stepforward in determining the dynamic heterogeneity from viscosity decoupling.
Research Interests:
Research Interests:
Vulnerability to atmospheric conditions and their associated toxicity limit the practical/industrial use of perovskites despite their tremendous promise in optoelectronics.
Research Interests:
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:...
Research Interests:
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:... more
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, 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
Research Interests: Structure-function relationships, Fluorescence Resonance Energy Transfer, Protein Stability, Macromolecular crowding, Fluorescence cross-correlation spectroscopy, and 13 moreProtein Structure and Function, FRET, Protein Domain, Secondary Structure, Polyethylene Glycol, Dextran, Enzymatic Activity, Human Serum Albumin (HSA), Excluded Volume Effect, Structure dynamics activity correlation, Single molecular biophysics, Active site dynamics, and Ficoll
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... more
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.
Research Interests:
A more compact structural conformation, higher active-site flexibility, lower viscosity, and higher solvent medium polarity are found to probably facilitate enzymatic activity in a hydrated deep eutectic solvent (DES).
Research Interests: Chemistry, Green Chemistry, Medicine, Ionic Liquids, Biocatalysis, and 10 moreFluorescence Correlation Spectroscopy, Physical sciences, Protein Function, Solvent Effect, CHEMICAL SCIENCES, Deep Eutectic Solvents, Protein Structure and Dynamics, Bromelain, Natural Deep Eutectic Solvent, and Physical chemistry and chemical physics
One of the main differences of intercellular environment compared to the laboratory condition is the presence of macromolecular crowder of various compositions, sizes, and shapes. In this contribution, we have contemplated a systematic... more
One of the main differences of intercellular environment compared to the laboratory condition is the presence of macromolecular crowder of various compositions, sizes, and shapes. In this contribution, we have contemplated a systematic shape dependency of macromolecular crowder on the thermodynamics and microsecond (µs) conformational fluctuation dynamics of protein unfolding taking human serum albumin (HSA) as the model protein and almost 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 stabilisation by dextran-40 and ficoll-70 occurs mainly through stabilising entropic effect, which is somewhat counteracted by destabilising enthalpic effect, in line with what is expected from the traditional interpretation of excluded volume and soft interaction. Surprisingly, the destabilising effect of PEG-35 is not through unfavourable interaction but through destabilising entropic effect, which is opposite to the excluded volume prediction. Our speculation is that the modulation of 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 gets 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 µs conformational dynamics of domain-III of HSA in all the states except in the intermediate state. Mesh-like PEG-35 in addition to hindering the µs conformational dynamics shifts the intermediate state from 40 ⁰C to 30 ⁰C. Overall, our result provides a new insight to decipher the mechanism of crowder induced changes of protein. Through our interpretation, we not only explain the unfavourable entropic contribution but also provides a physical basis to explain entropy-enthalpy compensation.
Research Interests:
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... more
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.
Research Interests:
Highlights: Dynamic nature is a crucial factor for optimal enzyme activity. Due to enormous complexity, it is difficult to directly correlate enzyme dynamics and activity. This question is addressed here taking an industrially crucial... more
Highlights:
Dynamic nature is a crucial factor for optimal enzyme activity.
Due to enormous complexity, it is difficult to directly correlate enzyme dynamics and activity.
This question is addressed here taking an industrially crucial proteolytic enzyme, bromelain.
We contemplated and correlated the structure, dynamics and activity of bromelain in versatile chemicals.
In all the cases, a robust relationship between active-site dynamics and activity emerges.
Microsecond active-site dynamics is probably a key factor for activity.
Abstract
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 nature is a crucial factor for optimal enzyme activity.
Due to enormous complexity, it is difficult to directly correlate enzyme dynamics and activity.
This question is addressed here taking an industrially crucial proteolytic enzyme, bromelain.
We contemplated and correlated the structure, dynamics and activity of bromelain in versatile chemicals.
In all the cases, a robust relationship between active-site dynamics and activity emerges.
Microsecond active-site dynamics is probably a key factor for activity.
Abstract
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.
Research Interests:
Aim: Selective and sensitive visual detection of Cu2+in aqueous solution at PPB level using easily synthesized compound. Background:The search for a chemosensor that can detect Cu2+ is very long owing to the fact that an optimum level... more
Aim: Selective and sensitive visual detection of Cu2+in aqueous solution at PPB level using easily synthesized compound.
Background:The search for a chemosensor that can detect Cu2+ is very long owing to the fact that an optimum level of Cu2+ is required for human health and the recommended amount of Cu2+ in drinking water is set to be 1-2 mgL-1 . Thus, it is very important to detect Cu2+ even at a very low concentration to assess the associated health risks.
