Connecting the Dots: Macromolecular Crowding and Protein Aggregation.

Proteins are one of the dynamic macromolecules that play a significant role in many physiologically important processes to sustain life on the earth. Proteins need to be properly folded into their active conformation to perform their function. Alteration in the protein folding process may lead to the formation of misfolded conformers. Accumulation of these misfolded conformers can result in the formation of protein aggregates which are attributed to many human pathological conditions including neurodegeneration, cataract, neuromuscular disorders, and diabetes. Living cells naturally have heterogeneous crowding environments with different concentrations of various biomolecules. Macromolecular crowding condition has been found to alter the protein conformation. Here in this review, we tried to show the relation between macromolecular crowding, protein aggregation, and its consequences.

The modulation of structural stability of horseradish peroxidase as a consequence of macromolecular crowding.

Macromolecular crowding plays an inevitable role in all biological processes influencing association, conformation, and other characteristics of proteins. Present study is based on the effect of macromolecular crowding on structure of horseradish peroxidase (HRP) enzyme. Concentration-dependent conformational changes induced by crowding agents, dextran 70 and polyethylene glycol (PEG)-4000, were monitored employing a range of biophysical techniques. The intrinsic fluorescence spectra showed transition of protein from native to unfolded state. Marked increase in 8-Anilino-1-naphthalene-sulphonoic acid and Thioflavin T fluorescence indicated presence of non-native moieties with 80 mg/mL dextran. Enhanced absorbance in turbidity, Soret, and Congo red in corroboration with scattering intensity at 350nm results revealed incidence of HRP aggregates. A new peak around 218 nm in CD spectra pointed towards change in secondary structure towards ß-sheets. Significant loss of enzyme activity upon structural disruption was seen. Comet assay demonstrated DNA damage and genotoxic nature of HRP aggregates, supporting spectroscopic, and fluorescence results. The normalized results were obtained with 120 mg/mL PEG-4000 close to that of native HRP implying no disruptive effect on structure. It can be hypothesized that macromolecular crowding is a vital element, which can have diverse effects. In this study, dextran 70 was observed to have pro-aggregatory effect while enhanced stability of native enzyme was witnessed with PEG. Hence, it can be stated that PEG has potentially better crowder as it helps retain the native enzyme structure. Routine addition of crowding agents is recommended if biological molecules are to be studied under more physiologically appropriate environments.

Refolding of Hemoglobin Under Macromolecular Confinement: Impersonating In Vivo Volume Exclusion.

Biomacromolecules evolve and function inside the cell under crowded conditions. The effect of macromolecular crowding and confinement on nature and interactions of biomacromolecules cannot be ruled out. This study demonstrates the effect of volume exclusion due to macromolecular crowding on refolding rate of Gn-HCl induced unfolded hemoglobin. The in vivo like crowding milieu was created using dextran 70. Unfolding of Hb was followed by the absorbance at 280 nm and intrinsic fluorescence intensity along with a bathochromic shift that shows the destabilization of Hb in the presence of the denaturing agent. This was supported by a decrease in soret absorbance, increased hydrodynamic radii and loss in secondary structure, evidenced from dynamic light scattering and circular dichroism experiments respectively. Refolding process of Hb was followed by an increase in soret absorbance, decrease in intrinsic fluorescence intensity with a hypsochromic shift, decreased hydrodynamic radii and gain in secondary structural content. The results revealed that the effect of confinement and volume exclusion is insignificant on the process of Hb refolding.

Aggregation of intrinsically disordered fibrinogen as the influence of backbone conformation.

