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Urea-induced protein denaturation is widely used to study protein folding and stability; however, the molecular mechanism and driving forces of this process are not yet fully understood. In particular, it is unclear whether either hydrophobic or polar interactions between urea molecules and residues at the protein surface drive denaturation. To address this question, here, many molecular dynamics simulations totalling ca. 7 µs of the CI2 protein in aqueous solution served to perform a computational thought experiment, in which we varied the polarity of urea. For apolar driving forces, hypopolar urea should show increased denaturation power; for polar driving forces, hyperpolar urea should be the stronger denaturant. Indeed, protein unfolding was observed in all simulations with decreased urea polarity. Hyperpolar urea, in contrast, turned out to stabilize the native state. Moreover, the differential interaction preferences between urea and the 20 amino acids turned out to be enhanced for hypopolar urea and suppressed (or even inverted) for hyperpolar urea. These results strongly suggest that apolar urea–protein interactions, and not polar interactions, are the dominant driving force for denaturation. Further, the observed interactions provide a detailed picture of the underlying molecular driving forces. Our simulations finally allowed characterization of CI2 unfolding pathways. Unfolding proceeds sequentially with alternating loss of secondary or tertiary structure. After the transition state, unfolding pathways show large structural heterogeneity.
Author Summary
To perform their physiological function, proteins have to fold into their characteristic three-dimensional structure. While the folded state is stable under physiological conditions, changes in the solvent can destabilize the folded state and even induce denaturation. One of the most commonly used denaturants is urea. Despite its widespread use to study protein folding and stability, however, the molecular mechanism and particularly the driving forces of urea-induced protein denaturation are not yet understood. Two mechanisms have been suggested, according to which denaturation is driven either by polar interactions via hydrogen bonds or by hydrophobic interactions with apolar amino acids. By systematically varying urea polarity and quantifying the interactions of the solvent molecules with all amino acids of the protein, the present simulation study reveals that it is mainly the apolar interactions that drive denaturation. Our results suggest a coherent microscopic picture for urea-induced denaturation and bear more general implications for protein stability in other environments, e.g., in chaperone-assisted folding.
Citation: Stumpe MC, Grubmüller H (2008) Polar or Apolar—The Role of Polarity for Urea-Induced Protein Denaturation. PLoS Comput Biol 4(11): e1000221. doi:10.1371/journal.pcbi.1000221
Editor: Vijay S. Pande, Stanford University, United States of America
Received: July 10, 2008; Accepted: September 30, 2008; Published: November 14, 2008
Copyright: © 2008 Stumpe, Grubmüller. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: MCS gratefully acknowledges support from the Deutsche Volkswagen Stiftung (Grant I/78 839).
Competing interests: The authors have declared that no competing interests exist.
Protein denaturation by osmolytes such as urea or guanidinium is widely used to study protein folding and stability. The underlying mechanism, however, is not yet fully understood on the molecular level. Despite the large number of theoretical and experimental studies carried out in the past decades to shed light on the molecular details of this process, no clear picture has emerged yet. On the one hand, the microsecond to millisecond timescales at which individual folding and unfolding events occur, as well as the need for synchronization in ensemble measurements, and the structural heterogeneity of unfolding pathways renders it difficult to gain atomistic insight from experiments. On the other hand, for computer simulations, folding/unfolding processes are typically too slow or too rare to be accessible.
Two basic model classes have guided the study of the driving forces of urea-induced protein denaturation, and still set the framework for ongoing discussions. According to the first model, urea induces changes in the water structure, which in turn weaken the hydrophobic effect and thus cause protein denaturation [1]–[3]. In this model of indirect interactions, two alternative views have been put forward in which urea is regarded either to break [1],[2], or to enhance [3] water structure. The second model, in contrast, attributes the denaturing effect of urea to direct interactions between urea and the protein [4]–[6]. Also this model comprises different aspects: either the interaction of urea with polar residues or the peptide backbone, mainly via hydrogen bonding [5]—or hydrophobic interaction with apolar residues [4].
All of these possibilities, and various combinations thereof, have been suggested as the primary driving force of denaturation, and are still controversially discussed. Whereas some studies have provided support for the primacy of indirect effects [7]–[12], this concept has been challenged by many authors [6],[13],[14], and many recent studies provide increasing evidence for direct interactions as the primary driving force for denaturation [14]–[25]. Within this framework, however, it is controversially discussed whether either polar [11],[12],[17],[22],[23],[25] or apolar [4], [15], [16], [18]–[21],[24],[26] interactions between urea and the protein dominate.
Here we address this question by studying the relevance of direct polar and apolar contacts with all-atom molecular dynamics (MD) simulations. We have chosen the chymotrypsin inhibitor 2 (CI2) protein as an example, the folding kinetics and thermodynamics of which have been extensively studied experimentally [27]. We consider the CI2 in water as well as in aqueous urea solution, and perform a thought experiment ("Gedankenexperiment"), in which urea polarity is varied by scaling its partial atomic charges. The rational of this computer experiment is as follows. If polar contacts such as hydrogen bonds between urea and the protein constituted the determinant interaction for denaturation, one would expect hyperpolar urea to be an even stronger denaturant than real urea. If, in contrast, apolar contacts played the major role for denaturation, one would expect hypopolar urea to be the stronger denaturant. Therefore, by monitoring the respective denaturation strengths in the simulations, we will be able to decide which of the two interaction types drives urea-induced unfolding.
Simulation Setup
All simulations were performed using the Gromacs [28]–[30] program suite, versions 3.2.1 and 3.3, with the OPLS-all-atom force-field [31],[32]. The TIP4P water model [33] was used, and the urea force field was adopted from Smith et al. [34], which is a refined version of the original OPLS parametrization by Duffy et al. [18]. A cutoff of 1.0 nm was used for short-range Coulomb as well as Lennard-Jones interactions. Particle Mesh Ewald summation (PME) [35],[36] was used to calculate the long-range electrostatic interactions with a grid-spacing of 0.12 nm and an interpolation order of 4. All simulations were performed in the NpT-ensemble using Berendsen-type temperature-coupling [37] with a coupling coefficient of τT = 0.1 ps and Berendsen-type pressure-coupling [37] at 1 bar with a coupling coefficient of τp = 1 ps. To allow comparison with the simulations reported in [11], the simulation temperature was set to 333 K (except for one simulation at 300 K), and the same CI2 double mutant (E33A, E34A) was used. An integration timestep of 2 fs was used together with the LINCS constraint solver [38] for all covalent bonds.
