We have cloned MKBP/human HspB2 and expressed it in E. coli. It was obtained mainly in the soluble fraction (). HspB2 was precipitated out with 20% saturated (NH4)2SO4. By a simple, one-step method of hydrophobic interaction chromatography, we could purify HspB2 in good yields and high homogeneity ().
SDS-PAGE pattern showing purification of HspB2 from the soluble fraction of E.coli BL21(DE3) cells over-expressing human HspB2.
We studied the secondary structure of HspB2 by far-UV Circular Dichroism (CD) spectroscopy. The far-UV CD spectrum of HspB2 exhibits a minimum at ~215 nm () and represents characteristic features indicating that the protein has significant β-sheet structural elements. Analysis of the CD spectrum using the CDNN program shows that the protein contains 3.4% helix, 50.5% β-sheet and 53.9% random coil.
Far-UV (a) and near-UV (b) CD spectra of HspB2.
The tertiary structure of HspB2 was investigated by near-UV CD spectroscopy. shows that the near-UV CD spectrum of HspB2 exhibits chirality in the region between 250–280 nm representing the chiral structure of phenylalanine and tyrosine residues and at 286 nm representing tryptophan residue, indicating a compact tertiary structure of HspB2. We further probed the tertiary structural aspects around its single tryptophan residue (at the 130th
position) by fluorescence spectroscopy. The intrinsic tryptophan fluorescence spectrum of HspB2 shows an emission maximum at 348 nm (), indicating that the sole tryptophan residue of HspB2 is exposed to the solvent. We have measured the accessibility of the tryptophan residue by fluorescence quenching by KI (). From the Lehrer plot 
shown in , the fractional accessibility of the Trp residue obtained was 0.8, indicating that the tryptophan residue is highly accessible to the quencher.
Figure 3 (a) Intrinsic fluorescence spectrum of HspB2. The excitation wavelength was 295 nm. The excitation and the emission band passes were set at 2.5 nm. The concentration of the proteins was 0.2 mg/ml. Fluorescence intensity is represented in arbitrary units. (more ...)
In order to understand the quaternary structure of HspB2, we have performed sedimentation velocity experiment using analytical ultracentrifugation (). Analysis of the sedimentation profile of the protein at 0.125 mg/ml using the program SEDFIT shows three populations of HspB2 with S20,w
values of 3.6, 5.2 and 6.5 S () corresponding to molecular masses of ~56, 97 and 134 kDa, obtained from fitting the profiles to Lamm's equation (See Materials and Methods
). However, when the experiment was performed at higher protein concentrations of 1 and 3 mg/ml the profile changed significantly. At a concentration of 1 mg/ml, the 56 kDa peak decreased and a significantly larger peak corresponding to 101 kDa was observed in addition to peaks corresponding to 149 and 205 kDa. Similarly at 3 mg/ml peaks corresponding to molecular masses of 114, 192 and 297 kDa were observed. (). As the sample preparation of different protein concentration involved dilution from the stock of higher concentration of the protein, it appears that the different species reported by the sedimentation experiments may be in equilibrium. Thus, the sedimentation velocity measurement shows that HspB2 exhibits polydisperse populations of multimeric species. Interestingly, oligomer sizes and the relative concentrations of the species were concentration-dependent.
Sedimentation velocity profile of HspB2.
We have investigated the hydrophobic surfaces on HspB2 using the hydrophobic probe, bis-ANS. Upon binding to the hydrophobic surface of a protein, the fluorescence intensity of bis-ANS is known to increase several-fold, accompanied by a blue shift in its emission maximum 
. shows that upon binding to HspB2 the fluorescence intensity of bis-ANS increases significantly accompanied by a shift in the emission maximum to ~505 nm, indicating that HspB2 exhibits accessible hydrophobic surfaces. Since hydrophobic interactions are important for molecular chaperone-like activity 
, we have compared its bis-ANS binding property with that of a known molecular chaperone, αB-crystallin. The extent of binding of bis-ANS to HspB2 is significantly less compared to that in the case of αB-crystallin (). In addition, it is also seen that while αB-crystallin exhibits saturable bis-ANS binding, HspB2 binding to bis-ANS does not seem to be saturable (the fluorescence intensity gradually increases without saturation behavior even up to the bis-ANS concentration of 200 µM (data not shown)). The reason for this unsaturable binding of bis-ANS to HspB2 is not clear. The observed lack of saturation in bis-ANS binding to HspB2 could be due to bis-ANS-binding-induced changes in the conformation as observed in the case of binding of ANS to carbonic anhydrase 
. However, the data in the presence of lower concentrations of bis-ANS (2–10 µM) suggests that HspB2 has much lesser exposed hydrophobic surfaces than αB-crystallin.
