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Chaperones Skp and SurA dynamically expand unfolded outer membrane protein X and synergistically disassemble oligomeric aggregates

Periplasmic chaperones Skp and SurA are essential players in outer membrane protein (OMP) biogenesis. They prevent unfolded OMPs from misfolding during their passage through the periplasmic space and aid in the disassembly of OMP aggregates under cellular stress conditions. However, functionally important links between interaction mechanisms, structural dynamics, and energetics that underpin both Skp and SurA association with OMPs have remained largely unresolved. Here, using single-molecule fluorescence spectroscopy, we dissect the conformational dynamics and thermodynamics of Skp and SurA binding to unfolded OmpX, and explore their disaggregase activities. We show that both chaperones expand unfolded OmpX distinctly and induce microsecond chain reconfigurations in the client OMP structure. We further reveal that Skp and SurA bind their substrate in a fine-tuned thermodynamic process via enthalpy–entropy compensation. Finally, we observed synergistic activity of both chaperones in the disaggregation of oligomeric OmpX aggregates. Our findings provide an intimate view into the multi-faceted functionalities of Skp and SurA and the fine-tuned balance between conformational flexibility and underlying energetics in aiding chaperone action during OMP biogenesis.


Introduction
Molecular chaperones are key cellular components that play fundamental roles in maintaining cellular proteostasis 1,2 . Essential activities of chaperones include the assistance of de novo protein folding, the stabilization of non-native proteins in folding competent or unfolded states, and the rescue of misfolded and aggregated proteins [3][4][5] .
Chaperones are an integral part of a wide range of protein quality control systems and their activities are intimately coupled to the biogenesis networks that aid the structural and functional maturation of proteins from their site of cellular synthesis to their target cellular compartments.
One biogenesis network where chaperone activity is of particular relevance is the biogenesis network of outer membrane proteins (OMPs) 6,7 . OMPs are a diverse group of β-barrel membrane proteins found in the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts. They fulfil a plethora of functions in cell signaling, metabolism, and transport [8][9][10] , are indispensable to the survival of bacteria 10,11 , and constitute important virulence factors and drug targets [12][13][14] . The OMP biosynthesis pathway is highly complex and conserved across all kingdoms of life 15 , and involves the coordinated action of a multicomponent protein machinery that aids in overcoming the many hurdles that these proteins have to surmount on their way to their target outer membrane 4,7,16 .
In Gram-negative bacteria, OMPs are translated in the cytoplasm, from where they are translocated across the inner bacterial membrane via the Sec machinery to the periplasmic space 17,5 . Within this aqueous compartment, OMPs are escorted to the outer membrane in an unfolded state (denoted as the uOMP state) with the aid of various chaperones that maintain the largely insoluble and aggregation prone uOMP polypeptide chains in a protected, partially unfolded state 18,19 . At the outer membrane, uOMPs are transferred to the β-barrel assembly machinery (BAM), which facilitates their native folding and insertion into the membrane 20 . Noteworthy, the periplasm is devoid of any known source of energy-providing molecules, such as ATP; hence, all chaperones as well as the entire folding machinery likely operates without the aid of external energy, following thermodynamic principles 21 . Two chaperones which have been shown to be indispensable for the biogenesis of bacterial OMPs are the seventeen-kilodalton protein (Skp) 22 and survival factor A (SurA) 23 . Skp and SurA, both located in the periplasm, exhibit anti-folding activity (also known as holdase activity), whereby they sequester uOMP substrates to prevent aggregation until they reach the bacterial outer membrane [24][25][26] . Their interaction with uOMPs is thermodynamically modulated due to the lack of energy-carrying molecules in the periplasm [27][28][29] . Depletion studies of periplasmic chaperones identified SurA as an essential chaperone for OMP biogenesis, leading to a drastic decrease in OMP density in the outer membrane due to the loss of SurA 30 . Skp depletion, on the other hand, led to an accumulation of misfolded OMPs and the activation of the cellular stress response 30 , while OMP density in the outer membrane remained the same. Interestingly, recent studies suggest substrate selectivity amongst the two chaperones 31 . Hence, it is of importance to understand the functional mechanisms underlying both Skp and SurA association with uOMPs.
