Extrinsic Probe Research Articles

Effect of Chain Branching on Orientational Ordering in Glycolipid Self-assembly by 2H-NMR using Extrinsic Probes

The structure and dynamics of molecular solids, liquid crystals, and thin films of membrane lipids may be elucidated from the use of deuterium NMR (2H-NMR) spectroscopy. The 2H-NMR spectrum of a deuterated material (either selectively or fully) is rather simpler to interpret compared with the corresponding proton NMR (1H-NMR). Here, we apply this technique to understand the effect of chain branching on orientational order of the glycolipid self-assembly by using deuterated probes. The 2H-NMR resonance of n-octanol-d17 in n-octyl-β-D-glucopyranoside (β-GlcOC8) and the branched-chain glycolipid 2-hexyldecyl-β-D-glucopyranoside (β-GlcOC10C6) were measured at different temperatures. We also measured the 2H-NMR spectrum of 2-ethyl-n-hexanol-α,α-d2, a branched-chain alcohol which is selectively deuterated at α-position in β-GlcOC8. All systems gave quadrupolar splittings in their respective 2H-NMR spectra, which are attributed to the formation of anisotropic phase. The n-octanol-d17 dissolved in β-GlcOC8 give us spectra in the lamellar phase and when used with a branched-chain lipid, i.e. β-GlcOC10C6, gave us new spectra in the inverse hexagonal phase. The magnitude of the quadrupolar splittings of the n-octanol-d17 is indicative of the degree of orientational ordering in the given system. The results show that the quadrupolar splitting is larger in β-GlcOC8 lipid than in β-GlcOC10C6.

Effects of nerolidol and limonene on stratum corneum membranes: A probe EPR and fluorescence spectroscopy study

The sesquiterpene nerolidol and the monoterpene limonene are potent skin-permeation enhancers that have also been shown to have antitumor, antibacterial, antifungal and antiparasitic activities. Because terpenes are membrane-active compounds, we used electron paramagnetic resonance (EPR) spectroscopy of three membrane spin labels combined with the fluorescence spectroscopy of three lipid probes to study the interactions of these terpenes with stratum corneum (SC) intercellular membranes. An experimental apparatus was developed to assess the lipid fluidity of hydrated SC membranes via the fluorescence anisotropy of extrinsic membrane probes. Both EPR and fluorescence probes indicated that the intercellular membranes of neonatal SC rats undergo a main phase transition at approximately 50°C. Taken together, the results indicated that treatment with 1% nerolidol (v/v) caused large fluidity increases in the more ordered phases of SC membranes and that these effects gradually decreased with increasing temperature. Additionally, compared with (+)-limonene, nerolidol was better able to change the SC membrane dynamics. EPR and fluorescence data suggest that these terpenes act as spacers in lipid packaging and create increased lipid disorder in the more ordered regions and phases of SC membranes, notably leading to a population of probes with less restricted motion.

In Vitro Protein Stability of Two Naturally Occurring Thiopurine S-Methyltransferase Variants: Biophysical Characterization of TPMT*6 and TPMT*8.

Thiopurine S-methyltransferase (TPMT) is a polymorphic enzyme involved in the metabolism and inactivation of thiopurine substances administered as immunosuppressants in the treatment of malignancies and autoimmune diseases. In this study, the naturally occurring variants, TPMT*6 (Y180F) and TPMT*8 (R215H), have been biophysically characterized. Despite being classified as low and intermediate in vivo enzyme activity variants, respectively, our results demonstrate a discrepancy because both TPMT*6 and TPMT*8 were found to exhibit normal functionality in vitro. While TPMT*8 exhibited biophysical properties almost indistinguishable from those of TPMTwt, the TPMT*6 variant was found to be destabilized. Furthermore, the contributions of the cofactor S-adenosylmethionine (SAM) to the thermodynamic stability of TPMT were investigated, but only a modest stabilizing effect was observed. Also presented herein is a new method for studies of the biophysical characteristics of TPMT and its variants using the extrinsic fluorescent probe 8-anilinonaphthalene-1-sulfonic acid (ANS). ANS was found to bind strongly to all investigated TPMT variants with a Kd of approximately 0.2 μM and a 1:1 binding ratio as determined by isothermal titration calorimetry (ITC). Circular dichroism and fluorescence measurements showed that ANS binds exclusively to the native state of TPMT, and binding to the active site was confirmed by molecular modeling and simulated docking as well as ITC measurements. The strong binding of the probe to native TPMT and the conformity of the obtained results demonstrate the advantages of using ANS binding characteristics in studies of this protein and its variants.