Fib having intrinsically disordered αC domains is involved in coagulation cascade and thrombosis. Fib molecules forms prefibrillar oligomers at 30%, and associate in 40 and 50% TFE to proceed α to ß transition, suggesting the formation of an intermolecular ß-structure. AFM images confirmed the nature of Fib aggregates at 40 and 50% TFE to be prefibrillar and fibrillar respectively. These aggregates possess high thioflavin T fluorescence with a shifted Congo red absorbance. Kinetics of Fib aggregation data at 50% TFE supports nucleation-dependent polymerization mechanism. At 60 and 70% TFE, no aggregation was observed. The inhibition of protein aggregation appears due to weakening of the hydrophobic interactions that were initially stabilizing the intermolecular ß-sheet structure in the protein aggregation. The loss of hydrophobic contacts seems to favor the formation of intramolecular hydrogen bonds over intermolecular hydrogen bonds leading to helix formation. To conclude, protein aggregation is accompanied by the formation of ß-sheet conformation, and induction of non-native helical segments in the protein inhibits aggregation. The discrepancy of the secondary structures on aggregation is proposed to stem from the disparity in the nature of the hydrogen bonds and packing of hydrophobic residues of the side chains in the ß-sheet and α-helix conformation.

Aggregation as a consequence of glycation: insight into the pathogenesis of arthritis.

Advanced glycation end products (AGEs) as a result of Maillard reaction are currently at the heart of the pathogenesis of several diseases and hence are the objective of numerous investigations. Glycation of proteins has been an implication in long-term complications. Collagen is the most abundant protein in the human body. The purpose of this study was to monitor and characterize the oligomeric aggregates and AGEs of human collagen on addition of glyoxal using ultraviolet, fluorescence, circular dichroism (CD) spectroscopy, docking studies, ITC, and microscopy. Collagen was incubated for varying time periods up to 21 days with three different concentrations (5, 20, and 40 mM) of glyoxal. Collagen exists as molten globule at day 6, evident from native-like secondary structure, altered tryptophan, and high ANS fluorescence due to surface-exposed hydrophobic residues. Glycated collagen as AGEs and aggregates was observed at day 18 and 21, respectively. Formation of AGE and aggregates were confirmed by UV and fluorescence spectroscopy. The obtained AGEs were characterized with respect to the extent of side chain modifications (lysine and arginine) forming the Schiff base, the carboxymethyl lysine, and carbonyl content. Non-tryptophan fluorescence for AGEs was also monitored as the emission peak at 400 and 440 nm, respectively. SEM and TEM confirmed the oligomeric nature of aggregates. Glyoxal at 40 mM shows maximum alterations in protein structure followed by 20 and 5 mM concentration. In the present paper, we propose that a high concentration of glyoxal for a prolonged time results in the formation of harmful aggregates and AGEs.

Exploring the Transition of Human α-Synuclein from Native to the Fibrillar State: Insights into the Pathogenesis of Parkinson's Disease.

The etiology of Parkinson's disease involves the interplay between the environmental and genetic factors. Here in this study human α-synuclein upon exposure to 100 µM pendimethalin for 12 h in vitro passes through a partially folded state which proceeds to the aggregated state and terminally ends in the fibrillar phase. Variations in the ANS fluorescence intensities led to the detection of intermediate and aggregated states at 6 and 10 h respectively. Far-UV CD analysis depicted significant α-helical content for intermediate state at 6 h in presence of 100 µM pendimethalin. Further increasing the incubation time to 12 h resulted in a predominant ß-sheet content which was confirmed to be fibrillar by TEM. Turbidity, Rayleigh scattering analysis, Congo red assay and ThT measurements supported the TEM data i.e. the formation of fibrillar structure of human α-synuclein upon 12 h incubation. Thus, our observation could suggest a possible underlying molecular basis for Parkinson's disease. Graphical Abstract Schematic elucidation of the factors involved in the fibrillation of α-Synuclein during Parkinson's pathogenesis.

Analysing Cytochrome c Aggregation and Fibrillation upon Interaction with Acetonitrile: an in Vitro Study.