Objective: We are still seeking for the easiest, cheapest, fastest and greenest sensor that can selectively, sensitively and accurately detect Cu2+ with lowest detection limit. Our objective of this work is to find one such Cu2+ sensor.
Methods: We have synthesized a quinoline derivative following very easy synthetic procedures and characterize the compound by standard methods. For sensing study, we used steady state absorption and emission spectroscopy.
Results: Our sensor can detect Cu2+ selectively and sensitively in an aqueous solution instantaneously, even in the presence of an excess amount of other salts. The pale-yellow color of the sensor turns red on the addition of Cu2+. There is no interference from other cations and anions. A 2:1 binding mechanism of the ligand with Cu2+ is proposed using Jobs plot with binding constant in the order of 109 M-2. We calculated the LOD to be 18 ppb, which is quite low than what is permissible in drinking water.
Conclusions: We developed a new quinoline based chemosensor following a straightforward synthetic procedure from very cheap starting materials that can detect Cu2+ visually and instantaneously in an aqueous solution with ppb level sensitivity and zero interference from other ions.
Background:The search for a chemosensor that can detect Cu2+ is very long owing to the fact that an optimum level of Cu2+ is required for human health and the recommended amount of Cu2+ in drinking water is set to be 1-2 mgL-1 . Thus, it is very important to detect Cu2+ even at a very low concentration to assess the associated health risks.
Objective: We are still seeking for the easiest, cheapest, fastest and greenest sensor that can selectively, sensitively and accurately detect Cu2+ with lowest detection limit. Our objective of this work is to find one such Cu2+ sensor.
Methods: We have synthesized a quinoline derivative following very easy synthetic procedures and characterize the compound by standard methods. For sensing study, we used steady state absorption and emission spectroscopy.
Results: Our sensor can detect Cu2+ selectively and sensitively in an aqueous solution instantaneously, even in the presence of an excess amount of other salts. The pale-yellow color of the sensor turns red on the addition of Cu2+. There is no interference from other cations and anions. A 2:1 binding mechanism of the ligand with Cu2+ is proposed using Jobs plot with binding constant in the order of 109 M-2. We calculated the LOD to be 18 ppb, which is quite low than what is permissible in drinking water.
Conclusions: We developed a new quinoline based chemosensor following a straightforward synthetic procedure from very cheap starting materials that can detect Cu2+ visually and instantaneously in an aqueous solution with ppb level sensitivity and zero interference from other ions.
Research Interests:
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... more
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...
Research Interests:
A simple analytical model was constructed and validated to understand and predict viscosity decoupling and dynamic heterogeneity in solvent media. We assumed that the Stokes-Einstein relationship is locally satisfied but their spatial... more
A simple analytical model was constructed and validated to understand and predict viscosity decoupling and dynamic heterogeneity in solvent media. We assumed that the Stokes-Einstein relationship is locally satisfied but their spatial average shows a breakdown.
Research Interests:
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... more
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.
Research Interests:
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... more
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.
Research Interests:
Research Interests:
Research Interests:
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... more
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.
Research Interests: Protein Dynamics, Macromolecular crowding, Fluorescence Correlation Spectroscopy, Single-molecule biophysics, Protein folding/unfolding, and 5 moreBiological Macromolecules, Protein Structure and Dynamics, Human Serum Albumin (HSA), Biochemistry and cell biology, and Thermodynamics of protein unfolding
Research Interests:
Research Interests:
Highlights • Under the action of GnHCl monomerization followed by denaturation of monomers take place. • At around ~ 308 K formation of monomers has been noticed. • Upto 338 K reversible formation of small aggregates (diameter ~ 77 Å)... more
Highlights
• Under the action of GnHCl monomerization followed by denaturation of monomers take place.
• At around ~ 308 K formation of monomers has been noticed.
• Upto 338 K reversible formation of small aggregates (diameter ~ 77 Å) takes place.
• Above 338 K irreversible formation of larger aggregates (diameter ~ 117 Å) is observed.