Chaotropes trigger conformational rearrangements differently in Concanavalin A

Concanavalin A (ConA) is a plant lectin having industrial and biological applications. Concanavalin A changes conformation upon exposure to different stress conditions, like exposure to sodium dodecyl sulphate, guanidine hydrochloride, varying hydronium ion potential, etc. The conformational changes were studied using circular dichroism spectroscopy and the structural rigidity of ConA was explored using fluorescence spectroscopy, taking tryptophan as an intrinsic and 8-anilino-1-naphthalenesulfonic acid as an extrinsic fluorescence probes, in different stress conditions. ConA loses the quaternary structure in all the studied stress conditions, which further leads to denaturation of the protein. However, the mechanism of denaturation varied with the studied conditions, like different SDS concentrations and hydronium ion potentials, wherein the protein undergoes a conformational rearrangement from $$\upbeta $$ -sheet to $$\upalpha $$ -helix. Moreover, GdnHCl triggered complete denaturation of ConA into a predominantly random coil conformation. The results suggested that denaturation of ConA follows different pathways depending on the chemical properties and concentrations of the denaturants used. SynopsisGuanidine hydrochloride shows complete unfolding of Concanavalin A (Con A) above 2 M concentration, whereas GdnHCl between 1–2 M triggered the molten globule-like conformation. At basic pH, ConA adopts all- $$\upbeta $$ -sheet conformation, and at acidic pH it retained demetallized monomeric jelly roll motif. Additionally, in the presence of SDS at >2 mM, ConA undergoes conformational rearrangement into a predominantly $$\upalpha $$ -helix conformation, rather a leticn-like all- $$\upbeta $$ -sheet conformation at lower concentrations of SDS.

Stepwise unfolding of Ribonuclease A by a biosurfactant

The interaction of Ribonuclease A (RNase A) with the bile salt, sodium deoxycholate (NaDC) is meticulously investigated using various spectroscopic techniques. The binding isotherm constructed from the modulation of intrinsic tyrosine fluorescence of the protein following interaction with NaDC conspicuously reveals an intrinsically complex stepwise interaction process which proceeds through the formation of distinct conformational states of the protein. The conformational transitions are found to occur at c1=2.2mM, and c2=7.2mM of NaDC. These results are subsequently corroborated from the studies of excited-state relaxation dynamics of the intrinsic tyrosine residues of RNase A, and the modulations in fluorescence behavior of an extrinsic probe (ANS) bound to the protein. The far-UV circular dichroism spectral analyses unveil only nominal influence on the secondary structural element of the protein up to [NaDC]=c1=2.2mM, which is then followed by marked disruption of the secondary structure of RNase A following further addition of NaDC. This clearly accounts for differential interaction behaviors of RNase A with the monomeric and micellar forms of NaDC (CMC of NaDC in aqueous buffer is estimated to be ∼3.0mM). In Region-I (up to c1=2.2mM), the protein:surfactant interaction is argued to be predominantly governed by electrostatic/ionic interaction force. Subsequently, in Region-II (up to c2=7.2mM) the RNase A:NaDC interaction accompanies major denaturation of the protein (∼17% loss of the secondary structure of RNase A at c2=7.2mM) resulting in significant exposure of hydrophobic surfaces of the protein. However, the tertiary structure of the protein remains essentially unperturbed within the concentration regime of NaDC under study.

Determination of the Critical Micelle Concentration of Triton X-100 Using the Compound 3-(benzoxazol-2-yl)-7-(N,N-diethylamino)chromen-2-one as Fluorescent Probe

In the present study we estimated the Critical Micelle Concentration (CMC) of Triton X-100 in an aqueous buffered medium (PBS buffer, pH 7.4), using 3-(benzoxazol-2-yl)-7-(N,N-diethylamino)chromen-2-one (BDC), a push-pull compound, as extrinsic fluorescence probe. The CMC value found, 0.33 mmol L -1 , shows good agreement with data from literature obtained using the fluorescence probe technique. Additionally, the polarity, in terms of the E T (30) scale, and the viscosity of the micelle microenvironment in which the probe is preferentially allocated, was estimated as being 46.9 kcal mol -1 and 70.3 cP, respectively, confirming that this microdomain is polar and highly viscous, with characteristics of polyethylene glycol groups consisting in the palisade layer at the micelle-water interface. DOI: http://dx.doi.org/10.17807/orbital.v0i0.923