The propensity of native state to form aggregated and fibrillar assemblies is a hallmark of amyloidosis. Our study was focused at analyzing the aggregation and fibrillation tendency of cytochrome c in presence of an organic solvent i.e. acetonitrile. In vitro analysis revealed that the interaction of cytochrome c with acetonitrile facilitated the oligomerization of cytochrome c via the passage through an intermediate state which was obtained at 20 % v/v concentration of acetonitrile featured by a sharp hike in the ANS fluorescence intensity with a blue shift of 20 nm compared to the native state. Oligomers and fibrils were formed at 40 and 50 % v/v concentration respectively as indicated by a significant hike in the ThT fluorescence intensity, red shift of 55 nm in congo red binding assay and an increase in absorbance at 350 nm. They possess ß-sheet structure as evident from appearance of peak at 217 nm. Finally, authenticity of oligomeric and fibrillar species was confirmed by TEM imaging which revealed bead like aggregates and a meshwork of thread like fibrils respectively. It could be suggested that the fibrillation of bovine cytchrome c could serve as a model protein to unravel the general aggregation and fibrillation pattern of heme proteins. Graphical abstract ᅟ.

Deciphering aggregates, prefibrillar oligomers and protofibrils of cytochrome c.

Aggregation of protein into insoluble intracellular complexes and inclusion bodies underlies the pathogenesis of human neurodegenerative diseases. Importance of cytochrome c (cyt c) arises from its involvement in apoptosis, sequence homology and for studying molecular evolution. A systemic investigation of polyethylene glycol (PEG) and trifluoroethanol (TFE) on the conformational stability of cyt c as a model hemeprotein was made using multi-methodological approach. Cyt c exists as molten globule (MG) at 60% PEG-400 and 40% TFE as confirmed by far-UV CD, attenuated total reflection Fourier transform infrared spectroscopy, Trp environment, 8-anilino-1-naphthalene-sulfonic acid (ANS) binding and blue shift in the soret band. Q-band splitting in MG states specifies conformational changes in the hydrophobic heme-binding pocket. Aggregates were detected at 90% PEG-400 and 50% TFE as confirmed by increase thioflavin T and ANS fluorescence and shift in Congo red absorbance. Detection of prefibrils and protofibrils at 90% PEG-400 and 50% TFE was possible after 72-h incubation. Single cell gel electrophoresis of prefibrils and protofibrils showed DNA damage confirming their toxicity and potential health hazards. Scanning electron microscopy and XRD analysis confirmed prefibrillar oligomers and protofibrils of cyt c.

Bovine serum albumin prevents human hemoglobin aggregation and retains its chaperone-like activity.

This study investigates the ability of bovine serum albumin (BSA) to act as an extracellular chaperone (EC) on human hemoglobin (Hb) at a pH of 7.4. The best temperature for studying this behavior was determined by analyzing Hb's aggregation kinetics at multiple temperatures. 55 °C was chosen as the optimal temperature for forming Hb amyloids. BSA was then tested at various concentrations (20-100 µM) to assess its chaperone-like activity on Hb at 55 °C. At a concentration of 100 µM, BSA exhibits chaperone-like activity with a client protein:BSA ratio of 1:10. The high ratio implies that the chaperone activity of BSA is favored by the effects of macromolecular crowding. The results showed that BSA has the potential to inhibit Hb's dissociation into alpha and beta subunits and protein aggregation by inhibiting secondary nucleation. BSA also causes the depolymerization of fibrils over time. The results were validated using molecular docking and all-atom molecular dynamics simulations. MD analysis such as RMSD, RMSF, Rg, SASA, Hydrogen bond, PCA, Free energy landscape (FEL) revealed that the stability of hemoglobin is greater when it is bound to BSA compared to unbound state. The study suggests that BSA can potentially bind to Hb dimers and reduce excitonic interactions, which reduces Hb aggregation. These results are consistent with the aggregation kinetics experiments.Communicated by Ramaswamy H. Sarma.

Methotrexate for Drug Repurposing as an Anti-Aggregatory Agent to Mercuric Treated α-Chymotrypsinogen-A.

