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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
FEBS J. Author manuscript; available in PMC 2017 August 1.
Published in final edited form as:
PMCID: PMC4992417

Protein folding on Biosensor tips: Folding of Maltodextrin glucosidase monitored by its interactions with GroEL


Protein folding has been extensively studied for past four decades by employing solution based experiments such as solubility, enzymatic activity, secondary structure analysis, and analytical methods like FRET, NMR and HD exchange. However, for rapid analysis of the folding process, solution based approaches are often plagued with aggregation side reactions resulting in poor yields. In this work we demonstrate that a Bio-Layer Interferometry (BLI) chaperonin detection system can be potentially applied to identify superior refolding conditions for denatured proteins. The degree of immobilized protein folding as a function of time can be detected by monitoring the binding of the high-affinity nucleotide-free form of the chaperonin GroEL. GroEL preferentially interacts with proteins that have hydrophobic surfaces exposed in their unfolded or partially folded form so a decrease in GroEL binding can be correlated with burial of hydrophobic surfaces as folding progresses. The magnitude of GroEL binding to the protein immobilized on Bio-layer interferometry biosensor inversely reflects the extent of protein folding and hydrophobic residue burial. We demonstrate conditions where accelerated folding can be observed for the aggregation prone protein Maltodextrin glucosidase (MalZ). Superior immobilized folding conditions identified on the Bio-layer interferometry biosensor surface were reproduced on Ni-NTA sepharose bead surfaces and resulted in significant improvement in folding yields of released MalZ (measured by enzymatic activity) compared to bulk refolding conditions in solution.

Keywords: Immobilized protein folding, Biolayer interferometry, Protein folding on Biosensor Tips, GroEL binding to monitor protein folding


Numerous experimental techniques are available to study protein folding and the interactions between chaperonins and substrate proteins. In vitro studies have been done by monitoring the changes in secondary structure during the unfolding and refolding of substrates utilizing circular dichroism [1, 2] and FTIR [3]. Where applicable, fluorescence spectroscopy can also monitor folding collapse near the micro-environment of the fluorophore and reports global folding kinetics (intrinsic or extrinsic) provided that more global orthogonal methods mirror the folding kinetics [4]. Many techniques like FRET [5], NMR [6], Cryo-EM [7] and HD exchange based mass spectroscopy [8] have been previously employed for detailed analysis of protein folding process. Single molecule studies are now employed widely for studying protein folding [9]. In vivo experiments analysing soluble protein content in cells and activity from whole cell lysates can also be used [10]. These techniques usually require relatively high protein concentrations or need extensive preparation, expertise, and time to run the experiments. Thus there is room for employing a rapid, inexpensive, easy to use analytical tool to rationally identify superior conditions to aid in the protein folding process. In the present study, a Bio-Layer Interferometry – GroEL chaperonin protein folding detection system has been utilized to monitor protein folding on the Bio-layer interferometry biosensor surface. The use of the chaperonin as a detector for partially folded protein intermediates comes from the well-known observation that the nucleotide-free high-affinity GroEL chaperonin tightly binds to protein folding intermediates to form complexes that then can be detected using simple gel filtration or immunoprecipitation assays [11]. In essence, the nucleotide-free high-affinity GroEL can be thought of as a folding quencher provided that the folding reaction is sufficiently slow. The Bio-layer interferometry technique is a label free method that can monitor protein-protein interactions with similar outputs (i.e. kinetic readouts and signal amplitudes) to surface plasmon resonance (Figure 1). A histidine-tagged version of maltodextrin glucosidase (MalZ), an aggregation prone protein was selected as a model system for this study since this is a large protein and should easily interact with the large GroEL binding site without steric interference from the Ni-NTA biosensor surface. On its own, unfolded MalZ does not refold very well due to deleterious self-aggregation. In the presence of the chaperonin GroEL alone, self-aggregation is prevented and refolding can be initiated by adding ATP and GroES [12]. Since the chaperonin does not bind to properly folded proteins, the high-affinity nucleotide-free chaperonin should only bind to partially folded conformers of MalZ [13] that are present on the Bio-layer interferometry biosensor surface. The results presented herein show that as the immobilized protein refolds, the GroEL chaperonin binding signal progressively diminishes as a function of time.