The structure of the CI2 protein was taken from the Protein Data Bank [39], PDB-code 1YPC [40]. Unresolved side chain atoms for residue MET40 (residue number 59 in the pdb file) were added using the program WHAT IF [41]. The box-size was chosen such that a minimum distance of 1.5 nm between protein atoms and the box was kept. For the solvation of the protein, pre-equilibrated structures of water and 8 M urea were used (taken from [42]). Sodium and chloride ions were added to yield a 150 mM ion concentration and mimic physiological conditions. Prior to each simulation, a 200 step steepest descent energy minimization and a 500 ps equilibration run with position restraints on the protein heavy atoms were carried out.
To avoid over-interpretation of possibly anecdotal events, multiple simulation runs were carried out for each parameter set (Table 1). Two simulations of CI2 in water, three simulations with regular urea charges, two simulations with 25% urea charges, five simulations with 50% urea charges, four simulations with 75% urea charges, two simulations with 150% urea charges and two simulations with 200% urea charges were performed, each at 333 K. In addition, one simulation in water at 300 K was performed to define native contacts and native secondary structure (see below). The total simulation time of all simulations was ca. 7 µs.
We note that a computational thought experiment not dissimilar to the one performed here was conducted by Sorin et al. [43], who investigated the relationship between solvent and protein structure in a "hydrophobic titration" experiment employing different TIP3P variants.
Solvent accessible hydrophobic surface areas (SAS) were calculated using the double cubic lattice method [44] with a 0.14 nm probe radius. Native contacts and native secondary structure were defined using the simulation at 300 K in water (W300 K), rather than the crystal structure. This approach has the advantage that fluctuations of the native state were captured which allowed a more direct comparison with the unfolding simulations. Residues were defined to be in contact if the distance between the closest atom pair was not larger than 0.4 nm. Contacts were defined as native if they were present during more than 50% of the time in simulation W300K. Contacts between neighboring residues were not considered for the calculation of the native contact fraction.
Secondary structure was classified using DSSP [45]. The native secondary structure was defined as the most frequently occurring structure type for each residue seen in simulation W300K, which was similar to that of the crystal structure. Helix, β-sheet, and turn-elements were considered to calculate the fraction of native secondary structure content.
Contact Coefficient
To quantify the frequency of interactions between urea and the amino acids, we used the contact coefficient CUW [46] for a particular amino acid X,(1)where NX–U and NX–W are the numbers of atomic contacts of amino acid X with urea and water molecules, respectively. Atoms were defined to be in contact if close than 0.35 nm. CUW is normalized using the total numbers of urea atoms (MU) and water atoms (MW). Accordingly, a residue with a contact coefficient of CUW = 1.0 has no interaction preference for either urea or water. Values above 1.0 indicate preferential interaction with urea, values below 1.0 indicate preferential interaction with water.
The Native State in Water/Urea
As a reference, we first analyzed the dynamics and stability of the folded CI2 protein as well as its protein-solvent interactions both in water and in 8 M aqueous urea solution. Figure 1 shows the Cα root-mean-square-deviation (RMSD, panel A) and the solvent accessible hydrophobic surface area (SAS, panel B) for the simulations in water (W1,2, blue) and in 8 M urea solution (, green). As can be seen, the Cα-RMSD of the protein in both solvents shows similar fluctuations with an average value of 0.3 nm, and no significant differences between both solvents are seen. In particular, no unfolding is observed, which is expected from the measured millisecond time scale for CI2 denaturation [47].
(A) Cα-RMSD. (B) SAS for the two simulations in water (blue) and the 3 simulations in aqueous urea with regular charges (green). The solid bold lines show traces smoothed by a running average over 500 ps; dim lines show raw data.
In contrast, and perhaps unexpectedly, the average SAS in aqueous urea is 2–3 nm2 larger than in water. As can be seen in Figure 1B, this difference is significantly larger than the SAS fluctuations of single trajectories. Closer inspection reveals that this difference results mainly from few specific residues whose side chains are more solvent-exposed in aqueous urea than in water. In particular, MET1, LEU32, ILE44 and PHE50 contribute dominantly to this difference (0.22 nm2, 0.17 nm2, 0.30 nm2 and 0.29 nm2, respectively). With only a few exceptions (e.g., ARG43), however, also the side chains of almost all other residues are slightly more exposed in aqueous urea solution than in water. Because these amino acids are among those which were found to have particularly strong contact preferences for urea (see [46]), we expect that the increased exposure of these side chains is caused by favorable interactions with urea molecules.
To check whether this trend holds not only for tripeptides [46], but also for the whole protein, we quantified these interactions using the contact coefficient CUW. Figure 2C shows the CUW values for each amino acid type in the CI2, averaged over time and over the three simulations in aqueous urea solution (). Indeed, the obtained contact coefficients are largely similar to those calculated for the individual amino acids in tripeptides [46]. In particular, apolar and aromatic amino acids, as well as the backbone, have pronounced contact preferences for urea, whereas charged amino acids have preferences for water contact. This finding confirms that polarity/apolarity is clearly a determining factor for the specific interactions of urea with the CI2 protein residues, and provides further motivation for our approach to investigate protein stability in solutions of urea with modified polarity.
The four panels show CUW for the different urea partial charge scalings (A: 50%, B: 75%, C: 100%, D: 150%). The color characterizes the amino acids. Red: charged, yellow: polar, gray: aliphatic, blue: aromatic, green: apolar. For better comparability, all CUW are sorted according to CUW in urea50%.
We note that the remaining differences between the contact coefficients of tripeptides versus those observed here for CI2—quantified by a correlation coefficient of r2 = 0.69—suggests that effects from sequence and structure of the folded CI2 protein account for ca. 30% of the contact preferences.
Protein Stability in Hypo- and Hyperpolar Urea
To investigate the denaturation strengths of hyper- or hypopolar urea, the partial charges of urea were scaled to values of 25%, 50%, 75%, 150%, and 200%. For each of these modified degrees of polarity, the CI2 protein was simulated in aqueous solution. Urea with partial charge scaling of x% will be denoted as "ureax%".
Since it is a priori not clear that upscaling or downscaling urea partial charges does in fact enhance polar or apolar, respectively, interactions with the protein, we investigated the contact coefficients of each amino acid type in the CI2 for hypo- and hyperpolar urea. As can be seen in Figure 2A and 2B, hypopolar urea indeed shows less interactions with charged and polar amino acids, and enhanced interactions with less polar residues. Hyperpolar urea150%, in contrast, exhibits fewer interactions with those amino acids preferentially interacting with "regular" urea100% (Figure 2D). Interactions with charged residues are even preferred by urea150% over interactions with less polar residues. In summary, lowering the polarity of urea enhances its interaction preferences: less preferred interactions become even less frequent, and preferred interactions become even more frequent. An exception is ARG, which does not show enhanced interactions for urea150%. We attribute this effect to the fact that ARG contains large polar as well as apolar parts.