Protein aggregation is related to numerous pathological conditions like Alzheimer's and Parkinson's disease. In our study, we have shown that an already existing FDA-approved drug; methotrexate (MTX) can be reprofiled on preformed α-chymotrypsinogen A (α-Cgn A) aggregates. The zymogen showed formation of aggregates upon interaction with mercuric ions, with increasing concentration of Hg2Cl2 (0-150 µM). The hike in ThT and ANS fluorescence concomitant with blue shift, bathochromic shift and the hyperchromic effect in the CR absorbance, RLS and turbidity measurements, substantiate the zymogen ß-rich aggregate formation. The secondary structural alterations of α- Cgn A as analyzed by CD measurements, FTIR and Raman spectra showed the transformation of native ß-barrel conformation to ß-inter-molecular rich aggregates. The native α- Cgn A have about 30% α-helical content which was found to be about 3% in presence of mercuric ions suggesting the formation of aggregates. The amorphous aggregates were visualized by SEM. On incubation of Hg2Cl2 treated α- Cgn A with increasing concentration of the MTX resulted in reversing aggregates to the native-like structure. These results were supported by remarkable decrease in ThT and ANS fluorescence intensities and CR absorbance and also consistent with CD, FTIR, and Raman spectroscopy data. MTX was found to increase the α-helical content of the zymogen from 3 to 15% proposing that drug is efficient in disrupting the ß-inter-molecular rich aggregates and reverting it to native like structure. The SEM images are in accordance with CD data showing the disintegration of aggregates. The most effective concentration of the drug was found to be 120 µM. Molecular docking analysis showed that MTX molecule was surrounded by the hydrophobic residues including Phe39, His40, Arg145, Tyr146, Thr151, Gly193, Ser195, and Gly216 and conventional hydrogen bonds, including Gln73 (bond length: 2.67Å), Gly142 (2.59Å), Thr144 (2.81Å), Asn150 (2.73Å), Asp153 (2.71Å), and Cys191 (2.53Å). This investigation will help to find the use of already existing drugs to cure protein misfolding-related abnormalities.

Investigating the binding interaction of quinoline yellow with bovine serum albumin and anti-amyloidogenic behavior of ferulic acid on QY-induced BSA fibrils.

Protein aggregation induces profound changes in the structure along with the conformation of the protein, and is responsible for the pathogenesis of a number of neurodegenerative conditions such as Huntington's, Creutzfeldt-Jacob, Type II diabetes mellitus, Alzheimer's, etc. Numerous multi-spectroscopic approaches and in-silico experiments were utilized to investigate BSA's biomolecular interaction and aggregation in the presence of quinoline yellow. The present research investigation evaluated the interaction of BSA with the food colorant (QY) at two different pH (7.4 and 2.0). The development of the BSA-QY complex was established with UV visible and fluorescence spectroscopy. The quenching of fluorescence upon the interaction of BSA with QY revealed the static nature of quenching mechanism. The Kb value obtained from our result is 4. 54 × 10-4 M-1. The results from the competitive site marker study infer that quinoline yellow is binding with the sub-domain IB of bovine serum albumin, specifically on site III. Three-dimensional fluorescence and synchronous fluorescence spectroscopy were applied for monitoring the alterations in the microenvironment of BSA upon the addition of quinoline yellow. The results from turbidity and RLS studies showed that higher concentrations of QY (80-400 µM) triggered bovine serum albumin (BSA) aggregation at pH 2.0. At pH 7.4, QY couldn't manage to trigger bovine serum albumin aggregation, perhaps because of the repulsion between negatively charged dye (QY) and anionic bovine serum albumin. The results from far-UV CD, Congo Red, and scanning electron microscopy implicate that the QY-induced aggregates exhibit amyloid fibril-like structures. Molecular docking results revealed that hydrophobic interactions, hydrogen bonding, and Pi-Sulfur interactions contribute to QY-induced aggregation of BSA. Further, the amyloid inhibitory potential of ferulic acid (FA), a phenolic acid on QY-induced aggregation of BSA, has also been assessed. The QY-induced amyloid fibrils are FA-soluble, as confirmed by turbidity, RLS, and far-UV CD studies. Far-UV CD results showed that FA retains α helix and inhibits cross ß sheet formation when the BSA samples were pre-incubated with increasing concentrations of FA (0-500 µM). Our findings conclude that QY dye successfully stimulates BSA aggregation, but ferulic acid inhibits QY-induced aggregation of BSA. Thus, FA can serve as a therapeutic agent and can help in the treatment of various amyloid-related conditions.