Structural studies of the eight β-stranded protein outer membrane protein X (OmpX) in the presence of Skp using nuclear magnetic resonance (NMR) spectroscopy have found that unfolded OmpX (uOmpX) shows sub-millisecond backbone dynamics 32 in complex with Skp. For binding of SurA to unfolded outer membrane protein A (uOmpA), both fluid globular 32 and expanded states 31 have been proposed. Recent studies have located various interaction sites of SurA and uOmpX using cross-linking, suggesting that SurAbound uOmpX populates multiple conformations 31,33 . Yet, long-range polypeptide chain dynamics and conformational heterogeneity of unfolded OMPs upon binding to chaperones remain elusive. In particular, how SurA-and Skp-bound OMP dynamics and heterogeneities differ, given their differential roles in regulating protein folding in the periplasmic space. Dynamic aspects are hypothesized to be important for chaperone-uOMP interactions, particularly to fine-tune energetics of the binding reaction through a reduction of the entropic costs upon binding 28,29,[34][35][36][37] . Yet, the enthalpic and entropic changes that determine Skp-OMP or SurA-OMP interactions and affect the conformations of the denatured substrate proteins are largely undefined.
In addition to the well-described holdase activities of Skp and SurA that protect OMPs from misfolding or aggregating, a recent study has suggested that Skp disaggregates oligomeric uOMP structures 38 . While SurA has not been directly implicated as a disaggregase, modelling studies propose a synergistic interaction amongst these chaperones especially under conditions of stress 18,39 , thus raising the question of the role of both chaperones played in disassembling OMP aggregates.
To gain insights into the multi-faceted functionalities of Skp and SurA and their action mechanisms, we study here the conformational dynamics and thermodynamics of the eight β-stranded protein OmpX in the presence of the chaperones, and explore their disaggregation activities. Using single-molecule Förster resonance energy transfer (smFRET), we resolve the heterogeneities, structural dynamics, and thermodynamics underlying the different states of uOmpX at near-native conditions. Strikingly, we find that both chaperones expand the unfolded polypeptide chain upon binding. The degree of expansion is concentration dependent for SurA, but not for Skp. Probing structural changes and chaperone interaction at different temperatures, we gain insights into the enthalpic and entropic contributions of complex formation and find that the interaction modes of both chaperones differ strongly and are dictated by entropy-enthalpy compensation. Finally, we use fluorescence correlation spectroscopy (FCS) to probe the disaggregation capabilities of Skp and SurA and find synergistic activity of both chaperones in the disassembly reaction of oligomeric OmpX aggregates. Our findings provide fundamentally new insights into the structural and energetic mechanisms underlying Skp and SurA chaperone-OMP interactions and their role in OMP biogenesis.

Studying chaperone effects on uOmpX conformational dynamics under native-like conditions by smFRET
Probing chaperone effects and conformational dynamics of uOMPs under native-like conditions (i.e., in the absence of or at very low denaturant concentrations) has been difficult to achieve due to the aggregation prone nature of OMPs in aqueous environments 7,40,41 . Single-molecule methods, in particular single-molecule fluorescence techniques such as smFRET, operate at very low concentrations (i.e., in the pM range), and thus alleviate protein aggregation and precipitation challenges [42][43][44] . smFRET measurements further provide access to subpopulation-specific conformational heterogeneity, even at very low denaturant concentrations or the absence thereof 45 , and allow probing of long-range polypeptide chain dynamics across a wide spectrum of timescales in the presence of binding partners and without the need of synchronizing the ensemble with perturbation/relaxation techniques [46][47][48] . We therefore set out to develop an smFRET assay that allowed us to study the conformational dynamics of single uOmpX polypeptide chains in native-like aqueous environments and the effect that the chaperones Skp and SurA have on the structural and dynamic properties of uOmpX.
To begin, we prepared an N-and C-terminal fluorescently labelled double-cysteine OmpX variant (OmpX1,149) furnished with donor and acceptor dyes ( Fig. 1a   Fluorescence lifetime and sufficient rotational averaging allowed us to exclude the possibility that the broadening originates from changes in dye quantum yields or a restricted rotational freedom of the dyes (see Supplementary Table 1 likely constitutes a misfolded state, denoted as uOmpXcompact throughout the article.