Having shown that upscaling or downscaling urea partial charges has the desired effect on the interaction strengths between urea and the different amino acids, we can now turn our attention to the influence on protein stability. Accordingly, we monitored the SAS for the different urea partial charge scalings (Figure 3). As can be seen, for hypopolar urea, the protein unfolds in all nine simulations (urea75% and urea50%, magenta and orange lines, respectively). In contrast, for hyperpolar urea150%, the SAS remains close to the native value and the protein remains stable in all simulations (black lines). In fact, the SAS is even smaller for hyperpolar urea than for regular urea, which suggests that hyperpolar urea compacts the folded state. Furthermore, this result suggests that urea150% would actually be a weaker denaturant than urea100%.
Blue: water, orange: urea50%, magenta: urea75%, green: urea100%, black: urea150%. The lines show traces smoothed by a running average over 500 ps. The histogram in the right panel shows the frequency of the respective SAS.
In summary, enhanced apolar interactions between urea and the protein destabilize the native state and induce unfolding of the CI2. Strengthening apolar interactions yields a stronger denaturant, while strengthening polar interactions yields a weaker denaturant.
We have also performed simulations with "extreme" urea25% and urea200%. However, these simulations exhibit artifacts which render them irrelevant for the present purpose and are therefore not shown in Figure 3. For partial charges scaled down to 25%, on the one hand, urea shows a strong tendency to self-aggregate to a hydrophobic layer in the periodic simulation box, which does not any more interact with the protein. Urea200%, on the other hand, induces a glass transition in the solvent, with drastically reduced urea diffusion coefficients (from ≈2.2·10−5 cm2/s to <0.001·10−5 cm2/s). As a result of the vanishing mobility, the urea molecules do not interact with the protein either. Similar underestimations of the diffusion coefficients in common force-fields has previously been observed for high ion concentrations [48].
We note that these two side-effects, urea aggregation and reduced diffusion coefficients, were also observed for the simulations with urea50% and urea150%, respectively, albeit to a (much) lesser extent. Care has to be taken, therefore, that these side-effects do not affect our main conclusions. In particular, one might argue that protein unfolding in urea50% is not necessarily a direct consequence of reduced urea polarity. Rather, it might be caused by inhomogeneities of urea concentration. However, since the observed unfolding events are quite similar to those observed for urea75%, where no significant aggregation is seen, we do not expect locally enhanced urea concentration to play a significant role.
For the simulations with hyperpolar urea150%, one might object that not enhanced protein stability, but reduced urea diffusion coefficient for urea150% (from ≈2.2·10−5 cm2/s to ≈0.1·10−5 cm2/s) is the reason that no unfolding is observed. To address this concern, two effects of this reduced urea mobility need to be considered. First, the reduced mobility of urea molecules implies much slower thermodynamic equilibration. And therefore, the thermodynamic equilibrium distribution at the protein surface might not be reached within the available simulation time. However, the diffusion time for a urea molecule to cross the whole box length is well within the simulations time (≈75 ns for urea150%), such that this effect can be excluded.
Second, the reduced mobility of urea molecules might slow down conformational changes of the protein due to higher solvent viscosity. Note, however, that conformational changes are seen on the simulation timescale, which lead to the observed compaction. Furthermore, as can be seen from the fast 10 ns SAS jumps in urea75% and urea50%, even a 20-fold enhanced viscosity is unlikely to prevent motions on a 500 ns timescale. This observation, together with the fact that other proteins, e.g. the Cold Shock protein, are observed to undergo large conformational changes in hyperpolar urea150% (data not shown) strongly suggests that the increased solvent viscosity does not compromise our interpretation.
The extent of both side-effects, self-diffusion slowdown and urea aggregation, is shown in the Supporting Information (Text S1).
Unfolding Pathways in Hypopolar Urea
In our simulations, the CI2 protein unfolds reproducibly in urea75% (all four simulations) and urea50% (all five simulations) which allows us to analyze unfolding pathways in more detail. To this aim, Figure 4 shows the unfolding pathways observed for the four simulations in urea75%. Here, the unfolding pathway is characterized by the fraction of native secondary structure versus fraction of native tertiary structure, measured by the fraction of native contacts. (The respective data for urea50% is provided as Figure S1 in the Supporting Information). In each of the nine cases, starting from the folded state (top right), the protein undergoes conformational changes eventually leading to denaturation and unfolding in all nine trajectories.
The numbers next to the protein structures denote the respective time of the snapshot in ns.
We first describe one unfolding trajectory () in detail, and subsequently discuss common features and differences of all nine unfolding trajectories. In simulation , reversible fluctuations of the secondary-structure (β-strand 3, ILE57-ARG62) trigger the first unfolding step. After 29 ns, a part of the coil region between β-strand 1 and β-strand 2 reorients. In particular, the sidechains of THR36, ILE37 and VAL38 rotate by about 180°, which apparently triggers, at 30 ns, a subsequent flip of the turn region formed by residues 22–25. This irreversible and fast unfolding step implies significant loss of native contacts and is followed by a longer phase of 80 ns during which the α-helix (res. 13–22) unfolds, with the ALA-rich region (ALA14, ALA15, ALA16) unfolding last at 110 ns. Subsequently, the turn (and former α-) region between residues 18–25 detaches from the protein core, while the ALA-region of the helix undergoes several partial refolding and unfolding events. Between 140 ns and 150 ns, further global unfolding rearrangements of the tertiary structure occur. At 150 ns, unfolding is completed with the disruption of β-strands 2 (res. 46–52) and 3 (res. 56–62).
Common Unfolding Features
Whereas the sequence and all the details of the described unfolding events are not necessarily similar in all unfolding trajectories, several common features emerge. In all simulations, unfolding proceeds stepwise, with alternating phases of loss of secondary and tertiary structure. In none of the simulations, both structure levels are seen to break down simultaneously; also not seen is the complete loss of one structure level before the other. Often, meta-stable parts of the trajectory, each sampled for a longer time (typically 100 ns and longer) and characterized by reversible fluctuations, are connected by fast transitions (about 5 ns), during which irreversible loss of native contacts occurs. Such alternating stepwise unfolding pattern is consistent with the nucleation-condensation mechanism of folding for the CI2 protein which has been derived from φ-value analysis [49].
In summary, a sequence of alternating unfolding steps is observed, which supports unfolding models that assume a strong coupling between tertiary and secondary loss of structure. We would like to emphasize that the sequence of meta-stable states seen in our simulations is consistent with the fact that CI2 is a two-state folder [27], because the observed transient states are both too short-lived and too heterogeneous to be resolved in current ensemble- or equilibrium-unfolding experiments.