Figure 1
The Bio-Layer Interferometry method to analyse protein-GroEL interaction


Construction of a MalZ biosensor and analysis of GroEL interactions

The construction of a Bio-Layer Interferometry MalZ biosensor was accomplished by binding native folded functionally active His-tagged MalZ onto Ni-NTA Biosensor tips. Binding of MalZ to the tip surface was observed by an increase in the Δλ (Recorded as Δnm) signal, and retention of this signal was observed upon washing with buffer containing ATP. The effect of nucleotide was tested on immobilized MalZ because it is a potential chelator that was used in subsequent experiments to dissociate GroEL from immobilized MalZ. As shown in Figure 2a, ATP addition did not affect the MalZ loading. The shaker speed of the equipment was kept at 2200 RPM to avoid mass transfer errors during the MalZ loading and GroEL binding. The Ni-NTA biosensors are used because denaturation of immobilized protein with GdnHCl on the biosensor tip is possible without any significant dissociation of the His-tagged protein within the time frame of the experiments (Figure c - step 11). MalZ binds to the Biosensor with a magnitude of binding reaching up to 3 Δnm in a 300 second loading step with 0.5 µM concentration of MalZ. (Figure 2a). The initial runs were used as control and the loading concentration and magnitude were further optimised in subsequent experiments. GroEL does not tightly bind to the biosensor tip in absence of MalZ. However, a low nonspecific adherence of 0.2 Δnm magnitude was recorded with a high concentration (1µM) of GroEL in 300 Seconds which rapidly reduced up to a magnitude of 0.1 on washing with buffer. This rapid return suggests that whatever GroEL binding magnitudes were recorded in the experiments were because of its binding to MalZ and the adherence of GroEL to biosensor is negligible (Figure 2b). Background GroEL adherence to the tip was analysed as a control in each experiment according to buffer and concentrations of GroEL used and was subtracted while analysing the results. GroEL binding to native MalZ was analysed and no increase in signal was observed upon addition of GroEL to native protein, showing that GroEL does not bind to native MalZ, which suggests that no exposed hydrophobic surface is present on the native protein. However, an increase in GroEL binding signal was observed when GroEL was added after denaturation of immobilised protein. Denaturation was done by 4 M Guanidine hydrochloride for 5 minutes. The loss of activity and loss of intrinsic fluorescence of MalZ within 5 minutes of 4 M GdnHCl treatment was confirmed through previous experiments. The experiment suggested that GroEL does not bind to the native MalZ, but associates with non-native conformations of MalZ (Figure 2c). GroEL binding was also observed when MalZ was denatured prior to the immobilization. The monitoring of the folding of denatured MalZ on biosensor tips is based on the observation that GroEL does not bind to native MalZ but binds to the non-native or unfolded form. The GroEL binding and ATP induced release steps could be monitored in real time with the output recorded on the sensogram.

