Bhaswati Sengupta

Conformational fluctuation dynamics of domain I of human serum albumin in the course of chemically and thermally induced unfolding using fluorescence correlation spectroscopy.

The present study elucidates the involvement of conformational fluctuation dynamics during chemically and thermally induced unfolding of human serum albumin (HSA) by fluorescence correlation spectroscopic (FCS) study, time-resolved fluorescence measurements, and circular dichroism (CD) spectroscopic methods. Two fluorescent probes, tetramethylrhodamine-5-maleimide (TMR) and N-(7-dimethylamino-4-methylcoumarin-3-yl) iodoacetamide (DACIA) were used to selectively label the domain I of HSA through the reaction with cys-34 for these studies. The guanidine hydrochloride (GnHCl) induced global structural change of HSA is monitored through its hydrodynamic radius (r(H)) and CD response, which is found to be two step in nature. In FCS experiment, along with the diffusion time component we have observed an exponential relaxation time component (τ(R)) that has been ascribed to the concerted chain dynamics of HSA. Unlike in the global structural change, we found that the τ(R) value changes in a different manner in the course of the unfolding. The dependence of τ(R) on the concentration of GnHCl was best fitted with a four state model, indicating the involvement of two intermediate states during the unfolding process, which were not observed through the CD response and r(H) data. The fluorescence lifetime measurement also supports our observation of intermediate states during the unfolding of HSA. However, no such intermediate states were observed during thermally induced unfolding of HSA.

Elucidation of the local dynamics of domain-III of human serum albumin over the ps–μs time regime using a new fluorescent label

The ps-μs dynamics of domain-III of human serum albumin (HSA) has been investigated using a new fluorescent marker selectively labeled to the Tyr-411 residue. The location of the marker has been confirmed using Förster resonance energy transfer (FRET) study. Steady state, time-resolved and single molecular level fluorescence techniques have been employed to understand the dynamics within the domain-III of HSA. It is found that solvent reorganization dynamics in domain-III is 1.7 times faster than that in domain-I. The timescale of the local rotational dynamics of domain-III is found to be 2.3 times faster than that of domain-I. Fluorescence correlation spectroscopic experiments reveal that domain-III of HSA has more conformational flexibility than domain-I. Together, the results deliver useful details of the local environment around the domain-III of HSA, which have not been explored earlier, mainly because of a lack of a suitable fluorescent marker for domain-III. The newly synthesized probe serves well as a site specific fluorescent marker for HSA, and can be used for further investigation of the ligand binding properties and enzymatic activity of domain-III of HSA.

Effect of sucrose on chemically and thermally induced unfolding of domain-I of human serum albumin: Solvation dynamics and fluorescence anisotropy study

The present study is devoted to understand the effect of sucrose on the hydration dynamics and rotational relaxation dynamics within the domain-I of HSA during chemically as well as thermally induced unfolding. It has been observed that the average solvation time become slower in the presence of sucrose for the lower concentrations of GnHCl, however at higher concentrations of GnHCl the effect of sucrose is almost negligible. From the time resolved fluorescence anisotropy it has been observed that in the lower concentration region of GnHCl the sucrose induced stabilization is small as compared to the higher concentrations of GnHCl. We have concluded that the hydration dynamics plays an important role in the sucrose induced stabilization process at the low concentration region; whereas environmental restriction is responsible at the higher concentration of GnHCl. However, we have observed a negligible stabilizing effect of sucrose towards the temperature induced unfolding.

Dynamical response in methanol–chloroform binary solvent mixture over fs–μs time regime

ABSTRACT The present study elucidates the extended nature of the weak interaction between methanol and chloroform in its mixture using steady-state, ultrafast and single molecule spectroscopic methods. UV–vis absorption spectroscopy using a solvatochromic dye indicates a synergistic solvation in the methanol–chloroform binary solvent mixture, which causes the solvatochromic dye to sense increased polarity compared to the bulk counterparts. Such synergism was not observed in the emission study and is explained by the weak nature of the interaction between methanol and chloroform. Fluorescence anisotropy and single molecule fluorescence spectroscopy are employed to understand the nature of such weak interaction on femtosecond to microsecond time scale. Retardation of both the rotational and translational diffusion of the reporter molecule indicates that the observed weak interaction is extended over large dimension in the condensed phase.

Single Molecular Level Probing of Structure and Dynamics of Papain Under Denaturation

INTRODUCTION Papain is a cysteine protease enzyme present in papaya and known to help in digesting peptide. Thus the structure and function of the active site of papain is of interest. OBJECTIVE The objective of present study is to unveil the overall structural transformation and the local structural change around the active site of papain as a function of chemical denaturant. METHODS Papain has been tagged at Cys-25 with a thiol specific fluorescence probe N-(7- dimethylamino-4-methylcoumarin-3-yl) iodoacetamide (DACIA). Guanidine hydrochloride (GnHCl) has been used as the chemical denaturant. Steady state, time-resolved, and single molecular level fluorescence techniques was applied to map the change in the local environment. RESULTS It is found that papain undergoes a two-step denaturation in the presence of GnHCl. Fluorescence correlation spectroscopic (FCS) data indicate that the size (hydrodynamic diameter) of native papain is ~36.8 Å, which steadily increases to ~53 Å in the presence of 6M GnHCl. FCS study also reveals that the conformational fluctuation time of papain is 6.3 µs in its native state, which decreased to 2.7 µs in the presence of 0.75 M GnHCl. Upon further increase in GnHCl concentration the conformational fluctuation time increase monotonically till 6 M GnHCl, where the time constant is measured as 14 µs. On the other hand, the measurement of ellipticity, hence the helical structure, by circular dichroism spectroscopy is found to be incapable to capture such structural transformation. CONCLUSION It is concluded that in the presence of small amount of GnHCl the active site of papain takes up a more compact structure (although the overall size increases) than in the native state, which has been designated as the intermediate state.