Onset of Unfolding
Next, we investigated whether regions of the CI2 exist where unfolding is particularly likely to start. To this end, the RMSD per residue was calculated for the initial phase of unfolding (defined by a significant increase in the SAS from the native value) for each of the simulations U75% and U50% (Figure 5). For comparison, the top row shows the root-mean-square-fluctuations per residue in the native state (simulation W300K). Many initial unfolding steps are seen to occur in regions that exhibit large fluctuations already in the native state in water at 300 K. Examples are the C-terminal end of the α-helix (res. Q22) and the adjacent turn-region (res. D23–E26, simulations ), as well as the coil- and turn-regions between β-strands 2 and 3 (simulations ). In contrast, regions that show only small fluctuation in the native state, e.g. res. 5–18 in simulations , tend to unfold later. In summary, no unique unfolding "hot-spot" is found, but rather several regions where unfolding likely begins.
Blue corresponds to low, red to high RMSD. The numbers on the left denote the start and end times of the respective displayed trajectory segment in ns. Top row: root-mean-square-fluctuations per residue in the native state. In the one-letter sequence code below, red marks the α-helix and blue β-strands.
Common Transient Structures
This observation led us to investigate whether common transient structures or putative intermediates exist in the unfolding pathways. To this end, for every unfolding trajectory i, the RMSD was calculated with respect to every structure Xj (t) (with a time resolution of Δt = 100 ps) of each of the other unfolding trajectories j (data not shown). In this analysis, conformations which occur in trajectory i as well as in trajectory j, would be revealed by a minimum in the respective RMSD. Unexpectedly, no pronounced minima were found, which indicates that no pair of trajectories shares common global structures, and that unfolding proceeds structurally different in all nine cases. Rather, during unfolding as well as after complete denaturation, the protein explores quite different regions of phase space. This finding further implies that the transition state ensemble consists of conformations which are structurally more heterogeneous than the thermal fluctuations of the native state. These results are consistent with the previous observations of a broad transition state ensemble [50] and the fact that the CI2 protein is a two-state folder without pronounced intermediates [27].
Transition State Ensemble
We therefore attempted to analyze the transition state (TS) ensemble in more detail. Although the TS ensemble can not be rigorously defined from the nine trajectories at hand, a reasonable estimate can be given. To that end, we calculated the (non-equilibrium) density ρ of states for the SAS as reaction coordinate for each of the nine unfolding trajectories, which served to provide a rough free energy estimate, −kT log ρ. In all nine trajectories, the native state showed up as a minimum at low SAS values, with an adjacent clear maximum, which served to locate the TS (data not shown). In most simulations, this maximum was consistently seen at an increased SAS of 3–5 nm2. This agreement suggests that our approach provides a reliable estimate for the TS.
In all nine simulations, the overall structure of the TS is found to be similar to that of the native state (about 70% native contacts), but more expanded, in agreement with previous experimental [51]–[53] and simulation results [11],[54]. The α-helix is still intact, albeit with its central region bent away from the molecule's center in most simulations, whereas the β-sheet is already partially disrupted in most cases. In agreement with a previous simulation study [11], we find the TS ensemble to be heterogeneous with respect to the loops, turns, and terminal regions. After the TS, unfolding proceeds in six out of the nine trajectories with disruption of the β-structure before unfolding of the α-helix; conversely, in the remaining three simulations, the α-helix unfolds before the β-strands. In all cases, the time span between α- and β-disruption was rather short; therefore, no defined sequence of the two processes was established.
Residual Structure in the Denatured State
We finally focus on the residual structure in the denatured state. In particular, we investigate a possible polyproline II helix structure (PPII, φ = −75°, ψ = 150°) which has been suggested as prevalent configuration for the denatured ensemble from CD-spectroscopy results [55]. Recently, this suggestion has gained considerable attention due to accumulating evidence for residual structure of denatured proteins [56]–[59]. We note that the sampling of the denatured state is very limited in our simulations (≈650 ns in total for urea75%), such that we expect this analysis to provide rough estimates rather than accurate numbers.
Figure 6 shows the Ramachandran plot for the folded CI2 protein (averaged over all simulations with urea100%, panel A), and the denatured protein (averaged over the denatured ensemble in all simulations with urea75%, panel B). Similar distributions of the folded or unfolded ensemble were seen in the other simulations (data not shown).
The white circles show the areas which have been used for the calculation of populations densitites, which are shown in the lower panel for antiparallel β-sheet ("apβ"), PP2 or parallel β-sheet ("pp2/pβ"), and helical (α) configurations.
As expected, the native state predominantly occupies three regions in (φ, ψ) space; the α region around (−70°, −27°, "α"), as well as the β-sheet regions around (−83°, 128°, parallel or PPII, "pp2/pβ") and (−142°, 149°, antiparallel, "apβ").
For the denatured protein, the same three regions are populated, although with different occupancies (Figure 6B). In particular, the PPII/pβ region becomes the most populated one, particularly when the denatured protein is very extended (SAS >40 nm2), which supports the pronounced role of this secondary structure element. However, the other two regions remain populated: the population of the antiparallel-β region increases from 11% to 17%, while the population of the α-region decreases significantly from 27% to 13%. Note that the presence of these backbone angle configurations does not imply correctly formed secondary structure elements in the denatured state.
The unambiguous classification of PPII from (φ,ψ) is complicated by the fact that other secondary structure elements share similar backbone configurations. Therefore, several different definitions to calculate PPII content have been developed and applied in the past. Integration over the pp2/p β-peak in Figure 6 yields an increase of 26% to 32% relative population. With the definition from Jha et al [60] (−100°<φ<0°, 50°<ψ<280°), we found a PPII population of ca. 35% in the denatured structures as compared to ca. 30% for the folded CI2. A third definition (−120°<φ<60°, 120°<ψ<240°), from Makowska et al [59], yields similar results with increase from 35% to 40% PPII. Hence, for all three definitions, we observed a pronounced, but not absolute prevalence of PPII configuration in the denatured ensemble. This finding corroborates recent results by Makowska et al. [59], who argued that PPII might be one of several possible backbone conformations in the denatured state.
To elucidate whether polar or, in contrast, apolar urea-protein interactions are the key driving force for urea-induced denaturation, thought experiment simulations were performed, in which the respective denaturation strengths of hyperpolar urea (with strengthened polar interactions) or hypopolar urea (with strengthened apolar interactions) were compared. To this end, the CI2 protein was simulated in water, in regular urea, and in hypo- and hyperpolar urea, which was realized by scaling the partial charges of the urea force field.