In conclusion, we find that uOmpXaq adopts a broad, heterogeneous ensemble of structures and populates a rugged energy landscape leading to a multi-tier dynamical behavior with both fast sub-millisecond chain reconfiguration dynamics and slow conformational interconversion. An overall expansion of the uOmpXaq polypeptide chain is observed, in line with a conformational loosening mechanism that likely serves to alleviate uOMP aggregation 40 .

Skp-and SurA-bound uOmpX is dynamically expanded
Next, we probed the effects that the chaperones Skp and SurA have on the structural and dynamic properties of the uOmpX polypeptide chain. To this end, we subjected denatured OmpX to a multi-step dilution and denaturation protocol to transfer the protein into nativelike buffer in the presence of the chaperones Skp or SurA, and subsequently recorded E histograms (Fig. 1a).
In a first set of experiments, we complexed uOmpX with Skp at 37ºC, which binds unfolded OMPs as a trimer 56,57 , denoted thereafter as Skp3. As reference, we first suggests that Skp3 interacts with uOmpX and forms a chaperone-bound state, in the following denoted as Skp3-uOmpX. This state is in equilibrium with and appears structurally distinct and more expanded than the uOmpXaq and uOmpXcompact states.
We used Gaussian fitting to determine the three peak positions observed in the E histogram series (see Supplementary Fig. 2 and Methods). This analysis showed that the peak positions of the two populations in the high FRET regime exhibited E values of Ê ≈ 0.75 and Ê ≈ 0.92, which are in very good agreement with the E values of the uOmpXaq (Êaq ≈ 0.77) and uOmpXcompact (Êcompact ≈ 0.92) states. This suggests that the presence of Skp3 does not affect the conformational properties of these states and implies that these do not directly interact with Skp3. We therefore identify these states, even in presence of chaperones, as uOmpXaq and uOmpXcompact. The FRET efficiency peak of Skp3-bound uOmpX was centered around ÊSkp 3-uOmpX ≈ 0.45, a considerably larger interfluorophore distance than the unbound uOmpX (i.e., uOmpXaq) state. A careful analysis of the donor lifetime, the fluorophore anisotropies, and the rotational correlation times of the fluorophores reassured that the lower E values reflect a global conformational change arising from the expansion of the uOmpX polypeptide chain upon binding to Skp3 (Supplementary Table 1). Moreover, Gaussian fitting revealed that, at [Skp3] = 2.5 µM, ~80% of uOmpX molecules were in a Skp3-uOmpX complexed state, suggesting that the presence of Skp shifts the equilibrium from the unbound to the bound form.
In a second set of experiments, we diluted uOmpX in the presence of the chaperone SurA at 37ºC. Upon addition of SurA, we observed, similarly to Skp3, an additional FRET efficiency peak at lower E values (Fig. 2c). Increasing [SurA] from 46 nM to 25 µM, lead to an increase of the low E state population (i.e., SurA-uOmpX). Gaussian fitting allowed us to identify the peak position and fraction of SurA-bound uOmpX with ÊSurA-uOmpX ≈ 0.6 at a concentration of ~6 µM, which is close to the intracellular concentration of SurA 18,59 .
At this concentration, ~87% of uOmpX molecules are bound to the chaperone. The lower Ê value of SurA-uOmpX indicated also an expanded conformation as compared to uOmpXaq, yet a less expanded conformation as compared to Skp3-SurA. The distinct peak positions of the two chaperone-bound states (ÊSkp 3-uOmpX ≈ 0.45 and ÊSurA-uOmpX ≈ 0.6) thus suggest that they themselves occupy a distinguishable configurational space likely due to the difference in the interaction mechanisms between the chaperone and its substrate 7,16 .