Diphenylhexatriene membrane probes DPH and TMA-DPH: A comparative molecular dynamics simulation study

Fluorescence spectroscopy and microscopy have been utilized as tools in membrane biophysics for decades now. Because phospholipids are non-fluorescent, the use of extrinsic membrane probes in this context is commonplace. Among the latter, 1,6-diphenylhexatriene (DPH) and its trimethylammonium derivative (TMA-DPH) have been extensively used. It is widely believed that, owing to its additional charged group, TMA-DPH is anchored at the lipid/water interface and reports on a bilayer region that is distinct from that of the hydrophobic DPH. In this study, we employ atomistic MD simulations to characterize the behavior of DPH and TMA-DPH in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and POPC/cholesterol (4:1) bilayers. We show that although the dynamics of TMA-DPH in these membranes is noticeably more hindered than that of DPH, the location of the average fluorophore of TMA-DPH is only ~3–4Å more shallow than that of DPH. The hindrance observed in the translational and rotational motions of TMA-DPH compared to DPH is mainly not due to significant differences in depth, but to the favorable electrostatic interactions of the former with electronegative lipid atoms instead. By revealing detailed insights on the behavior of these two probes, our results are useful both in the interpretation of past work and in the planning of future experiments using them as membrane reporters.

Bile salt induced solubilization of methylene blue: Study on methylene blue fluorescence properties and molecular mechanics calculation

Methylene blue (MB) is a hydrophobic drug molecule, having importance both as a staining reagent and pharmaceutical agent. MB is strongly fluorescent, with an emission peak at 686nm (λex 665nm). In the study, the possibility of MB as an extrinsic fluorophore to study the micellization behavior of bile salts (BSs) was carried out. Since BSs are drug delivery systems, the solubilization of hydrophobic MB drug molecule by BSs was achieved and the nature of association of MB with BS media, namely sodium cholate (NaC) and sodium deoxycholate (NaDC) was evaluated. Change in the photophysical properties of MB is monitored through fluorescence intensity and fluorescence anisotropy at emission peak, 686nm of MB. Molecular mechanics calculations were carried out to evaluate the MB–BS association. The estimated heat of formation, ΔHf values are –625.19kcal/mol for MB–NaC and –757.48kcal/mol for MB–NaDC. The photophysical study also revealed that MB reports the step-wise aggregation pattern of BSs media, as an extrinsic fluorescence probe.

Infrared and Fluorescence Assessment of Protein Dynamics: From Folding to Function.

While folding or performing functions, a protein can sample a rich set of conformational space. However, experimentally capturing all of the important motions with sufficient detail to allow a mechanistic description of their dynamics is nontrivial since such conformational events often occur over a wide range of time and length scales. Therefore, many methods have been employed to assess protein conformational dynamics, and depending on the nature of the conformational transition in question, some may be more advantageous than others. Herein, we describe our recent efforts, and also those of others, wherever appropriate, to use infrared- and fluorescence-based techniques to interrogate protein folding and functional dynamics. Specifically, we focus on discussing how to use extrinsic spectroscopic probes to enhance the structural resolution of these techniques and how to exploit various cross-linking strategies to acquire dynamic and mechanistic information that was previously difficult to attain.

Vibrational and Electronic Stark Effects in Green Fluorescent Protein

The use of unnatural amino acids to probe non-covalent interactions, dynamics, and folding in proteins is growing rapidly. Specifically, para-cyanophenylalanine (pCNF) is an attractive probe of electrostatics because of its sensitivity to electric fields and its minimal structural perturbation when replacing phenylalanine or tyrosine. However, due to the potential for hydrogen bonding to the nitrile moiety there is potential for the introduction of nonnative interactions, which can cloud our understanding of native proteins. Usual attempts to quantify the extent of nitrile perturbation include functional assays or structural measurements. While structure and function are undoubtedly linked to electrostatic environment, they are not direct reporters of it. An intrinsic reporter of electrostatic environment would allow for the direct comparison of environments with and without an extrinsic nitrile probe. Boxer and coworkers have shown that the intrinsic fluorophore of green fluorescent protein (GFP) is sensitive to electric fields in a manner consistent with a linear Stark effect. Here we incorporate multiple pCNF probes into buried sites near the GFP fluorophore and measure changes in the electric field environment from both the nitriles and the fluorophore in response to a series of amino acid mutations. Our results show that pCNF probes can be incorporated near the GFP fluorophore without affecting the electric field changes experienced by the fluorophore. Additionally, changes in electric field measured from the nitrile are in agreement with those measured from the fluorophore. These results suggest that, for this model system, nitrile probes do not significantly perturb the native electrostatic environment. The agreement of changes in electric field from vibrational and electronic Stark effect probes inside the beta barrel of GFP suggests nitrile frequencies can be interpreted in terms of the Stark effect even when they participate in hydrogen bonding.