Abstract: β-Lactoglobulin is one of the major components of bovine milk and it remains in a dimeric form under physiological conditions. The present contribution elucidates the structural change of β-lactoglobulin at pH 7.4 under the action of guanidine hydrochloride (GnHCl) and heat at the single molecular level. The only free cysteine (Cys-121) of β-lactoglobulin has been tagged with 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) for this purpose. The dimeric structure of β-lactoglobulin found to undergoes a monomerization prior to the unfolding process upon being subjected to GnHCl. The hydrodynamic diameter of the native dimer, native monomer and the unfolded monomer has been estimated as ~ 55 Å, ~ 29 Å and ~ 37 Å, respectively. The free energy change for the monomerization and denaturation are respectively 1.57 kcal mol− 1 and 8.93 kcal mol− 1. With change in temperature, development of two types of aggregates (small aggregates and large aggregates) was observed, which is triggered by the formation of the monomeric structure of β-lactoglobulin. The hydrodynamic diameters of the smaller and larger aggregates have been estimated to be ~ 77 Å and ~ 117 Å, respectively. The formation of small aggregates turns out to be reversible whereas that of larger aggregates is irreversible. The free energy associated with these two steps are 0.69 kcal mol− 1 and 9.09 kcal mol− 1. Based on the size parameters, the smaller and larger aggregates have been proposed to contain ~twenty and ~sixty monomeric units. It has also been concluded that the monomeric subunits retain their native like secondary structure in these aggregates.
• Under the action of GnHCl monomerization followed by denaturation of monomers take place.
• At around ~ 308 K formation of monomers has been noticed.
• Upto 338 K reversible formation of small aggregates (diameter ~ 77 Å) takes place.
• Above 338 K irreversible formation of larger aggregates (diameter ~ 117 Å) is observed.
Abstract: β-Lactoglobulin is one of the major components of bovine milk and it remains in a dimeric form under physiological conditions. The present contribution elucidates the structural change of β-lactoglobulin at pH 7.4 under the action of guanidine hydrochloride (GnHCl) and heat at the single molecular level. The only free cysteine (Cys-121) of β-lactoglobulin has been tagged with 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) for this purpose. The dimeric structure of β-lactoglobulin found to undergoes a monomerization prior to the unfolding process upon being subjected to GnHCl. The hydrodynamic diameter of the native dimer, native monomer and the unfolded monomer has been estimated as ~ 55 Å, ~ 29 Å and ~ 37 Å, respectively. The free energy change for the monomerization and denaturation are respectively 1.57 kcal mol− 1 and 8.93 kcal mol− 1. With change in temperature, development of two types of aggregates (small aggregates and large aggregates) was observed, which is triggered by the formation of the monomeric structure of β-lactoglobulin. The hydrodynamic diameters of the smaller and larger aggregates have been estimated to be ~ 77 Å and ~ 117 Å, respectively. The formation of small aggregates turns out to be reversible whereas that of larger aggregates is irreversible. The free energy associated with these two steps are 0.69 kcal mol− 1 and 9.09 kcal mol− 1. Based on the size parameters, the smaller and larger aggregates have been proposed to contain ~twenty and ~sixty monomeric units. It has also been concluded that the monomeric subunits retain their native like secondary structure in these aggregates.
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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... more
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...
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The local structural dynamics and denaturation profile of domain-III of HSA against guanidine hydrochloride (GnHCl) and temperature has been studied using a coumarin based solvatochromic fluorescent probe p-nitrophenyl coumarin ester... more
The local structural dynamics and denaturation profile of domain-III of HSA against guanidine hydrochloride (GnHCl) and temperature has been studied using a coumarin based solvatochromic fluorescent probe p-nitrophenyl coumarin ester (NPCE), covalently tagged to Tyr-411 residue. By the steady state, time-resolved and single molecular level fluorescence studies it has been established that the domain-III of HSA is very sensitive to GnHCl but somewhat resistant to temperature and the domain specific unfolding proceeds in an altered way as compared to the overall unfolding of HSA. While the overall denaturation of HSA is a two-state process for both GnHCl and heat, domain-III adopts two intermediate states for GnHCl induced denaturation and one intermediate state for temperature induced denaturation. Fluorescence correlation spectroscopic investigation divulges the conformational dynamics of domain-III of HSA in the native, intermediates and denatured state.
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Deep eutectic solvents (DESs) have emerged as a new kind of solvent and an excellent alternative to ionic liquids, thanks to its simplicity in preparing and availability of a large number of potential constituents to tune the properties.... more
Deep eutectic solvents (DESs) have emerged as a new kind of solvent and an excellent alternative to ionic liquids, thanks to its simplicity in preparing and availability of a large number of potential constituents to tune the properties. However, most of the reported DESs are ionic, and to the best of our knowledge, only a few non-ionic DESs are available, which are liquid at room temperature. Here, we rationally design and report a new ternary non-ionic DES comprising of acetamide, urea, and sorbitol, which is liquid at room temperature. We also report some of the important physical properties like refractive index (n D), sound velocity (u), density (q), and dynamic viscosity (g).