We have investigated whether HspB2 oligomers exhibit dynamic subunit exchange property. Subunit exchange has been studied in αA-crystallin by fluorescence resonance energy transfer using AIAS-labeled αA-crystallin as a donor and LYI-labeled αA-crystallin as an acceptor 
. HspB2 has a sole cysteine residue at the 118th
position in its sequence. We have labeled the cysteine residue of HspB2 by AIAS and LYI and studied whether the labeled protein exhibits FRET upon mixing. shows a decrease in the fluorescence intensity of AIAS with some increase in the fluorescence intensity of LYI upon incubating the mixture of AIAS- and LYI-labeled HspB2 at 37°C for 15 min. This result indicates a FRET between the fluorophores and hence the subunit exchange process between the AIAS- and LYI-HspB2 oligomers. shows the fluorescence spectrum of the mixture of AIAS- and LYI-labelled HspB2 incubated at 4°C. The spectrum exhibits the fluorescence spectral characteristics only of AIAS without any change with respect to incubation time, indicating that HspB2 does not exhibit subunit exchange at 4°C. However, we have compared the subunit exchange process of HspB2 at 37°C and 20°C as a function of time and found that the subunit exchange kinetics are not significantly different at these two temperatures (). Thus, our study shows that HspB2 exhibits the property of subunit exchange among its oligomeric species.
Subunit exchange studies of HspB2 at different temperatures.
We have investigated whether HspB2 exhibits chaperone-like activity towards amorphous aggregation of various target proteins. Upon reduction of the disulfide bonds, the B-chain of insulin aggregates 
, leading to increase in light scattering (). In the presence of HspB2, the aggregation of insulin decreased as a function of HspB2 concentration (, indicating that it exhibits chaperone-like activity against the DTT-induced aggregation of insulin. At insulin to HspB2 ratio of 1
0.125 (w/w), HspB2 exhibited ~37% prevention of aggregation, which increased to ~71% at insulin to HspB2 ratio of 1: 0.25. With further increase in HspB2, the extent of protection remained at ~72.5% ().
Chaperone-like activity of human HspB2 towards DTT-induced aggregation of insulin.
We have also performed the chaperone assay of HspB2 using thermal aggregation of citrate synthase at 43°C as a model system (-(c)). Surprisingly, unlike in the case of insulin, HspB2 did not exhibit concentration-dependent increase in prevention of aggregation of citrate synthase. At a citrate synthase to HspB2 ratio of 1: 0.25 (w/w), the light scattering decreased partially, indicating partial protection by HspB2. However, as the concentration of HspB2 increased, there was a gradual but slight increase in light scattering, indicating that percent protection decreased with increase in HspB2 concentration (). Even at a citrate synthase to HspB2 ratio of 1
8 (w/w), the percent protection decreased (data not shown). When the assay samples were centrifuged to separate the soluble protein and precipitate and the samples analyzed on SDS-PAGE, the amount of HspB2 in the precipitate increased with increase in HspB2 concentration, showing that HspB2 co-precipitated with citrate synthase (). Thus, it appears that HspB2 is unable to effectively prevent the aggregation of citrate synthase, whereas it prevents the aggregation of insulin to the extent of ~75%. It is possible that HspB2 is active at 37°C, the temperature at which the insulin aggregation assay was carried out, whereas it is ineffective at the elevated assay temperature of 43°C, the assay temperature for citrate synthase aggregation; hence the lack of activity against citrate synthase. We have, therefore, investigated the chaperone-like activity of HspB2 towards the aggregation of yeast alcohol dehydrogenase (ADH) at 48°C.
Effect of HspB2 on the thermal aggregation of citrate synthase (CS) at 43°C.