A simple pendulum borehole tiltmeter based on a triaxial optical-fibre displacement sensor

Sensitive instruments like strainmeters and tiltmeters are necessary for measuring slowly varying low amplitude Earth deformations. Nonetheless, laser and fibre interferometers are particularly suitable for interrogating such instruments due to their extreme precision and accuracy. In this paper, a practical design of a simple pendulum borehole tiltmeter based on laser fibre interferometric displacement sensors is presented. A prototype instrument has been constructed using welded borosilicate with a pendulum length of 0.85 m resulting in a main resonance frequency of 0.6 Hz. By implementing three coplanar extrinsic fibre Fabry-Perot in-terferometric probes and appropriate signal filtering, our instrument provides tilt measurements that are insensitive to parasitic deformations caused by temperature and pressure variations. This prototype has been installed in an underground facility (Rustrel, France) where results show accurate measurements of Earth strains derived from Earth and ocean tides, local hydro-logic effects, as well as local and remote earthquakes. The large dynamic range and the high sensitivity of this tiltmeter render it an invaluable tool for numerous geophysical applications such as transient fault motion, volcanic strain and reservoir monitoring.

Allosteric regulation: Illuminating allosteric conformational change with an environmentally sensitive fluorescent probe

Allosteric regulation was a hot topic in the 1960s, but there was very limited structural data on allosteric equilibria, and no solid information on the rates of allosteric conformational changes. In this Biochemical Journal Classic paper from 1969 George Radda and his first D.Phil. student, George Dodd determined the rate of allosteric transition in the regulatory enzyme glutamate dehydrogenase by a method new in the 1960s, the fluorescence of an environmentally sensitive extrinsic probe.

Measuring electric fields and noncovalent interactions using the vibrational stark effect.

Over the past decade, we have developed a spectroscopic approach to measure electric fields inside matter with high spatial (<1 Å) and field (<1 MV/cm) resolution. The approach hinges on exploiting a physical phenomenon known as the vibrational Stark effect (VSE), which ultimately provides a direct mapping between observed vibrational frequencies and electric fields. Therefore, the frequency of a vibrational probe encodes information about the local electric field in the vicinity around the probe. The VSE method has enabled us to understand in great detail the underlying physical nature of several important biomolecular phenomena, such as drug-receptor selectivity in tyrosine kinases, catalysis by the enzyme ketosteroid isomerase, and unidirectional electron transfer in the photosynthetic reaction center. Beyond these specific examples, the VSE has provided a conceptual foundation for how to model intermolecular (noncovalent) interactions in a quantitative, consistent, and general manner. The starting point for research in this area is to choose (or design) a vibrational probe to interrogate the particular system of interest. Vibrational probes are sometimes intrinsic to the system in question, but we have also devised ways to build them into the system (extrinsic probes), often with minimal perturbation. With modern instruments, vibrational frequencies can increasingly be recorded with very high spatial, temporal, and frequency resolution, affording electric field maps correspondingly resolved in space, time, and field magnitude. In this Account, we set out to explain the VSE in broad strokes to make its relevance accessible to chemists of all specialties. Our intention is not to provide an encyclopedic review of published work but rather to motivate the underlying framework of the methodology and to describe how we make and interpret the measurements. Using certain vibrational probes, benchmarked against computer models, it is possible to use the VSE to measure absolute electric fields in arbitrary environments. The VSE approach provides an organizing framework for thinking generally about intermolecular interactions in a quantitative way and may serve as a useful conceptual tool for molecular design.

Dual-sensor fluorescent probes of surfactant-induced unfolding of human serum albumin.