Protective role of chlorogenic acid in preserving cytochrome-c stability against HFIP-induced molten globule state at physiological pH.

Numerous neurodegenerative disorders are characterized by protein misfolding and aggregation. The mechanism of protein aggregation is intricate, and it is very challenging to study at cellular level. Inhibition of protein aggregation by interfering with its pathway is one of the ways to prevent neurodegenerative diseases. In the present work, we have evaluated the protective effect of a polyphenol compound chlorogenic acid (CGA) on the native and molten globule state of horse heart cytochrome c (cyt c). A molten globule state of this heme protein was achieved in the presence of fluorinated alcohol 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) at physiological pH, as studied by UV-Vis absorption, circular dichroism, intrinsic and ANS fluorescence. We found that at 50 % (v/v) HFIP, the native cyt c transformed into a molten globule state. The same techniques were also used to analyze the protective effect of CGA on the molten globule state of cyt c, and the results show that the CGA prevented the molten globular state and retained the protein close to the native state at 1:1 protein:CGA sub molar ratio. Molecular dynamics study also revealed that CGA retains the stability of cyt c in HFIP medium by preserving it in an intermediate state close to native conformation.

Detection and analysis of protofibrils and fibrils of hemoglobin: implications for the pathogenesis and cure of heme loss related maladies.

TFE induces structural alterations of proteins similar to the lipid environment of biological membranes, implicating these studies worthy of analyzing protein conformation in membranes such as red blood cells (RBCs). Heme loss occurs on rupturing of RBCs as found in diseases namely haemophilia, haemolytic anaemia, diabetes mellitus. TFE can be implied in discovering therapeutic targets, as it mimics the biological membrane environment. A global transition of hemoglobin (Hb) in presence of TFE was studied by using multi-methodological approach. The presence of partially folded state of Hb at 15% v/v TFE was confirmed by altered tryptophan environment, and retention of native-like secondary and tertiary structure. Molten globule state was observed at 20% v/v TFE as detected by increase tryptophan and high ANS fluorescence, slight alterations in Soret band relative to native. TFE on increasing concentration induced protofibrils at 25% v/v and fibrils at 45% v/v as depicted by altered tryptophan environment, heme loss, increase in non-native ß-sheet secondary and tertiary structure, large hydrodynamic radii of heme-protein, high ANS, thioflavin T fluorescence and shift in Congo Red absorbance. Comet assay showed that protofibrils are cytotoxic to lymphocytes. SEM and XRD confirmed these aggregates to be fibrillar in nature.

Consequential secondary structure alterations and aggregation during prolonged casein glycation.

Non-enzymatic glycosylation (glycation) of casein is a process used not just to ameliorate the quality of dairy products but also to increase the shelf life of canned foods, including baby milk supplements. Incubation of κ-casein with reducing sugars for 15 days at physiological temperature showed the formation of a molten globule state at day 9 and 12 during fructation and glucation respectively. This state exhibits substantial secondary structure and maximum ANS binding. Later on, glycation resulted in the formation of aggregates at day 12 in presence of fructose and day 15 in presence of glucose. Aggregates possess extensive ß-sheet structure as revealed by far-UV CD and FTIR. These aggregates showed altered tryptophan environment, decrease ANS binding relative to molten globule state and increase in Thioflavin T fluorescence. Aggregates were also accompanied by the accumulation of AGEs, indicative of structural damage to the protein and formation of potentially harmful species at the physiological level. Fructose was more reactive than glucose and thus caused early and significant changes in the protein. From our studies, we conclude that controlling the extent of the Maillard reaction in the food industry is essential to counter its negative effects and expand its safety spectrum.