Figure 2
Monitoring of protein folding process on the Biosensor

MalZ folding on Biosensor tip monitored through GroEL binding

In order to clearly understand the concept of monitoring protein folding in terms of its interaction with GroEL, a demonstrative experiment was performed. GroEL binding to native MalZ, denatured MalZ, refolded MalZ and further denatured MalZ has been measu red and compared to demonstrate the method. This was a demonstrative experiment and not a quantitative experiment so higher protein concentrations were used for the ease of displaying the sensogram. Native MalZ was first bound to the biosensor to a magnitude of 5.5 Δnm in step 1 (Figure 2c). As a control, binding of GroEL to native MalZ was attempted in step 2 of this experiment but as observed previously there was no binding on addition of GroEL to native protein. In the next step (step 3) MalZ was denatured by 4M GdnHCl in the immobilised state only. Initial GroEL binding to the denatured MalZ (step 4) was observed with a considerable magnitude which was about 1.3 Δnm. ATP was added at a final concentration of 10mM. ATP mediates a conformational change in GroEL which leads to protein release. In this case the signal observed was the release of GroEL from refolded or partially folded immobilized MalZ and the release event is reflected through a decrease in signal amplitude (step 5 and 6) and total release could be confirmed by the return of the GroEL binding signal to the original baseline prior to GroEL addition. The GroEL binding step was repeated (step 7) and it was observed that the GroEL binding magnitude decreased to about 0.8 Δnm suggesting that a fraction of MalZ was folded. The percentage or fraction of folding has been determined as the total decrease in terms of extent of GroEL binding, and thus the total folding comes out to be ~38% as the GroEL rebinding to MalZ (step 7) after ATP mediated release is about 62% of the original binding between denatured protein and GroEL (step 4). The GroEL was then removed by dipping the biosensor in 10mM ATP solution (Step 8, 9). GroEL dissociates completely and the baseline could be traced back to the same magnitude as it was just before GroEL association. Now the refolded MalZ was denatured again (step 10) with the same concentration of GdnHCl used for the first denaturation. At this stage (step 11) the extent of GroEL binding was compared to the GroEL binding in the initial binding step (step4). The binding magnitude in this step was about 1.25 Δnm, which was similar to the initial GroEL binding just after denaturation of MalZ (Figure 2c). This suggests that the decrease in GroEL binding to MalZ (in step 7) was due to refolding of a fraction of immobilised MalZ. Thus, this experiment shows refolding of MalZ depicted by decrease in signal of GroEL binding in step 7. It also shows that GroEL binding and ATP mediated release of GroEL from substrate proteins can be monitored in real time and finally it confirms through the final step 11 that decrease in magnitude can be correlated to protein folding as it clearly rules out the possibility that the decrease in GroEL association signal during the step 7 after refolding is because of loss of immobilised MalZ from the biosensor This experiment demonstrates the concept to study folding of a protein on the basis of chaperone interactions.

Optimization of MalZ loading concentrations and comparison with GroEL binding

In order to find out a proper concentration of MalZ which does not saturate the biosensor tip and to maintain a linearity in binding of MalZ to GroEL for more quantitative experiments, an optimization of MalZ loading concentration was carried out. The advantage of the method as a screening tool can be only proposed when least protein concentrations can be used. Hence, lower concentrations giving reliable signals were optimized. It was found that MalZ loading signal on the biosensor follows a linear relationship from 12.5nM to 0.25µM concentration and then it starts to saturate at higher concentrations (Figure 3a), when the loading was done in a 60s step. The optimum concentration of MalZ protein was chosen at 50nM as this will ensure that overloading of the biosensor surface will be avoided. Optimal loading also prevents intra-surface aggregation from interfering with further analysis. The ratio of GroEL binding is linear to the MalZ load at lower concentrations (Figure 3b). This experiment also further consolidated the concept that the GroEL binding to MalZ on the biosensor is proportional to the amount of unfolded or denatured protein loaded on the biosensor. This optimization was also important as normalization could be done in terms of MalZ loading if any minor differences in MalZ loading is observed in comparative experiments. Further, GroEL binding to 50nM MalZ load at different GroEL concentrations were plotted (Figure 3c). Based on these results, GroEL concentration of 0.2µM was used for further experiments with a MalZ loading concentration of 50nM, apart from minimizing the steric hindrances, requirement of such low concentrations of protein and GroEL are definitely an advantage in this screening tool. These results also proved the linear dependency of GroEL binding to MalZ protein load on the biosensor in the range which was used for further experiments.

Figure 3
Optimization of MalZ and GroEL concentrations for the folding studies

Identification of superior refolding condition using temperature in the presence a physiologically relevant buffer (potassium glutamate)

The folding of MalZ protein was measured using GroEL binding as a parameter. It was evident from the previous experiment where a GroEL binding and release cycle is shown (Figure 2c) that GroEL binding decreases as the protein is allowed to fold while in an immobilised state. Refolding of MalZ at different time points of 1, 2, 5, 10, 30 and 60 minutes was assessed using the GroEL quench protocol at three different temperatures 12°C, 24°C and 37°C. The magnitude of GroEL binding (i.e. GroEL quench) to MalZ was plotted against the refolding time of the immobilized protein (Figure 4a). The folding profile at 12°C showed slow MalZ refolding with a final recovery of 36% native signal after one hour. The refolding profile does not reach a plateau value within this time frame suggesting that the refolding is incomplete after 1 hour. As the temperature is increased to 24°C, the folding after 1 hour was recorded to be 77% with little hint that the reaction is approaching a plateau value. At 37°C, the folding magnitude detected by the GroEL quench protocol indicates that refolding saturates at around 86% as the values are similar at 30 and 1 hour refolding. Based on reversibility experiments (ATP addition to release any bound GroEL and denaturation) on the biosensor tip (Figure 2c), >90% of the originally loaded MalZ remains attached to the biosensor surface at 37°C throughout the immobilized refolding reaction even after 1 hour.