In all nine simulations with reduced urea polarity, the CI2 protein unfolded within 300 ns. In contrast, the protein remained stable in the simulations with increased urea polarity, and the folded state was found to be even slightly more compact than in water. These results provide strong evidence that interactions with less polar parts—rather than polar interactions—are the main driving force for urea-induced protein denaturation. Together with previous results [46], a coherent picture for urea-induced protein denaturation emerges. Urea molecules accumulate around less polar side chains and exposed backbone, forming an interface between less polar protein surface and water. The resulting displacement of water molecules from the protein surface into bulk water is entropically and enthalpically favorable and reduces the hydrophobic effect, such that unfolding of the protein becomes favorable. The ability of urea to form hydrogen bonds to the protein backbone is not the main driving force for denaturation, but contributes to the overall energetics by preventing unsatisfied hydrogen bond sites at the protein backbone. This view is also in agreement with recent spectroscopic results which provide evidence against the dominant role of polar interactions and hydrogen bonds [61].
It is interesting to note a relation to the mechanism of chaperone-mediated folding. Recent investigations of the chaperone GroEL [62] provide support for the suggestion that the hydrophobic environment of the open state of GroEL facilitates unfolding, whereas the hydrophilic environment of the closed state of GroEL facilitates folding [63],[64]. In our simulations, we also find a more hydrophobic environment (aqueous solution of hypopolar urea) to facilitate unfolding, and a more hydrophilic environment (aqueous solution of hyperpolar urea) to facilitate folding.
For regular urea, the preferences of the 20 natural amino acids for contacts with either urea or water were largely similar to those found previously for tripeptides [46]. In particular, less polar residues interacted preferentially with urea, whereas polar and particularly charged residues had stronger preferences for interaction with water. As expected, the characteristics of this interaction profile were amplified for hypopolar urea, and inverted for hyperpolar urea.
The observation that the CI2 protein does not unfold within several hundred microseconds in urea with regular charges is consistent with the measured millisecond unfolding time [47]. We could not reproduce the complete nanosecond-unfolding seen in previous simulations [11], which however employed a cutoff-approximation for the long-range electrostatics.
On the structure level, our simulations suggest that denaturation proceeds rather heterogeneously and not via narrow, distinct pathways. In particular, unfolding of the CI2 was observed to start in stochastically one of several regions rather than one. However, regions with large structural fluctuations already in the folded state often turned out to be primary unfolding regions. Moreover, the nine unfolding pathways in the simulations with urea75% and urea50% turned out to share no common conformations during unfolding, which is consistent with the fact that CI2 is a two-state-folder without meta-stable folding intermediates. This heterogeneity of unfolding pathways prompts us to suggest an "inverted funnel"-scenario for the unfolding energy landscape, with multiple pathways leading from the narrow mesa of the folded state down to the relatively flat and extended region of the denatured ensemble.
Whereas no shared conformations were found in the different unfolding pathways on the detailed level, more general common features of the unfolding process emerge. In particular, in most of the simulations unfolding was observed to proceed with alternating and sequential loss of secondary and tertiary structure. This finding is consistent with the coupling between secondary and tertiary structure formation in the nucleation-condensation folding process of the CI2 inferred from spectroscopic and mutation studies [49],[65],[66]. It further suggests that the processes of structure-formation during folding and structure-loss during denaturation share common features.
Finally, our simulations allowed us to analyze the residual structure in the denatured state. Overall, relatively little residual secondary structure was seen, in agreement with previous CD studies [65]. Polyproline II turned out to be the most prominent, however not dominant residual structure in the unfolded ensemble. This finding supports the recent suggestion that polyproline II is one of several possible backbone conformations in the denatured state [59]. α-helical structure was found to be drastically reduced, whereas the population of β-sheet like backbone conformations was even slightly enhanced in the denatured state. Should such increase of β-sheet like backbone conformations turn out to be a common feature of unfolded protein ensembles, it might be relevant for the structural understanding of β-amyloid formation.
Supporting Information
Figure S1.
CI2 unfolding pathways in urea with 50% partial charge scaling.
Text S1.
Extent of self-diffusion slowdown and urea aggregation.
Author Contributions
Conceived and designed the experiments: MCS HG. Performed the experiments: MCS. Analyzed the data: MCS. Wrote the paper: MCS HG.
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text:
Hair Band
August 4th, 2013
It's time to consider a new barber.
(submitted by Deboarah in the UK)
Those are kitchen table cuts.
The translucent plastic frames on the kids also add a little pizzazz to the Bowl and Bed-head hair-dos.
WOOOO! that´s style! original hair and original teeth!!!!!!!
I am bowled over by this photo.
This is the handiwork of Mom using the orange-handled kitchen scissors, isn't it?
Either that, or kid gave herself a haircut the day before the photo appointment, and this was Mom's last-minute fix.

subset: commoncrawl


text:
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Seat Leasing Call Center
Business Process Outsourcing, Human Resource, business process
Just want to start with 2 to 100 seats?? Or you want to test the market? No worries! Seat Leasing is the best option...Available immediately and ready for use fully-equipped workstations for start-up…We can give you the highest quality technology and excellent English speaking agents at 50-70% Reduced price of the average Telemarketing Services.
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Tag - Photovine
Google shutters Slide; Photovine and other apps to be pulled
Google is reportedly set to shutter Slide, the social app developer that was acquired by the search giant just last year. The startup had continued to develop new apps after it was purchased, leading to the recent release of a new photo-sharing service, Photovine, that raised eyebrows when it was released on the iOS App Store without an equivalent launch on the Android Market.
Google opens up Photovine access to public
Photovine, Google's newest photo-sharing service, has opened up access to the general public via an iOS app update. Previously access to the service was limited to invites, much like other Google projects. Photovine distinguishes itself from most social networks by revolving not around individual users, but rather "vines," photo collections united by a single theme.
Google Photovine sharing service reaches iOS
Google has launched an iOS app for Photovine, its new photo sharing service. Access to the service is currently invite-only, so downloaders must either request entry in-app or visit the official Photovine website. The app is a free download and runs on any iOS device running iOS 4.2 or better.
Google confirms, teases Photovine sharing
The Photovine website went live on Tuesday, but is not yet ready for uploading. The site, the name of which Google trademarked in June, appears to be a photo sharing network run by the search giant. A teaser on the site simply states that it's a "fun way to learn more about your friends, meet new people, and share your world like never before."