–(f) shows the effect of HspB2 on the aggregation of ADH. At an ADH to HspB2 ratio of 1
0.1 (w/w), HspB2 shows ~12.5% protection(curve 2, ). As the concentration of HspB2 is increased, it shows a progressive increase in the chaperone-like activity and at an ADH to HspB2 ratio of 1
2 (w/w), it shows ~83% prevention of aggregation (curve 5, ). The samples of ADH heated at 48°C in the absence or the presence of various concentrations of HspB2 in the chaperone assay described above were centrifuged to separate the soluble protein and the precipitate. These samples were then run on SDS-PAGE. It is evident from that while almost all the ADH was obtained in the precipitate in the absence of HspB2, the amount of ADH recovered in the soluble fraction increased as the concentration of HspB2 in the assay increased. It is interesting to note that unlike in the citrate synthase protection assay, very little HspB2 was found in the precipitate even at the highest concentration of HspB2 employed in the assay (). Thus, these studies show that HspB2 can exhibit chaperone-like activity even at 48°C. HspB2 has the ability to prevent the aggregation of insulin and ADH, but not that of citrate synthase, indicating that it exhibits target protein-dependent chaperone-like activity. This property seems to be different from that of other members of the mammalian sHsp family such as αA-crystallin, αB-crystallin, Hsp27 and Hsp22 
We have investigated whether HspB2 exhibits chaperone activity in preventing well-ordered amyloid fibril formation. α-Synuclein is a natively unstructured presynaptic protein whose aggregation and amyloid fibril formation is involved in some neurodegenerative diseases such as Parkinson's disease, multiple system atrophy and other synucleinopathies including dementia with Lewy bodies 
. The anionic detergent SDS, which mimics membrane environment, has been shown to promote amyloid fibril formation in vitro 
. We have investigated the effect of HspB2 on the SDS-induced amyloid fibril formation of α-synuclein as a model system. shows the amyloid fibril formation of α-synuclein upon seeding with the sonicated preformed fibril seed as monitored by Thioflavin-T (ThT) fluorescence. At an α-synuclein to HspB2 ratio of 1
0.5 (w/w), HspB2 prevents the seeded amyloid fibril growth of α-synuclein to an extent of ~54%. The extent of prevention of amyloid fibril formation increases to ~63% and to ~78% at 1
1 and 1
1.5 (w/w) ratios respectively of α-synuclein to HspB2 (. This result indicates that HspB2 exhibits chaperone property in preventing the amyloid fibril formation of α-synuclein.
Prevention of α-synuclein amyloid fibril formation by HspB2.
Interestingly, HspB2 which is predominantly present in the cytosol of cells translocates to the surface of mitochondria upon heat stress and protect cells from heat-induced cell death 
. We have investigated temperature-induced conformational changes of HspB2, if any. shows the far-UV CD spectra of HspB2 at 45°C and 55°C. The overall negative ellipticity is increased, with the increase being more pronounced below 210 nm. shows the changes in the ellipticity at 214 nm as a function of temperature. The negative ellipticity gradually increases till 45°C and increases more sharply above this temperature. These results indicate that HspB2 does not undergo global unfolding and loss of its entire secondary structure in the temperature range studied. However, the increase in negative ellipticity which is more pronounced below 210 nm indicates a significant conformational alteration involving some local unfolding of its secondary structural elements.
Temperature-induced changes in the conformation and chaperone property of HspB2.
shows that the near-UV CD spectra of HspB2 at 25°C and at 45°C differ only marginally and hence marginal changes in its tertiary structural packing around the aromatic amino acid residues. The ellipticity at 286 nm as a function of temperature shows marginal changes till 45°C and then decreases sharply with further increase in temperature (). We have labeled the sole cysteine residue at 118 (in the “α-crystallin domain”) with the fluorescent probe AIAS and measured its fluorescence polarization as a function of temperature (). The polarization value increases gradually till 45°C, and decreases more sharply above this temperature resulting in the increased mobility of the probe above 45°C, indicating the increased flexibility of the region at higher temperature. Thus, all these results indicate (i) significant (local) conformational change of HspB2 around the heat shock temperatures (40–45°C) and (ii) that HspB2 loses its tertiary structure while having significant secondary structure at higher temperatures (eg. 55°C), suggesting that HspB2 exhibits a transition to a molten-globule-like state 
Having observed temperature-dependent conformational changes in HspB2, we have investigated the effect of temperature on its chaperone-like activity towards the aggregation of insulin. shows that the percentage protection offered by HspB2 to insulin increases significantly with increase in temperature, indicating that HspB2 exhibits higher chaperoning efficiency at temperatures relevant to heat shock.