Figure 4
Optimizing MalZ refolding conditions under immobilized conditions under different temperatures

To confirm that the on surface folding conditions can be scaled up, the folding of MalZ under different temperatures was also analysed using an equivalent Ni-NTA resin capture system where the readout for folding is assessed by measuring the activity of released MalZ at equivalent time points used in the chaperonin BLI refolding quench system. The efficiency of the immobilized Ni-NTA surface refolding was compared with the “in solution” refolding. In both cases the protein was allowed to refold for 15 minutes at temperatures of 12°C, 24°C and 37°C. As previously observed [12] spontaneous refolding of MalZ in the absence of the GroEL chaperonin in solution is inefficient because under these concentrations the protein aggregates during refolding. In contrast, the immobilized refolding scheme resulted in a significant improvement in regain of activity at all temperature conditions (Figure 4b). The refolding yields from resin based immobilization and release was highest at 37°C, in line with what was observed for the BLI GroEL quench protocol. Although the results cannot be exactly compared in a quantitative sense, the results were highly correlated. The conditions used here show that the rate of folding of MalZ is fastest at the physiological temperature of 37°C under native buffering conditions potassium glutamate [14, 15, and 16].

Conformational states of non-native MalZ required for GroEL binding on biosensor tip monitored through intrinsic fluorescence spectroscopy

It was evident from these experiments that GroEL binding to substrate protein is dependent on the availability of non-native proteins. To verify the relationship between the GroEL binding magnitude to MalZ and the extent of conformational changes during unfolding of the MalZ protein, a comparison was made between the in solution fluorescence monitored profile of MalZ unfolding with the MalZ biosensor BLI-GroEL quench profile. To measure the MalZ denaturation progression on the MalZ BLI biosensor, a number of identically loaded MalZ biosensor were incubated in increasing concentrations of GdnHCl at 25°C. The biosensor was then removed from the denaturant solution, washed for 5 second to remove any residual denaturant and then dipped into refolding buffer containing nucleotide free GroEL to assess the amount of unfolded/partially folded protein that is present on the BLI biosensor surface. The increase in magnitude of GroEL binding was compared to the change in fluorescence intensities of MalZ at a fixed emission wavelength (334 nm) as well as average emission wavelengths (Figure 5a) at increasing concentrations of GdnHCl. The BLI sensogram signal (due to GroEL binding to denatured MalZ) saturates above 2M GdnHCl concentration. Likewise, changes in fluorescence intensity also shows a similar plateau signal above 2 M GdnHCl. The rates of denaturation profiles at intermediate denaturant concentrations were remarkably matched. The denaturation experiments were repeated in the presences of known protein stabilizers such as glycerol (Figure 5b) and sucrose (Figure 5c). The presence of the stabilizer osmolytes in the denaturant solution delays the protein denaturation yielding denaturation profiles that are similar and are right shifted with respect to denaturant concentration for the GroEL binding BLI biosensor signal and the decrease of fluorescence. This particular comparative scheme is useful in assessing potential stabilizing conditions for folding and refolded protein systems.

