No abstract

Effect of ethanol on the size and structure of a protein cytochrome C (Cyt C) is investigated using fluorescence correlation spectroscopy (FCS) and molecular dynamics (MD) simulations. For FCS studies, Cyt C is covalently labeled with a fluorescent probe, alexa 488. FCS studies indicate that on addition of ethanol, the size of the protein varies non-monotonically. The size of Cyt C increases (i.e., the protein unfolds) on addition of alcohol (ethanol) up to a mole fraction of 0.2 (44.75% v/v) and decreases at higher alcohol concentration. In order to provide a molecular origin of this structural transition, we explore the conformational free energy landscape of Cyt C as a function of radius of gyration (R) at different compositions of water-ethanol binary mixture using MD simulations. Cyt C exhibits a minimum at R ∼ 13 Å in bulk water (0% alcohol). Upon increasing ethanol concentration, a second minimum appears in the free energy surface with gradually larger R up to χ ∼ 0.2 (44.75% v/v). This suggests gradual unfolding of the protein. At a higher concentration of alcohol (χ > 0.2), the minimum at large R vanishes, indicating compaction. Analysis of the contact map and the solvent organization around protein indicates a preferential solvation of the hydrophobic residues by ethanol up to χ = 0.2 (44.75% v/v) and this causes the gradual unfolding of the protein. At high concentration (χ = 0.3 (58% v/v)), due to structural organization in bulk water-ethanol binary mixture, the extent of preferential solvation by ethanol decreases. This causes a structural transition of Cyt C towards a more compact state.

Fluorescence dynamics in the endoplasmic reticulum (ER) of a live non-cancer lung cell (WI38) and a lung cancer cell (A549) are studied by using time-resolved confocal microscopy. To selectively study the organelle, ER, we have used an ER-Tracker dye. From the emission maximum (λmaxem) of the ER-Tracker dye, polarity (i.e. dielectric constant, ϵ) in the ER region of the cells (≈500 nm in WI38 and ≈510 nm in A549) is estimated to be similar to that of chloroform (λmaxem =506 nm, ϵ≈5). The red shift by 10 nm in λmaxem in the cancer cell (A549) suggests a slightly higher polarity compared to the non-cancer cell (WI38). The fluorescence intensity of the ER-Tracker dye exhibits prolonged intermittent oscillations on a timescale of 2-6 seconds for the cancer cell (A549). For the non-cancer cell (WI38), such fluorescence oscillations are much less prominent. The marked fluorescence intensity oscillations in the cancer cell are attributed to enhanced calcium oscillations. The average solvent relaxation time (<τs >) of the ER region in the lung cancer cell (A549, 250±50 ps) is about four times faster than that in the non-cancer cell (WI38, 1000±50 ps).

Unfolding/refolding of a plasma protein, human serum albumin (HSA), is studied using fluorescence correlation spectroscopy (FCS) and single molecule fluorescence resonance energy transfer (sm-FRET). Addition of cholesterol causes unfolding of HSA resulting in an increase in the hydrodynamic diameter (dH = 2rH) from 76 Å in the native state to 120 Å upon addition of 1 mM cholesterol. Addition of β-cyclodextrin to HSA (unfolded by cholesterol) restores the hydrodynamic diameter back to 78 Å. The cholesterol induced unfolding and β-cyclodextrin induced refolding are also monitored by measuring the distance between a FRET donor (CPM dye, D) and a FRET acceptor (Alexa 488, A) covalently attached to the protein (HSA). It is observed that the average D-A distance increases from 45 ± 1 Å at 0 mM cholesterol to 51 ± 1 Å at 1 mM cholesterol. Upon addition of β-cyclodextrin, the D-A distance is restored to 45 ± 1 Å. The binding study indicates that nearly 94% of HSA molecules remain bound to cholesterol in the absence of β-cyclodextrin and only 5% binds to cholesterol in the presence of β-cyclodextrin. As much as 57% of the HSA and 99% of the cholesterol molecules bind to β-cyclodextrin. Thus β-cyclodextrin removes cholesterol from HSA by hydrophobic binding to cholesterol ("strip off") and also, itself binds to HSA. The conformational dynamics results suggest that addition of β-cyclodextrin restores native like binding free energy and folding dynamics.

The abundance of protein dimers and multidomain proteins is a testimony to their importance in various cellular functions. Several mechanisms exist, explaining how they assemble. The energy landscape theory has shown that, irrespective of the mechanism followed, folding and binding of dimers and multidomain proteins are funneled processes. Using a structure based model, we have characterized the folding landscape and dimerization mechanism of the DNA binding domain (DBD) in a complex multidomain, homodimeric transcription factor, catabolite activator protein (CAP). The DBD is tethered to the nucleotide binding domain (NBD) of CAP. Our investigation revealed that, as the tethered DBD of CAP transitions from an unfolded to the folded state, complementary folding and backtracking occur between the individual subunits within the DBD. This redistributes the entropies of the DBDs in both the subunits and might play a role in consequently modulating the free energy surface to reduce the entropic folding barrier. This redistribution of entropies forms the basis of an unusual intersubunit assisted folding mechanism whereby each subunit acts as a chaperone for the other. We have also investigated the effect of tethering on the folding landscape of DBD and found that the folding landscape can change depending on the tethering conditions.