Google lands Photovine trademark, hints deeper photo sharing
Google may be planning to expand its photo sharing beyond just Picasa. The company obtained a trademark for "Photovine" on June 7 and used proxy firm MarkMonitor to acquire Photovine.com just a week later. Few clues exist as to what it would involve, although the trademark bills it as "transmission of visual images and data by telecommunications networks" and "on-line social networking services."
Now AAPL Stock: 135.72 ( + 0.38 )
Cirrus creates Lightning-headphone dev kit
Apple supplier Cirrus Logic has introduced a MFi-compliant new development kit for companies interested in using Cirrus' chips to create Lightning-based headphones, which -- regardless of whether rumors about Apple dropping the analog headphone jack in its iPhone this fall -- can offer advantages to music-loving iOS device users. The kit mentions some of the advantages of an all-digital headset or headphone connector, including higher-bitrate support, a more customizable experience, and support for power and data transfer into headphone hardware. Several companies already make Lightning headphones, and Apple has supported the concept since June 2014. http://bit.ly/29giiZj
Apple Store app offers Procreate Pocket
The Apple Store app for iPhone, which periodically rewards users with free app gifts, is now offering the iPhone "Pocket" version of drawing app Procreate for those who have the free Apple Store app until July 28. Users who have redeemed the offer by navigating to the "Stores" tab of the app and swiping past the "iPhone Upgrade Program" banner to the "Procreate" banner have noted that only the limited Pocket (iPhone) version of the app is available free, even if the Apple Store app is installed and the offer redeemed on an iPad. The Pocket version currently sells for $3 on the iOS App Store. [32.4MB]
Porsche adds CarPlay to 2017 Panamera
Porsche has added a fifth model of vehicle to its CarPlay-supported lineup, announcing that the 2017 Panamera -- which will arrive in the US in January -- will include Apple's infotainment technology, and be seen on a giant 12.3-inch touchscreen as part of an all-new Porsche Communication Management system. The luxury sedan starts at $99,900 for the 4S model, and scales up to the Panamera Turbo, which sells for $146,900. Other vehicles that currently support CarPlay include the 2016 911 and the 2017 models of Macan, 718 Boxster, and 718 Cayman. The company did not mention support for Google's corresponding Android Auto in its announcement. http://bit.ly/295ZQ94
Apple employees testing wheelchair features
New features included in the forthcoming watchOS 3 are being tested by Apple retail store employees, including a new activity-tracking feature that has been designed with wheelchair users in mind. The move is slightly unusual in that, while retail employees have previously been used to test pre-release versions of OS X and iOS, this marks the first time they've been included in the otherwise developer-only watchOS betas. The company is said to have gone to great lengths to modify the activity tracker for wheelchair users, including changing the "time to stand" notification to "time to roll" and including two wheelchair-centric workout apps. http://bit.ly/2955JDa
SanDisk reveals two 256GB microSDXC cards
SanDisk has introduced two 256GB microSDXC cards. Arriving in August for $150, the Ultra microSDXC UHS-I Premium Edition card offers transfer speeds of up to 95MB/s for reading data. The Extreme microSDXC UHS-I card can read at a fast 100MB/s and write at up to 90MB/s, and will be shipping sometime in the fourth quarter for $200. http://bit.ly/294Q1If
Apple's third-quarter results due July 26
Apple has advised it will be issuing its third-quarter results on July 26, with a conference call to answer investor and analyst queries about the earnings set to take place later that day. The stream of the call will go live at 2pm PT (5pm ET) via Apple's investor site, with the results themselves expected to be released roughly 30 minutes before the call commences. Apple's guidance for the quarter put revenue at between $41 billion and $43 billion. http://apple.co/1oi1Pbm
Twitter stickers slowly roll out to users
Twitter has introduced "stickers," allowing users to add extra graphical elements to their photos before uploading them to the micro-blogging service. A library of hundreds of accessories, props, and emoji will be available to use as stickers, which can be resized, rotated, and placed anywhere on the photograph. Images with stickers will also become searchable with viewers able to select a sticker to see how others use the same graphic in their own posts. Twitter advises stickers will be rolling out to users over the next few weeks, and will work on both the mobile apps and through the browser. http://bit.ly/29bbwUE


text:
Chief, Division of Medical Oncology
My name is Lowell Anthony; I joined the UK Markey Cancer Center with more than two decades of experience in oncology and a special focus in gastrointestinal and neuroendocrine oncology. My clinical interests include drug development ranging from Phase I to III clinical trials. Agents targeting specific cellular signaling pathways and injectable radiolabeled compounds make up my past and current clinical investigative interests. I bring a clinical correlative component to complement the ongoing translational activities at Markey Cancer Center.
I attended Vanderbilt University Medical School, where I also completed a medical residency and fellowship training in medical oncology and clinical pharmacology. I have previously been on the medical faculties at Vanderbilt University and the Louisiana State University Health Sciences Center in New Orleans.
It takes a village to treat cancer, so when the patient is initially evaluated at Markey Cancer Center, a care plan is developed across specialties. So it's more encompassing than just a single visit to one specialist who treats cancer. Right now in oncology, the most exciting development is immunotherapy. Our challenge is to find out how we integrate this into other forms of how we treat cancer; both from other medicine, other drugs and potentially other modalities such as radiation.
Markey Cancer CenterWhitney-Hendrickson BuildingFirst Floor800 Rose St.Lexington KY 40536-0098
Call 859-257-4488Call 866-340-4488
Directions to Markey Cancer Center - Whitney-Hendrickson Building »
Markey Cancer CenterWhitney-Hendrickson BuildingRoom 134800 Rose St.Lexington KY 40536-0098
Call 859-323-5275Call 859-323-8043Fax 859-257-3757
Kentucky ClinicSecond Floor, Room D211740 S. LimestoneLexington KY 40536-0284
Call 859-323-3778Fax 859-257-9813
AmenitiesDining, Internet/Wi-Fi, Information desk, Pharmacy services, Wheelchairs
Kentucky Clinic Map (PDF, 616 KB) » Directions to Kentucky Clinic Parking »
UK HealthCare accepts many forms of insurance.
See list of insurers »
Markey oncologist Lowell Anthony says it takes a village to treat cancer - Dec. 29, 2016
Clinging to Life, Markey Patient Saved by Clinical Trial - Nov. 22, 2016


text:
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text:
3 of the Pub Landlords best ever stand-up shows brought together in this boxset collection. Includes: One Man, One Guvnor, The Only Way is Epic and Barrel of Fun Live.