Figure 5
GroEL binding related to MalZ unfolding


In this work, we demonstrate that the BLI GroEL detection system can be used to detect the time it takes for an immobilized protein on a BLI biosensor to acquire a collapsed conformation that does not or minimally interacts with the nucleotide free high affinity GroEL chaperonin. The biosensor surface Ni-NTA chemistries can be used to immobilize his tagged protein where unfolding and refolding reactions can occur without aggregation interference. The refolding on the microscale BLI surfaces correlates with the scaled-up Ni-NTA bead resin release assay and provides an orthogonal method to readily screen superior folding conditions. Chaperonin binding to a folding protein indicates that the folding reaction proceeds through folding intermediates that expose hydrophobic surfaces. Conditions that cause rapid burial of hydrophobic surfaces through simple folding will show decreased interactions with the chaperonin. This GroEL-BLI folding detection system can potentially identify solution conditions and or ligands that can accelerate protein folding and diminish aggregation. The readout for extent of folding of a protein correlates with the reduction in signal intensity from GroEL binding to the folded protein as compared to the binding signal from fully denatured protein. Ready dissociation of GroEL from immobilized MalZ with ATP, but not with buffer alone, indicates that the binding of GroEL to partially folded MalZ is specific. Since the promiscuous GroEL chaperonin generally binds to virtually any hydrophobic surface, this approach can be utilized for an extremely wide variety of proteins. The results from various experiments reported in this manuscript demonstrate how GroEL binding with denatured MalZ decreases gradually, correlating with the refolding of the immobilized protein molecules and indicating that once refolded, the protein does not rapidly revert to its original unfolded form. The rate of folding of MalZ protein under immobilized condition on BLI tips clearly increases as the temperature increases from 12 to 37°C. Understandably, the close correlation of tryptophan fluorescence changes for MalZ exposed for 5 minutes with increasing denaturing solutions compared with the increasing GroEL binding signal to MalZ on the BLI biosensor under the same denaturing conditions indicates that assessing the folding status of a protein on the biosensor surface is a valid approach. It has been demonstrated that the binding of MalZ to GroEL is maximum at a denaturant concentration where the change in intrinsic fluorescence signal is also maximum. The addition of polyol folding stabilizers such as glycerol and sucrose to the denaturant solution understandably delays the unfolding reaction and concomitantly results comparable time dependent delays in solution unfolding (Figure 5).

The assessment of protein folding by employing a chaperone based method on BLI can provide significant advantage in terms of time and protein concentrations required when optimization for better folding conditions are required prior to the experiments with more precise methods. This method is a fast and easy way to optimise/ access the temperature and refolding buffer conditions for refolding while avoiding large scale protein aggregation. One could conceivably use other chaperone proteins besides GroEL. However as discussed in Naik et al. [17, 18], the nucleotide free GroEL has the tightest binding affinity for partially folded or hydrophobic protein, is stable, binding is reversible, the binding site is large and the binding is promiscuous. Any conformational alteration, destabilization in the structures of certain proteins in solution can also be identified by this same method [18]. This approach also works with a reversed orientation (GroEL on the Biosensor) BLI method [17]. Binding and detection of partially folded proteins can be done rapidly and with great ease. For example, with the single channel BLI unit (BLItz), minimum sample volumes of 4 µl at a concentration of 50nM are sufficient for loading stock of MalZ. From a biotechnology standpoint, this technique could be used as an easy initial method to screen the initial state of folding for protein substrates and aid in screening refolding conditions at very low concentrations before proceeding to scale up protocols that require larger protein concentrations.

In summary, the Bio-Layer Interferometry coupled with a GroEL detection system provides an easy, convenient, and fast method for rapidly monitoring refolding of immobilized proteins. Refolding of aggregation prone Maltodextrin glucosidase has been demonstrated using this BLI – GroEL method where a decrease in specific binding (i.e. reversible with ATP addition) correlates with the acquisition of a native active fold. This technique can prove to be a rapid, efficient and convenient method to determine superior folding conditions. The results were validated by conventional experimental method like enzymatic activity assay and fluorescence spectroscopy where the superior folding conditions can be determined using a micro-scale BLI chaperonin detection system can be replicated on scaled-up bead-based platforms.