Bioflavonoids ameliorate crowding induced hemoglobin aggregation: a spectroscopic and molecular docking approach.

The cellular environment is densely crowded, confining biomacromolecules including proteins to less available space. This macromolecular confinement may affect the physiological conformation of proteins in long-term processes like ageing. Changes in physiological protein structure can lead to protein conformational disorders including neurodegeneration. An intervention approach using food and plant derived bioflavonoids offered a way to find a treatment for these enervating pathological conditions as there is no remedy available. The bioflavonoids NAR (naringenin), 7HD (7 hydroxyflavanone) and CHR (chrysin) were tested for their ability to protect Hb (hemoglobin) against crowding-induced aggregation. Morphological and secondary structural transitions were studied using microscopic and circular dichroism experiments, respectively. The kinetic study was carried out using the relative thioflavin T assay. Molecular docking, AmylPred2, admetSAR and FRET were applied to understand the binding parameters of bioflavonoids with Hb and their drug likeliness. Isolated human lymphocytes were used as a cellular system to study the toxic effects of Hb aggregates. Redox perturbation and cytotoxicity were evaluated by DCFH-DA and MTT assays, respectively. This study suggests that bioflavonoids bind to Hb in the vicinity of aggregation prone amino acid sequences. Binding of the bioflavonoids stabilizes the Hb against crowding-induced structural alterations. Therefore, this study signifies the potential of bioflavonoids for future treatment of many proteopathies including neurodegeneration.Communicated by Ramaswamy H. Sarma.

Glycation promotes the formation of genotoxic aggregates in glucose oxidase.

This study investigates the effect of pentose sugars (ribose and arabinose) on the structural and chemical modifications in glucose oxidase (GOD) as well as genotoxic potential of this modified form. An intermediate state of GOD was observed on day 12 of incubation having CD minima peaks at 222 and 208 nm, characteristic of α-helix and a few tertiary contacts with altered tryptophan environment and high ANS binding. All these features indicate the existence of molten globule state of the GOD with ribose and arabinose on day 12. GOD on day 15 of incubation forms ß structures as revealed by CD and FTIR which may be due to its aggregation. Furthermore, GOD on day 15 showed a remarkable increase in Thioflavin T fluorescence at 485 nm. Comet assay of lymphocytes and plasmid nicking assay in presence of glycated GOD show DNA damage which confirmed the genotoxicity of advance glycated end products. Hence, our study suggests that glycated GOD results in the formation of aggregates and the advanced glycated end products, which are genotoxic in nature.

Existence of different structural intermediates and aggregates on the folding pathway of ovalbumin.

Structural modifications of ovalbumin in presence of different concentration of guanidine hydrochloride (Gdn HCl) and glucose were investigated by using intrinsic fluorescence, Fourier transform infra-red spectroscopy, circular dichroism and 8-anilino-1-naphthalene-sulphonic acid, to confirm that partially folded intermediates of ovalbumin lead to aggregation. The two partially folded intermediates of ovalbumin were observed one at 1 M Gdn HCl and another in the presence of 20 mM glucose at 3 M Gdn HCl. Both intermediates exist as compact states with altered intrinsic fluorescence, prominent ß-sheet secondary structure and enhanced ANS binding. Ovalbumin in the presence of glucose required more concentration of Gdn HCl (3 M) to exist as an intermediate state than control (1 M). Such alpha-helix/beta-sheet transition of proteins is a crucial step in amyloidogenic diseases and represents an internal rearrangement of local contacts in an already folded protein. Further, incubation for 24 h resulted in the formation of aggregates as detected by thioflavin T-assay. On further increasing the concentration of glucose to 50 mM and incubation time for various days resulted in the formation of molten globule state of ovalbumin at 6th day. Later on, at 10th day advanced glycated end products were observed.

Consequence of macromolecular crowding on aggregation propensity and structural stability of haemoglobin under glycating conditions.