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Trekking in the Everest Region
Practical Guide with 27 Detailed Route Maps & 52 Village Plans
By Jamie McGuinness | Paperback | 0 Review(s)
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Fully revised fifth edition of this practical guide with 27 detailed route maps covering not only the classic treks but also the wild routes: Everest expedition route Rolwaling Trekking from Lukla Salpa-Arun The Gokyo trek High passes Trekking peaks including Mera and Island Peak Information for all... read more budgets and trekking stylesRanging from lush terraced fields to the highest mountain on earth, the scenery is breath-taking. There are trekking possibilities to suit all budgets from independent trekkers on a shoestring staying in simple lodges with Sherpa families to travelers on all-inclusive guided treks with every luxury provided. Getting to Nepal from Europe, North America and Australasia Kathmandu trekking preparations and what to see Where to stay and eat Kathmandu and along the trails Employing a guide or porter in Nepal The environment how to minimize your impact on a fragile region and still take hot green showers Health and safety 27 route maps and village plans Plus 30 color photos.
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Lewis Hamilton has already opened talks with Mercedes with regard to signing a new deal.
Last week Mercedes confirmed they had signed Nico Rosberg to a new long-term contract, believed to run through to the end of 2016.
The German manufacturing giant is now eager to extend its partnership with Hamilton, who still has another 18 months to run of his initial three-year agreement.
Hamilton revealed last week ahead of the German Grand Prix he was "quite relaxed" with regard to his situation with Mercedes.
The 29-year-old stated he was "very, very happy in the team", adding he felt it would be "very difficult for me to see myself anywhere else" given his association with Mercedes since the age of 13.
Hamilton, however, kept the door slightly ajar for a move elsewhere as he said: "But you never know what the future may hold."
F1 German Grand Prix - In pictures
F1 German Grand Prix - In pictures
1/17 Grand Prix
Nico Rosberg celebrates his home win
2/17 Grand Prix
Rosberg drives to victory
3/17 Grand Prix
Fernando Alonso takes powers forward
4/17 Grand Prix
Felipe Massa crashes out early on
5/17 Grand Prix
People in historic dresses perform prior to the race in Hockenheim
6/17 Qualifying
Lewis Hamilton's Mercedes is taken away on the back of a lorry after crashing out of Q1
Getty Images
7/17 Qualifying
Lewis Hamilton's Mercedes is dragged out of the barrier at the Sachs hairpin
Getty images
8/17 Qualifying
Lewis Hamilton crashes out of Q1 for the Germand Grand Prix
9/17 Qualifying
Lewis Hamilton hits the barrier at the Sachs hairpin in Hockenheim
10/17 Third practice
A Red Bull mechanic awaits Daniel Ricciardo to come to pit lane
Getty Images
11/17 Third practice
Sebastian Vettel has his car wheeled into the garage by his Red Bull mechanics
Getty Images
12/17 Second practice
Nico Rosberg topped the timesheets once again at the end of second practice
Getty Images
13/17 Second practice
Williams test driver Susie Wolff was in action on Friday for the second Grand Prix weekend running
Getty Images
14/17 Second practice
Actor and Le Mans driver Patrick Dempsey is a guest driver in the supporting Porsche Supercup race at Hockenheim this weekend
Getty Images
15/17 First practice
Daniel Ricciardo against the Hockenheim forest backdrop
Getty Images
16/17 First practice
Fernando Alonso sweeps through turns two and three at Hockenheim
Getty Images
17/17 First practice
Nico Rosberg topped the timesheets after first practice
Getty Images
Hamilton, Rosberg and Mercedes are currently F1's dream ticket as the duo have won nine of the 10 races so far this season, whilst the team is firmly on course for the constructors' title.
Although there have been moments of consternation for motorsport boss Toto Wolff to contend with, the Austrian is eager to retain Hamilton beyond the end of 2015.
"We are already sitting together and negotiating with Lewis about a multi-year contract," said Wolff, speaking to Austria's Kronen Zeitung newspaper.
"Lewis is a fixed part of the team, and I would hope for a long time to come."
Any decision from Hamilton may be some way off yet, and much could depend on the outcome of this year's title fight with Rosberg.
Hamilton has fallen 14 points adrift after Rosberg's win on home soil at Hockenheim on Sunday, although the Briton deserves full credit for limiting the damage after a stirring drive from 20th to third.
A qualifying crash following a right-front brake disc failure again put Hamilton on the back foot in his duel with Rosberg.
Hamilton required acupuncture ahead of the race, and this week will also have physiotherapy prior to competing at a circuit he has made his own in recent years, the Hungaroring just outside Budapest.
Hamilton has won four Hungarian Grands Prix from seven attempts, and the last two in particular, so completing a hat-trick to go into the sport's summer break on a high would provide him with a huge tonic.
"I'm not going there with any concern about maintaining my record," said Hamilton.
"I'm simply going there to win, as was the case ahead of the weekend in Germany. I went into it with the right mentality and preparation.
"But things happened and got in the way. That's not to say I want luck or wish for luck. I never pray or wish for that. I just don't want anything like that.
"All I hope is the car sticks together so I can go out there and do the job."
To that end, Hamilton would love nothing more than three trouble-free days as he feels he deserves as much given his problems of late.
Qualifying especially has proven a thorn in his side of late as he has had to contend with issues or his own failings at the last five race Saturdays.
"A couple of times it's been down to me not getting the lap," said Hamilton.
"I think I would have got the lap in Germany, but then who knows. The car broke down, but that's okay, there are many more races to go."
With a laugh, Hamilton said: "I'm certainly not planning on starting at the back in Hungary!
"But a clean weekend is what I'm aiming for, to come away from there with some strong points ahead of the break when I'll get some rest going into the last part of the season.
With a huge grin he then added: "Maybe I'll go visit some Indians, or a Buddha and rub his belly or whatever.
"I'll try all the different religions to see if I can get some luck."
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Views 6038 05/11/2015
The Boeing 737 is a short- to medium-range twin-engine narrow-body jet airliner. Originally developed as a shorter, lower-cost twin-engine airliner derived from Boeing's 707 and 727, the 737 has developed into a family of nine passenger models with a capacity of 85 to 215 passengers. The 737 is Boeing's only narrow-body airliner in production, with the -600, -700, -800, and -900ER variants currently being built. A re-engined and redesigned version, the 737 MAX, is set to debut in 2017.
Originally envisioned in 1964, the initial 737-100 flew in 1967 and entered airline service in February 1968. Next the lengthened 737-200 entered service in April 1968. In the 1980s Boeing launched the -300, -400, and -500 models, subsequently referred to as the Boeing 737 Classic series. The 737 Classics added capacity and incorporated CFM56 turbofan engines along with wing improvements. In the 1990s Boeing introduced the 737 Next Generation with multiple changes including a redesigned wing, upgraded cockpit, and new interior. The 737 Next Generation comprises the four -600, -700, -800, and -900ER models, ranging from 102 ft (31.09 m) to 138 ft (42.06 m) in length. Boeing Business Jet versions of the 737 Next Generation are also produced.