Material and Methods

Maltodextrin glucosidase preparation

Plasmid PCS19 containing the malZ gene was a generous gift from Prof. Winfried Boos, (University of Konstanz, Germany). The plasmid contains malZ gene under control of a strong IPTG inducible T5 promoter and it contains ampicillin resistance for selection and imparts a C-terminal 6x His tag to the protein. E.coli BL-21 cells were transformed with the plasmid and grown at 37°C in Luria Bertani medium (HiMedia, Mumbai, India) containing 80µg/ml ampicillin (HiMedia, Mumbai, India). Induction for protein overexpression was done with 1mM IPTG (SRL, Mumbai, India) when O.D. at 600nm reached to a value of 0.6. The culture was kept for 8 hours post induction and protein expression was confirmed on a SDS PAGE gel. MalZ purification was carried out by Nickel affinity chromatography on a 5ml His-Trap column and Akta Purifier (GE Healthcare, Little Chalfont, U.K.) was used for the purification process. 20mM sodium phosphate buffer (Merck, Darmstadt, Germany) containing 500mM NaCl (Merck, Darmstadt, Germany) were used for lysis and column equilibration. MalZ was eluted near 200mM imidazole (Sigma Aldrich, Missouri, U.S.A.) through a linear gradient of imidazole from 20mM to 500mM.

Chaperone preparation

GroEL containing plasmid pACYC-EL was gifted by Prof. Hideki Taguchi (Tokyo Institute of Technology, Yokohama, Japan). This plasmid contains the groEL gene regulated by tac promoter and it uses chloramphenicol resistance marker for selection. E.coli BL-21 cells were transformed with this plasmid and were grown on Luria Bertani medium containing 20µg/ml chloramphenicol for selection. Induction was done with IPTG to a final concentration of 1mM when the O.D. at 600nm of the culture was around 0.6. Purification of GroEL was done following the protocol outlined in Wang et al. [19]. Q-Sepharose anion exchange column (GE Healthcare, Little Chalfont, U.K.) was used for anion exchange chromatography. A Source 15ISO column (GE healthcare, Little Chalfont, U.K.) was used for hydrophobic interaction chromatography to remove hydrophobic contaminants and the purified GroEL was further treated with affigel blue (BioRad, California, U.S.A.) for 18 hours to obtain GroEL free of tryptophan containing contaminants [20]. The purity of GroEL was checked by fluorescence spectra and tryptophan fluorescence was negligible after affigel blue treatment as compared to considerable fluorescence before affigel blue treatment [21, 22]. The second derivative wavelength scan in UV region did not show any indole absorbance in the 291 -295 nm, thus confirming high purity GroEL preparation [23]. The purified GroEL was concentrated to 25-30 µM concentration and was stored with 10% glycerol at 4°C.

Description of Bio Layer Interferometry

The basic principle of this technique is based on a light interference based signal. When molecules bind to the surface of the bio-sensor, the change in surface thickness on the Bio-layer interferometry biolayer is detected. This is based on developing an interference pattern from constructive and destructive wave patterns of a reference reflection compared with the reflection at the biolayer at the biosensor tip. Molecules binding to the biolayer portion results in both changes in thickness as well as local refractive index changes located at the end of the Bio-layer interferometry biosensor tip. Changes in the constructive/destructive λ interference pattern of these two reflected beams results in a wavelength shift interference pattern. This shift in wavelength (Δλ, recorded as a Δnm) is monitored and is proportional to the number and mass of molecules binding on the bio-sensor tip. This label free technique is comparable to Surface Plasmon Resonance and in all instances tested, yields similar quantitative and qualitative results. In the Bio-layer interferometry based method an increase in Δnm signal is observed upon loading the subject proteins on to the biosensor. A further increase in signal is observed when interacting molecules bind to the immobilized subject protein (Figure 1).Identification of structurally altered and aggregated states of proteins using GroEL as a probe on Bio-layer interferometry has been previously described [17] and validated with SPR [18]. Chaperone binding to immobilized protein and ATP mediated chaperone release can be monitored on Bio-layer interferometry similar to previously done work on surface plasmon resonance [24, 25].