Elucidation of μs dynamics of domain-III of human serum albumin during the chemical and thermal unfolding: A fluorescence correlation spectroscopic investigation

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.

Direct Observation of Intermediate State(s) in the Mechanistic Investigation of Domain Specific Protein-Surfactant Interaction

INTRODUCTION Interaction of surfactants with proteins can decipher important information regarding the stability and behavior of proteins. For multi-domain proteins, these interactions vary domain wise and these details are crucial in understanding the contribution of different domains of the protein in its overall activity. OBJECTIVE The objective of the present work is to study the interaction of surfactants with domain III of Human Serum Albumin (HSA) and to compare the same with the global interaction. METHODS Interaction of the anionic Sodium Dodecyl Sulphate (SDS) and the Cationic Cetyltrimethylammonium Bromide (CTAB) surfactants with domain III of Human Serum Albumin (HSA) has been studied using 8-Anilino-1-Naphthalene-Sulphonate (ANS) as a fluorescent marker. Circular Dichroism (CD) spectroscopy has been used to study the protein-surfactant interaction for the overall protein. RESULTS SDS is found to interact sequentially with domain III of HSA having two detectable intermediate states in the binding process. In case of CTAB, we have observed only one intermediate state for its interaction with domain III. Although Quantum yield measurement can reflect the presence of such intermediate state, the overall conformational change of the HSA on addition of surfactants, studied by Circular Dichroism (CD) spectroscopy, and the ANS-Trp distance measurement by FRET could not resolve the presence of such intermediate states. The esterase activity of HSA in presence of different amount of surfactants is also in accordance with our above observation. CONCLUSION The interaction of both the surfactants with HSA is found to be sequential in nature. The most important conclusion revealed from our study is that the nature of protein-surfactant interaction is not same throughout the entire protein. Our study reveals that different parts of the multi-domain HSA have different affinity to the surfactant molecules.

Detail Modes of Binding Assessed by Bulk and Single Molecular Level Fluorescence, MD Simulation, and Its Temperature Dependence: Coumarin 152 with Human Serum Albumin Revisited

This study describes the effect of temperature on binding parameters as well as mode of binding between coumarin 152 (C152) and human serum albumin (HSA). Site marker competitive experiment, Forster resonance energy transfer (FRET), and molecular docking study show that C152 binds to the digitoxin binding site in domain III of HSA. The binding constant calculated at the single molecular level experiment matches well with the ensemble average measurement, which indicate that at even low dye concentration the binding reaction proceeds with equal probability. Further, FRET and molecular dynamic simulation confirm that the binding location of C152 is independent on temperature (278 K to 323 K). It has been revealed that the binding affinity of C152 to HSA was almost unaffected until 298 K; afterward, it decreases continuously on increasing temperature forming two distinct regions. Thermodynamic parameters for association indicate that strong electrostatic and hydrophobic interactions are operational at lower temperature region, whereas hydrogen bonding predominates at higher temperate region.

Calmidazolium Chloride and Its Complex with Serum Albumin Prevent Huntingtin Exon1 Aggregation

Huntingtons disease (HD) is a genetic disorder caused by a CAG expansion mutation in Huntingtin gene leading to polyglutamine (polyQ) expansion in the N-terminus side of Huntingtin (Httex1) protein. Neurodegeneration in HD is linked to aggregates formed by Httex1 bearing an expanded polyQ. Initiation and elongation steps of Httex1 aggregation are potential target steps for the discovery of therapeutic molecules for HD, which is currently untreatable. Here we report Httex1 aggregation inhibition by calmidazolium chloride (CLC) by acting on the initial aggregation event. Because it is hydrophobic, CLC was adsorbed to the vial surface and could not sustain an inhibition effect for a longer duration. The use of bovine serum albumin (BSA) prevented CLC adsorption by forming a BSA-CLC complex. This complex showed improved Httex1 aggregation inhibition by interacting with the aggregation initiator, the NT17 part of Httex1. Furthermore, biocompatible CLC-loaded BSA nanoparticles were made which reduced the polyQ aggregates in HD-150Q cells.

Monomerization and aggregation of β-Lactoglobulin under adverse condition: A fluorescence correlation spectroscopic investigation

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 pH7.4 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 ~55Å, ~29Å and ~37Å, respectively. The free energy change for the monomerization and denaturation are respectively 1.57kcalmol-1 and 8.93kcalmol-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 ~77Å and ~117Å, 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 0.69kcalmol-1 and 9.09kcalmol-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.