Since temperature affected the conformation and reconfiguration dynamics of uOmpXaq,  Table 3). The FRET efficiencies of the unbound (uOmpXaq) and compact uOmpX (uOmpXcompact) states did not vary markedly across the chaperone concentration series performed at different temperatures ( Supplementary Fig. 4). For the chaperone-bound fraction of uOmpX, ÊSkp 3-uOmpX remained constant, within error, across nearly three orders of magnitude of [Skp3] at 25 ºC ( Fig. 2d, left panel). This suggests a concentration-independent, possibly 1:1 stoichiometry of Skp3 and uOmpX, in accordance with previous studies 32, 60 . Increasing the temperature of the complex, however, shifted ÊSkp 3-uOmpX to higher E values (Fig. 2d) Next, we modeled the structural ensemble of the states globally using a log-normal distribution and obtained a standard deviation of the donor-acceptor distance (sR) and the expected inter-dye distance áRinter-dyeñ (inset Fig. 2f  change, the emission-delay histograms for two initial microtime windows will start to separate. For uOmpXaq, all delay time windows DT ± DDT produced separated emissiondelay histograms (Fig. 3b). The amplitude of separation was greater at a lower time point DT = 5 µs as compared to that at higher time points of 5 µs or 240 µs. This reinforced our finding that uOmpXaq exhibits both fast and slow conformational dynamics by virtue of its rough energy landscape. For both Skp3-uOmpX (Fig. 3c) and SurA-uOmpX (Fig. 3d) complexes, by contrast, the emission-delay histograms started separating at lower time points DT = 2.5 µs and DT = 6 µs, respectively. Skp-or SurA-bound uOmpX thus shows a dominating microsecond conformational change, suggesting that both chaperones modulate the energy landscape of uOmpX such that the energy barriers between the sub-population of their bound substrate are reduced, leading to a dynamization of the polypeptide chain in the chaperone-bound state.

Entropic and enthalpic contributions of chaperone-uOmpX interactions
We next set out to obtain insights into the energetic contributions of Skp and SurA chaperone-uOmpX interactions. Interaction isotherms at different temperatures allow for the extraction of enthalpic, DH, and entropic, DS, contributions to the free energy change of interaction, DG, which are connected to the association constant, Ka, for a bimolecular association reaction via: In order to extract entropic and enthalpic contributions, we developed a global analysis approach that allows reconstruction of FRET efficiency distributions at different temperatures and chaperone concentrations ( Fig. 4a and Methods). Briefly, by varying the optimization parameters enthalpy (DH), entropy (DS), and considering the fraction of the compact state (fc(T)), which is likely unable to be chaperone complexed, as well as the maximum fraction of the bound state (fb,max(T)), and minimizing the reduced chi- More interestingly, for Skp3-uOmpX complex formation, the binding enthalpy and entropy changes were DH (Skp3-uOmpX) = -298 ± 19 kJ mol -1 and DS (Skp3-uOmpX) = -0.84 ± 0.06 kJ mol -1 K -1 (Fig. 4b), respectively, yielding an interaction free energy change of DG (Skp3-uOmpX) = (-38.3 ± 0.2) kJ mol -1 at 37 ºC. The change of enthalpy upon binding is large, in agreement with recent findings demonstrating rich interaction of the uOmpX polypeptide chain with the cavity formed by Skp3 32 . At the same time, the change in interaction entropy is strongly negative, suggesting a stark reduction of the overall configurational space for uOmpX upon its encapsulation by Skp3. Hence, Skp3 binding to uOmpX is entropically unfavorable, yet this entropic penalty is counter-weighted by a large enthalpic contribution, enabling the binding of Skp3 to the client OMP.
For SurA-uOmpX complex formation, the binding enthalpy and entropy changes were DH (SurA-uOmpX) = -132 ± 24 kJ mol -1 and DS (SurA-uOmpX) = -0.24 ± 0.08 kJ mol -1 K -1 (Fig. 4b), respectively, yielding an overall stabilization of the complex of DG (SurA-uOmpX) = (-57 ± 1) kJ mol -1 at 37 ºC, which is slightly more favorable than for Skp3. The change of enthalpy was two-fold lower than for Skp3, likely due to more transient binding as suggested in recent studies 31,33 . At the same time, the change of entropy of uOmpX upon SurA binding was three-fold lower than with Skp3, and close to zero, indicating only a minimal change of configurational space for the binding of SurA to uOmpX. This agrees well with our finding of an increased coefficient of variation (i.e., structural heterogeneity) for SurA-uOmpX as compared to Skp3-uOmpX. Thus, binding of SurA to its client uOmpX is markedly different in nature than Skp3 binding to uOmpX. For Skp3, the reduction of conformational freedom of uOmpX in the cavity of Skp3 needs to be compensated by an increased interaction enthalpy (Fig. 4c). By contrast, uOmpX binding to the surface of SurA reduces the conformational freedom to a lesser extent and thus a lower enthalpic contribution is sufficient to enable binding (Fig. 4c).