Protein loading on the Biosensor

Stock maltodextrin glucosidase was concentrated with an Amicon centrifugal concentrator to a final concentration of 80-100µM in 20mM sodium phosphate buffer containing an imidazole concentration of 50mM. The buffer used for this Bio-Layer interferometry control experiment consisted of 50mM HEPES, 20mM Magnesium acetate, 10mM Potassium acetate at pH7.4. MalZ was diluted in this buffer to a final concentration of 0.5µM thus reducing the final imidazole concentration to 0.25 mM which does not affect loading of MalZ on the biosensor. Tube mode of the Blitz instrument (ForteBIO, Pall Life Sciences, California, U.S.A.) was used for all readings. The shaker speed of the instrument was kept at 2200 RPM. After 30s initial baseline with the refolding buffer, MalZ was loaded on the tip for 300s. A 30s baseline was followed by ATP treatment for 300s. As a control experiment, binding of 1µM GroEL to the Ni-NTA biosensor was tested. The residual GroEL adherence to the free tip was measured during each experiment, and subtracted as a background from the actual readings during analysis. GroEL binding to MalZ in native state was analysed and subsequently immobilised MalZ was denatured in 4 M GuHCl for 5 minutes, denaturant was washed off the tip and binding of GroEL to the denatured MalZ was observed.

Protein folding on Bio-layer interferometry biosensor

MalZ protein at a concentration of 0.5 µM was loaded to the biosensor and GroEL binding was monitored after denaturation by 4M GdnHCl. Buffer and conditions were the same as used in the previous experiment and the complete cycle was done at 25°C. GroEL was used at 1µM concentration and magnitude of total GroEL binding was recorded. After a baseline step, the biosensor tip was immersed in 10mM ATP solution so that GroEL releases the substrate protein due to conformational transition mediated by ATP hydrolysis. Baseline step was added after each cycle to remove the excess MalZ, GdnHCl, GroEL or ATP.

The biosensogram construction begins with 1) loading His tagged MalZ onto a Ni-NTA biosensor, 2) followed by baseline loading of GroEL to check for binding to native MalZ, 3) GdnHCl mediated denaturation, 4) GroEL binding to denatured MalZ, 5-6) ATP mediated release of MalZ by GroEL, done twice with a baseline in between, 7) GroEL binding to partially refolded MalZ, 8-9) ATP mediated release of MalZ by GroEL, done twice with a baseline in between, 10) GdnHCl mediated denaturation, 11) GroEL binding to denatured MalZ.

Optimization of MalZ concentrations for loading and GroEL binding

To avoid overloading, steric effects and intramolecular aggregation, loading signals were plotted and compared at different MalZ concentrations to identify the quantitative linear load region and to identify lower protein requirements for screening method development. MalZ was diluted over a concentration range of 12.5nM to 2µM and the loading amplitude signal (at 60 seconds) onto the Ni-NTA biosensors were recorded for each concentration. The biosensor amplitude (Δnm) of MalZ loading was plotted against the respective concentration of MalZ. GroEL at 1µM was added to these MalZ concentrations to identify linearity of GroEL binding to MalZ loading. The MalZ buffer was containing 50mM HEPES at pH 7.4, 20mM Magnesium acetate and 10mM potassium acetate. In the next experiment, MalZ is loaded at different concentration and denatured by 4M GdnHCl, binding of 1µM GroEL is done to the loaded MalZ. In a further step, GroEL association at different GroEL concentrations was observed to the MalZ loaded at 50nM concentration. Any minimal nonspecific GroEL binding to the biosensor, when present, was subtracted in all cases to remove the background signal.

Folding of immobilized MalZ at different temperatures monitored through GroEL binding

The experiments were performed in chloride free buffer containing 20mM sodium phosphate, 20mM magnesium glutamate and 10mM potassium glutamate. Separate biosensors were used for each time point. MalZ was loaded on the biosensor at an optimized concentration of 50nM. MalZ was loaded for 60 seconds on the biosensor tip and then after a brief wash, it was denatured by 4M GdnHCl for 4 minutes. After denaturation, the biosensor was briefly washed with refolding buffer to remove denaturant, removed from the Bio-layer interferometry instrument, and was incubated for a given time point at mentioned temperatures using a controlled temperature block. The biosensor was then again washed for 5 seconds to normalize to room temperature and in all cases GroEL binding step was carried out at room temperature of 25°C only. The GroEL binding magnitudes at these different points were plotted and compared to find out the dependence of the folding pattern of MalZ on these specific temperatures.