The 737 series is the best-selling jet airliner in the history of aviation. The 737 has been continuously manufactured by Boeing since 1967 with 7,283 aircraft delivered and 2,759 orders yet to be fulfilled as of August 2012. 737 assembly is centered at the Boeing Renton Factory in Renton, Washington. Many 737s serve markets previously filled by 707, 727, 757, DC-9, and MD-80/MD-90 airliners, and the aircraft currently competes primarily with the Airbus A320 family. There are, on average, 1,250 Boeing 737s airborne at any given time, with two departing or landing somewhere every five seconds.


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Top Fund Manager Adds Banks After Stress Tests: Corporate India
Indian banks, which posted their biggest rally in four years yesterday, will defy predictions of worsening bad loans with the economy poised for a rebound, according to the nation's biggest money manager.
HDFC Asset Management Co.'s Mid-Cap Opportunities Fund is raising stakes in non-state lenders after the fund's stress tests showed they were "attractive," said Chirag Setalvad, who manages the portfolio. A Bloomberg gauge of 11 banks surged 9.9 percent yesterday after new central bank Governor Raghuram Rajan flagged measures to free up the industry. The index has declined 48 percent from a May high as the monetary authority raised interest rates to stem the rupee's unprecedented drop.
Setalvad forecasts banks' profitability, already at the lowest since 2009, will recover in two years as Rajan unwinds measures to support the currency that increased costs for banks, and as Asia's third-largest economy recovers. Overseas and local investors have been selling lenders since June as bad loans rose to the highest level in five years.
"The banking sector has got issues and when that happens it presents an opportunity," Setalvad, whose company manages $15.9 billion of assets, said in an interview, without identifying companies. "Even the ugly is getting priced in adequately. The general expectation is that the economy will recover therefore asset quality will improve."
Yes Bank Ltd. (YES) yesterday surged 22 percent to 287.50 rupees, paring this year's loss to 38 percent. HDFC Bank Ltd. (HDFCB), India's biggest lender by market value, rallied the most since May 2009. The BI Indian Banks index's 50 percent decline this year made it the worst performing among 222 Asian indexes tracked by Bloomberg.
'Underweight Banks'
Yesterday's "jump in bank stocks has to be short-covering; no long-only fund comes and buys in the manner that we saw," Prateek Agrawal, chief investment officer at ASK Investment Managers Pvt., said in an interview on Bloomberg TV India. "We are seriously underweight banks. The stress on their balance sheets in terms of bad loans will only increase."
Foreigners sold $1.4 billion of shares in lenders in the two months to July after buying $3 billion in the January to May period, latest data from the market regulator show. Indian fund managers cut their holdings in bank shares to 17 percent in July from 21 percent in January, data show.
Bad loans in the banking system rose to 3.92 percent of total lending as of June 30, the highest in at least five years, from 3.4 percent at the end of March, according to central bank data.
Bad Loans
The stressed-asset ratio, which measures bad loans and restructured assets as a percentage of loans, was at 10.02 percent at the end of June, central bank data show. The measure is approaching 10.4 percent, a level last seen in 2002, according to rating company ICRA Ltd.
Rajan's move this week to flag the need to cut the requirement for banks to invest in government securities and ensure lending to productive sectors of the economy, as well as allow some lenders to open branches without seeking RBI approval, will help improve the delinquent loan situation, said Manish Sonthalia, a Mumbai-based money manager at Motilal Oswal Asset Management Co.
The central bank will also look at ways to improve the recovery mechanism for bad loans and propose a database for large loans across lenders, Rajan, the ex-International Monetary Fund chief economist, said.
"The steps may not alleviate immediate stress," Seshadri Sen, a Mumbai-based analyst with JPMorgan Chase & Co. wrote in a report dated Sept. 4. "But some will have a major long-term impact on bank profitability and growth."
State Banks
HDFC Mid-Cap Opportunities Fund (HDFMCOG) added 100,000 shares of Yes Bank in July boosting its holding to 1.4 million shares and bought 30,000 shares of Axis Bank Ltd. (AXSB) to increase ownership to 500,000 shares, according to data compiled by Bloomberg. Holdings in state-owned Punjab National Bank (PNB) and Indian Bank (INBK) were unchanged in the period.
The cost to insure debt of State Bank of India (SBIN) surged 165 basis points in three months, according to data provider CMA. Setalvad is switching into non-state banks after his "call on public sector undertaking banks went wrong," he said. "I didn't anticipate the economic environment to get so adverse and as a result the asset quality to remain under pressure for as long as it has."
Rating Cut
India's $1.8 trillion economy expanded 4.4 percent in the three months through June from a year earlier, the slowest pace since 2009 amid weakening industrial production. The government's failure to cut its fiscal and current account deficits prompted Standard & Poor's this week to reiterate it may downgrade India's rating to junk.
The Reserve Bank raised two interest rates in July to support the currency, further imperiling economic expansion. The rate at which Indian banks lend to each other for three months surged to a five-year high of 11.59 percent on Sept. 4, compared with 8.52 percent at the end of June.
Easing of restrictions on banks and the government's move to attract overseas investments may help revive economic growth. The IMF forecasts India's economy will expand 6.3 percent in 2014. The RBI predicts a 5.5 percent growth in the year ending March 31.
India's lower house of parliament passed a bill allowing foreign investment in the country's pension funds for the first time. The government, which eased norms for retail companies and airlines, is also negotiating with opposition parties to raise the FDI cap in insurance to 49 percent from the current 26 percent.
"Expectations of earnings growth are low as investors are extrapolating low economic growth and low profit margins," Setalvad said. "There is a chance that we will be positively surprised because margins should revert and medium-term growth may be better than what is currently expected."
To contact the reporters on this story: Ameya Karve in Mumbai at firstname.lastname@example.com; Rajhkumar K Shaaw in Mumbai at firstname.lastname@example.com
To contact the editor responsible for this story: Arijit Ghosh at email@example.com


text:
The Sea
Our neighbour's frogs are finding each other very attracting this morning - they really making a row. Could've slept another 30 minutes or so... But a coffee does it already.
This shot is another one i digged out of the archives, it was taken somewhere near the northest point of the south island of new zealand.
The little car there is the one i bought when i was there, got me throughout the island and was well worth it. I sold again before i went home...
It is almost 2 years ago, since I saw the sea.
Music? Charles Trenet - La Mer :p
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Taken on March 27, 2007