SurA and Skp3 act synergistically as disaggregases on oligomeric OmpX structures
Aside from the known uOMP holdase activities of Skp and SurA, recent work has suggested that Skp also has the ability to act as a disaggregase to disassemble oligomeric OMP structures 38 . By contrast, only indirect evidence 18,39 suggests that SurA might be involved in synergetic interaction with other periplasmic chaperones, such as Skp, under cellular stress in the disassembly of aggregates. To address this question, we investigated the action of Skp3 and SurA towards aggregated OmpX (OmpXAgg) and devised an FCS-based assay that allows probing the disaggregase efficacy of the two chaperones. To this extent, we diluted labeled and denatured uOmpX at pM concentrations with unlabeled and denatured uOmpX in buffer to yield a final OmpX concentration of 1 µM (Fig. 5a). The residual GdmCl and LDAO concentrations were 12 mM and <200 nM, respectively. It is important to note, that due to the 100,000-fold concentration difference of labeled and unlabeled uOmpX, only a small fraction of potential aggregates will be fluorescently marked and thus visible to FCS measurements. We incubated the mixture for 10 min and subsequently performed FCS measurements at 37 ºC and obtained a multimodal auto-correlation function with two major components suggesting the presence of a fast and a slowly diffusing species (Fig. 5b, green). Comparison with an FCS curve recorded from a highly diluted sample of only labeled uOmpXaq (~10 pM) indicates that the fast-diffusing component in the correlation curve of the aggregation sample stems from monomeric uOmpXaq, with a characteristic diffusion time of tDiff = (0.27 ± 0.01) ms (Fig. 5b, grey, Supplementary Table   5, Supplementary Fig. 6a). The second, slowly diffusing species in the OmpXAgg measurements showed a tDiff = (31.6 ± 1.3) ms, about two orders of magnitude slower than single uOmpXaq molecules. Notably, OmpXAgg is possibly an ensemble of differently sized aggregates; hence, values reported here reflect average values. We determined that in our aggregation mixture, about 24 ± 2% of the labeled uOmpX molecules are part of aggregates (termed apparent fraction, Fig. 4d). Interestingly, at lower temperatures, the propensity of OmpX aggregation was reduced ( Supplementary Fig. 6e).  Fig. 6b and c). To quantify the fractional amounts of the three components (i.e., uOmpXaq, chaperone-bound monomeric uOmpX, and OmpXAgg), we fitted the autocorrelation data of our mixture experiment with three components. Here, the respective diffusion times were kept constant to extract the amplitudes of each component as they are inversely proportional to the concentrations and fractional amounts of the species. Addition of Skp3 to the aggregation mixture reduced the apparent fraction of OmpXAgg to 20 ± 2 % and addition of SurA even to 13 ± 1 %. Interestingly, in both cases the disaggregation of OmpX was found to be accompanied by an increase in the free uOmpXaq population (Supplementary Fig. 6f) and only negligibly in the chaperone-uOmpX complex. Assuming, uniform mixing of labelled and unlabeled uOmpX sample, the increase of the free uOmpX population suggests that the chaperones show a higher affinity towards OmpXAgg as compared to uOmpX. This finding is significant, as it suggests that careful adaptation of affinities is crucial during physiological stress conditions. In such a scenario, disaggregation activity might be more important than holdase activity to prevent the formation of large toxic OMP aggregates in the periplasm.
Finally, we asked the question whether there are synergistic effects on the disaggregation reaction, given that both chaperones are present in the periplasm. We performed our disaggregation assay in which both chaperones were added simultaneously to the mixture containing OmpXAgg and labelled uOmpX at pM concentrations after 10 min incubation at 37ºC. Strikingly, the fraction of OmpXAgg was reduced to ~5% (Fig. 5d). Taking into account our single-chaperone measurements, for an independent, additive reduction of the apparent OmpXAgg, we would have expected a reduction to ~9%. The increased disaggregation by a factor of two for both chaperones together suggests therefore a cooperative action of Skp3 and SurA.  suggesting that uOmpX interacts with Skp3 at multiple sites by spreading across the inner surface of the chaperone cavity (Fig. 6a). The expansion of uOmpX upon Skp3 binding was independent of the [Skp3], indicating that the valency of Skp3 likely does not change as expected for the small eight β-stranded OmpX 60 . The SurA-uOmpX complex (Fig. 6b) We further quantified the dynamics of both Skp3-and SurA-bound uOmpX using 2D-FLCS. We demonstrate that the intrachain dynamics of the chaperone-complexed uOmpX chain are on a timescale of 5 to 10 μs ( Fig. 6a and 6b), whereby the fast chain reconfiguration dynamics are possibly facilitated through local transient interactions with the chaperones. This is in contrast to the multi-tier (i.e., both slow conformational and fast reconfiguration chain) dynamics observed for the aqueous uOmpX state (Fig. 6c).