Two extrinsic fluorescent probes, 3-(dimethylamino)-8,9,10,11-tetrahydro-7H-cyclohepta[a]naphthalen-7-one (1) and 7-(dimethylamino)-2,3-dihydrophenanthren-4(1H)-one (2), are used to probe the unfolding of human serum albumin by sodium dodecyl sulfate (SDS). These probes respond separately to the polarity and H-bond-donating ability of their surroundings. Competitive binding experiments show that fluorophore 1 binds to site I (domain IIA) and 2 binds to site II (domain IIIA). The local acidity of 1 in site I is out of the sensing range of 1, whereas the local acidity of 2 in site II is calculated to be nearly zero on Catalan's solvent acidity index. Both probes show that the first two equivalents of bound SDS result in a decrease in the local polarity of the binding sites. Each subsequent equivalent of SDS gives rise to a dramatic increase in polarity until HSA is saturated with seven molecules of SDS at the end of the specific binding domain. Compound 2 experiences an increase of acidity of 0.10 on Catalan's solvent acidity index through seven equivalents of SDS, but the local acidity for 1 is still out of range. The increase in acidity experienced by 2 is greater than the increase in polarity. This result is consistent with greater exposure of the carbonyl group in 2, but not the bulk of 2, to the aqueous solvent in site II of the SDS-saturated HSA complex.

Site-specific infrared probes of proteins.

Infrared spectroscopy has played an instrumental role in the study of a wide variety of biological questions. However, in many cases, it is impossible or difficult to rely on the intrinsic vibrational modes of biological molecules of interest, such as proteins, to reveal structural and environmental information in a site-specific manner. To overcome this limitation, investigators have dedicated many recent efforts to the development and application of various extrinsic vibrational probes that can be incorporated into biological molecules and used to site-specifically interrogate their structural or environmental properties. In this review, we highlight recent advancements in this rapidly growing research area.

PH-dependent conformational changes in the HCV NS3 protein modulate its ATPase and helicase activities.

The hepatitis C virus (HCV) infects 170 to 200 million people worldwide and is, therefore, a major health problem. The lack of efficient treatments that specifically target the viral proteins or RNA and its high chronicity rate make hepatitis C the cause of many deaths and hepatic transplants annually. The NS3 protein is considered an important target for the development of anti-HCV drugs because it is composed of two domains (a serine protease in the N-terminal portion and an RNA helicase/NTPase in the C-terminal portion), which are essential for viral replication and proliferation. We expressed and purified both the NS3 helicase domain (NS3hel) and the full-length NS3 protein (NS3FL) and characterized pH-dependent structural changes associated with the increase in their ATPase and helicase activities at acidic pH. Using intrinsic fluorescence experiments, we have observed that NS3hel was less stable at pH 6.4 than at pH 7.2. Moreover, binding curves using an extrinsic fluorescent probe (bis-ANS) and ATPase assays performed under different pH conditions demonstrated that the hydrophobic clefts of NS3 are significantly more exposed to the aqueous medium at acidic pH. Using fluorescence spectroscopy and anisotropy assays, we have also observed more protein interaction with DNA upon pH acidification, which suggests that the hydrophobic clefts exposure on NS3 might be related to a loss of stability that could lead it to adopt a more open conformation. This conformational change at acidic pH would stimulate both its ATPase and helicase activities, as well as its ability to bind DNA. Taken together, our results indicate that the NS3 protein adopts a more open conformation due to acidification from pH 7.2 to 6.4, resulting in a more active form at a pH that is found near Golgi-derived membranes. This increased activity could better allow NS3 to carry out its functions during HCV replication.

Extrinsic Fabry–Pérot Underwater Acoustic Sensor Based on Micromachined Center-Embossed Diaphragm