Folding of immobilized MalZ at different temperatures monitored through activity assay

Enzymatic activity assay was utilized to analyse the refolding of MalZ protein in two sets. In one set the solution based refolding of MalZ was investigated and in the second case the effect of immobilization was analysed by immobilizing MalZ on Ni-NTA sepharose resin. Sodium phosphate buffer at a concentration of 20mM was used with 20mM magnesium glutamate and 10mM potassium glutamate which is similar to the composition used for the Bio-layer interferometry based experiments. MalZ enzymatic activity was used to normalize protein concentrations in both cases so that equal amount of protein is present for comparison. MalZ protein was loaded on the Ni-NTA sepharose resin and washed thoroughly to remove any unbound protein. After washing, the resin was re-suspended in imidazole free buffer. At this stage, we took 250 µl resin (containing native protein) and added it to 1 ml substrate solution. MalZ enzymatic activity was measured by the substrate p-nitrophenyl α-D maltoside [26] (Gold Biotechnology, Missouri, U.S.A.), which on hydrolysis by MalZ releases p-nitro phenol that gives a characteristic yellow colour and can be quantified by measuring absorbance at 405nm. The substrate was prepared at 5mM concentration in the mentioned refolding buffer, additionally containing 500mM imidazole (MalZ elutes near 200 mM imidazole). This reading was compared with solution based activity reading to normalize amount of protein for the solution based experiments. For the resin based experiments the resin was kept on mild shaking condition and equal volumes of resin suspension was taken for each set of experiment. For the refolding studies the protein denaturation was done by 4M GdnHCl and in the further steps the protein in solution and on beads were diluted by refolding buffer and incubated at the temperatures 12°C, 24°C and 37°C. A 15 min incubation in refolding buffer was chosen so as to clearly identify the differences (15 min show significant differences in the BLI data). Enzymatic activity of each set was measured and analysed to identify the refolding percentages by comparing to activity readings of native protein. There was no significant effect of imidazole concentration on the enzymatic activity of the protein in the given buffer conditions, as judged through measurement of MalZ activity at various imidazole concentrations.

Comparison of solution unfolding of MalZ using fluorescence, with biosensor immobilized MalZ using GroEL Binding detection

The same buffer was used as in previous experiments and MalZ at a final concentration of 0.5µM was used for the fluorescence based experiments and 50nM for Bio-layer interferometry based experiments. GdnHCl was used at concentrations of 0M, 0.5M, 1M, 1.5M, 2M, 2.5M, 3M, 3.5M and 4M. GdnHCl concentrations above 4M does not have further effect. For Bio-layer interferometry experiments, native MalZ was immobilized on separate biosensors and each one was treated with one of the mentioned GdnHCl concentrations for 5 minutes. After a 10 second buffer wash to remove the denaturant from the biosensor tip containing immobilized MalZ, the tip was transferred into a GroEL solution to monitor binding amplitudes. These were plotted against the respective GdnHCl concentrations. For intrinsic tryptophan fluorescence, MalZ at 0.5µM concentration was denatured in these GdnHCl concentrations. After a 5 min incubation with denaturant, emission spectra between 300 and 400nm was recorded using excitation at 290 nm on an Agilent cary eclipse fluorimeter (Agilent Technologies, California, U.S.A.). Both GroEL binding experiment on Bio-layer interferometry and fluorescence measurement were repeated in presence of 4M glycerol and 1M sucrose as stabilizers.


This work was supported by the research grants to T.K.C from Council of Scientific and Industrial Research, Govt. of India, Grant no. 37(1565)/12/EMR-II and by grant funds from NIH R01AI090085 (MTF) and a Proof of Concept Grant Kansas University Innovation Center grant (MTF). A.P. acknowledge Council of Scientific and Industrial Research, Govt. of India for doctoral fellowship award no. 09/086(1072)/2010-EMR-I. A.K.S. acknowledges CSIR for research fellowship. The authors acknowledge Indian Institute of Technology Delhi for infrastructural support.


Bio-Layer Interferometry
Maltodextrin glucosidase
Guanidine Hydrochloride
shift in wavelength interference
Optical Density


Author Contributions

A.P. and A.K.S. performed all the experiments. T.K.C. and M.T.F. designed the experiments. A.P., A.K.S., T.K.C. and M.T.F. analysed the data and wrote the manuscript.

Additional Information

Competing financial interests: The authors declare no competing financial interests.


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