Conclusions
Periplasmic chaperones thus increase conformational flexibility, despite the expansion of the unfolded state. We hypothesize that such a manipulation and dynamization of the energy landscape of the OMP substrate might be beneficial for BAM interactions 65 .
Notably, transient binding interactions and multi-valency interactions were also observed for the cytoplasmic chaperone Trigger Factor (TF), which is structurally homologous to SurA, and like the latter also involved in ATP-independent transfer of clients to the downstream chaperone machinery for folding 67 .
Given that the periplasm is deficient of ATP as an energy source, protein folding and chaperoning, involving binding to the client uOMPs, has to be driven entirely by a change in the free energy of the system. An analysis of binding isotherms at different temperatures revealed that the change of entropy upon binding to the client uOmpX substrate was low for Skp3 and close to zero for SurA, suggesting that the conformational freedom is affected only minimally. A low entropic change upon binding has been hypothesized to be favorable for locally transient chaperone-uOMP interactions, due to a reduced enthalpy-entropy compensation 29 . Strikingly, comparing Skp3 and SurA, we find that the chaperone-uOmpX interactions are fine-tuned exquisitely, meaning a higher entropic change of Skp3 is compensated by a higher enthalpic term, or vice versa in the case for SurA, to ensure a favorable, yet low change of free energy upon binding (Fig. 6).
Hence, the different contributions of enthalpy and entropy to both complexes suggests that the thermodynamics of the Skp3-uOmpX and SurA-uOmpX interaction are differently calibrated in accordance with their chaperoning mechanisms.
Lastly, we found that both chaperones have the ability to disassemble aggregated OmpX (Fig. 6d). Interestingly, both chaperones show a higher affinity towards aggregated OmpX than uOmpX. A higher affinity towards the aggregate might be particularly useful when aggregated OmpX emerges, for example, under stress conditions to avoid the accumulation of OMP aggregates. Surprisingly, we found that Skp3 and SurA act synergistically on uOmpX aggregates, suggesting higher order interactions between both periplasmic chaperones. Disaggregation mechanisms have also been observed in the ATP-dependent cytoplasmic chaperones of Hsp70 family 68,69 . However, both Skp and SurA disaggregate oligomeric structures in the absence of an external energy source, thereby supporting the known disaggregase DegP, with a yet to be determined mechanism.
In summary, both Skp and SurA binding of uOmpX expands the unfolded polypeptide chain distinctly. Interestingly, the unfolded polypeptide chain, in complex with both chaperones, exhibits a dynamization on the microsecond timescale. While the structural features of the bound polypeptide appear similar, the thermodynamic aspects of the Skp3-uOmpX and SurA-uOmpX interaction differ and SurA binding results in a reduced loss of entropy compared to Skp binding. In a biological context, expansion and fast chain reconfiguration of the bound polypeptide may serve to ensure that the uOMP remains accessible to the BAM complex. In energetic terms, the enthalpy-entropy compensations consent with the binding modes of the two chaperones to maintain their interaction with uOmpX energetically favorable. Lastly, both chaperones synergistically disaggregate OmpX aggregates, suggesting additional interactions between both chaperones that enhance the disaggregation. We hypothesize that the tightly regulated multi-faceted functionalities of both chaperones enable cellular vitality to be maintained and regulated under normal and stress conditions.

Protein production
A tag-free double-cysteine variant of OmpX (OmpX1,149) without signal sequence was produced as inclusion bodies in Escherichia coli BL21(DE3) cells and purified following standard procedures including anion exchange chromatography, as previously reported 70,71 . After refolding, the protein was labeled with FRET donor (ATTO532, Atto-

Sample preparation for single-molecule measurements
A multi-step dilution protocol, as shown in Fig. 1a, was followed to prepare samples for