Cell interiors are extremely congested with biological macromolecules exerting crowding effect, influencing various physiognomies of protein life. Present work deals with effect of crowding on folding behaviour of haemoglobin (Hb) under glycating conditions. Macromolecular crowding was mimicked by concentrated solutions of dextran 70. Hb with 0.2 M fructose and ribose was incubated separately for 96 h in dilute and crowded solution to analyse conformational changes. Reduced intrinsic and ANS fluorescence, decreased Soret absorbance, enhanced turbidity, browning of protein, red shift in ThT and Congo red spectra significantly unveiled protein aggregation. FTIR and CD results revealed transition from α-helix to ß-sheets confirming aggregation. Transmission electron microscopy exhibited incidence of aggregates. Macromolecular crowding was witnessed to defend conformational stability of native Hb under stress condition at 100 mg/ml dextran, noticeably indicating deceleration of aggregation. Stabilising effect of crowding was marginally better in fructosylated Hb than with ribose due to difference in their glycation potential. Contrarily, in over-crowded solution where dextran concentration was 500 mg/ml, heightened aggregation was perceived implying concentration dependant, dual nature of macromolecular crowding. The novelty of this study lies in idea of considering macromolecular crowding as a key player in regulation of protein stability which was safely ignored previously.

Macromolecular crowding stabilises native structure of α-chymotrypsinogen-A against hexafluoropropanol-induced aggregates.

Cell interior is extremely congested with tightly packed biological macromolecules that exerts macromolecular crowding effect, influencing biophysical properties of proteins. To have a deeper insight into it we studied consequences of crowding on aggregation susceptibility and structural stability of α-chymotrypsinogen-A, pro-enzyme of serine protease family, upon addition of co-solvent reported to exert stress on polypeptides crafting favourable conditions for aggregation. Hexafluoropropan-2-ol (HFIP), a fluorinated alcohol caused structural disruption at 5% v/v unveiled by reduced intrinsic intensity and blue shifted ANS spectra. Significantly enhanced, red-shifted ThT and Congo red spectra sustained conformational changes concomitant with aggregation. FTIR and CD results confirmed transition of native structure to non-native extended, cross-linked beta-sheets. Transmission electron micrographs visibly exhibited incidence of amorphous aggregates. Macromolecular crowding, typically mimicked by concentrated solutions of dextran 70, was noticeably witnessed to defend conformational stability under denaturing condition. The native structure was retained maximally in presence of 100 mg/ml followed by 200 and 300 mg/ml dextran indicating concentration dependent deceleration of aggregate formation. It can be established that explicit consideration of crowding effects using relevant range of inert crowding agents must be a requisite for presumptions on intracellular conformational behaviour of proteins deduced from in vitro experiments.

Aggregation of globular protein as a consequences of macromolecular crowding: A time and concentration dependent study.

The living cells show profoundly crowded condition, called as macromolecular crowding. Crowding essentially impacts on protein structure and lead to its aggregation. Protein aggregates have been involved in a wide range of diseases including Parkinson, Alzheimer's, and Huntington's. Increased in normal physiological macromolecular crowding because of increasing age can be implicated as one of the leading cause of proteopathies. In the present study, we have demonstrated the effect of macromolecular crowding on native structure of hemoglobin using bovine serum albumin as a crowding agent. Conformational changes of Hb at different concentrations of BSA were monitored using intrinsic fluorescence and ATR-FTIR spectroscopy. These results showed the transition of native Hb to a non-native form. Thermodynamic parameters were analyzed by isothermal titration calorimetry. The measurements of turbidity, thioflavin T, congo red and intrinsic fluorescence revealed the formation of significant protein aggregates with time. The kinetics of protein aggregation using relative ThT and 8-anilino-1-naphthalenesulphonic acid spectra clearly showed acceleration of the process with time and in concentration dependent manner. The spectra at 80g/l of BSA incubated for 64h showed formation of maximum Hb aggregates. Transmission electron microscopy results clearly showed the formation of amyloid aggregates structures, thus supporting the spectroscopic data.