Most research works emphasized on the acoustic sensor miniaturization are not optimized for applications under high surrounding pressure. For underwater acoustic sensing, measuring the small acoustic pressure from the huge hydrostatic pressure makes the design of the sensor challenging. In this paper, we attempt to solve the problem by adopting a center-embossed diaphragm as the sensitive structure. The deformation angular error is defined to evaluate the optical performance degradation and the optical sensitivity reduction under hydrostatic pressure. Simulations indicate that the central embossment is beneficial to maintain optics-related properties, although the structural sensitivity is reduced. Then, the diaphragm design guideline for underwater acoustic sensing is regulated. An optical fiber extrinsic Fabry-Perot interferometer probe based on the diaphragm, which was micromachined by double-side etching of the silicon on insulator, was designed and assembled. Experimental results show that the interferometric fringe preserves similar shapes at any tested depth from 0 (in air) to 50 cm. The recovered signal detected by the sensor coincides well with the corresponding transmitted signal. The pressure sensitivity response is flat in frequency range from 10 to 2 kHz, of which value is about -154.6 dB re. 1/μPa. It agrees well with the theoretical predication. These results demonstrated that the designed sensor according to the guideline can be used as an underwater acoustic sensor. Moreover, the sensor has potential applications in smart unmanned platforms and swiftly deployable arrays.

Glycosaminoglycan-protein interaction studies using fluorescence spectroscopy.

Fluorescence spectroscopy is a quantitative analytical tool that has been extensively used to provide structural and dynamical information on GAG-protein complexes. It possesses major advantages including high sensitivity, relative ease of applicability, and wide range of available fluorescence labels and probes. It has been applied to practically every protein-GAG system through the use of either intrinsic (e.g., Trp) or extrinsic (e.g., a non-covalent fluorophore) probe. For studies involving GAGs, it forms the basis for measurement of dissociation constant of complexes and the stoichiometry of binding, which helps elucidate many other thermodynamic and/or mechanistic parameters. We describe the step-by-step procedure to measure the affinity of GAG-protein complexes, parse the ionic and nonionic components of the free energy of binding, and identify the site of GAG binding through competitive binding experiments.

Binding phenomena and fluorescence quenching. II: Photophysics of aromatic residues and dependence of fluorescence spectra on protein conformation

The three amino acids with aromatic ring side chains—phenylalanine (Phe), tyrosine (Tyr), and especially tryptophan (Trp) have played a long and productive role in helping unlock the secrets of protein behavior by optical spectroscopy (absorption, fluorescence, circular dichroism, etc.) In principle, an appropriately placed Trp will undergo fluorescence wavelength and/or intensity changes upon whatever functional process a protein performs. Although perceived to be enigmatic and not well understood, Trp is arguably now better understood than many of the extrinsic probes currently in use. Basic principles of intrinsic tryptophan fluorescence quenching and wavelength shifts in proteins are presented, with strong emphasis on the importance of electrostatics. The condensed description of findings from recent experiments and simulations of tryptophan fluorescence and intrinsic quenching in proteins is designed to help authors in planning and interpreting experimental results of ligand binding studies.

Revisiting Structural Hieracrchy: A Fluorescence Investigation of Unfolding of an Oligomeric Protein

α-crystallin is a multimeric lens protein with chaperone-like function and is responsible for maintaining lens transparency. The structural stability and unfolding-refolding properties of this protein are believed to be important for its function. We undertook a multi-probe based fluorescence approach to explore the changes in the various levels of organization of α-crystallin at different urea concentration region. Steady-state fluorescence emission and quenching studies using extrinsic and intrinsic fluorescence probes reveal that at 0.6 M urea a compact structural intermediate is formed which has a native-like secondary structure with minimal yet detectable tertiary structure perturbation associated with enhanced surface exposure of hydrophobic groups, possibly arising from interfacial structure meltdown. At 2.8 M urea the tertiary interactions undergo almost complete collapse with partial disintegration of secondary and quaternary structure. Investigation of surface solvation with picosecond resolved fluorescence transients of acrylodan covalently tagged to α-crystallin reveals a dry native-like core of α-crystallin at 0.6 M urea compared to enhanced water penetration at 2.8 M urea and extensive solvation at 6 M urea. Temperature dependent subunit exchange kinetics reveal decrease of activation energy for the subunit exchange process by 22 kJ mol-1 on changing urea concentration from 0 to 0.6 M compared to over 75 kJ mol-1 on changing urea concentration from 0 to 2.8 M. Dynamic light scattering study indicates swelling at 0.6 M urea, however oligomerization is retained as observed from sedimentation equilibrium experiment. At 2.8 M urea the oligomeric size is reduced and a monomer is produced at 6 M urea. These data clearly demonstrates that tertiary structure dissolution precedes oligomeric degradation. Such non-hierarchical structure dissolution indicates the possibility of tertiary contact formation to be a rather later folding event in case of large oligomeric proteins likes α-Crystallin.