Colorimetric Assay Reports on Acyl Carrier Protein Interactions

The ability to produce new molecules of potential pharmaceutical relevance via combinatorial biosynthesis hinges on improving our understanding of acyl-carrier protein (ACP)-protein interactions. However, the weak and transient nature of these interactions makes them difficult to study using traditional spectroscopic approaches. Herein we report that converting the terminal thiol of the E. coli ACP 4′-phosphopantetheine arm into a mixed disulfide with 2-nitro-5-thiobenzoate ion (TNB−) activates this site to form a selective covalent cross-link with the active site cysteine of a cognate ketoacyl synthase (KS). The concomitant release of TNB2−, which absorbs at 412 nm, provides a visual and quantitative measure of mechanistically relevant ACP-KS interactions. The colorimetric assay can propel the engineering of biosynthetic routes to novel chemical diversity by providing a high-throughput screen for functional hybrid ACP-KS partnerships as well as the discovery of novel antimicrobial agents by enabling the rapid identification of small molecule inhibitors of ACP-KS interactions.


Results
Purification of holo-ACPs and FabF. AcpP, ACT ACP and FabF were expressed as His 6 -tagged constructs in BAP1 10 or BL21-DE3 competent cells. We originally intended to convert the terminal thiol of the ACP Ppant arm to the thiocyanate for subsequent mechanistic cross-linking and monitoring via infrared spectroscopy, and thus it was essential to obtain the ACPs of interest in the holo-form (in which the Ppant arm is installed). Despite the capability of BAP1 cells to co-express the Sfp phosphopantethienyl transferase that catalyzes the transfer of a 4′-Ppant moiety from CoA to the conserved serine residue at the N-terminus of helix II of ACP to afford holo-ACP, we obtained mixtures of apo-and holo-ACPs from protein expression in this cell line. The observed mixtures could be due to inefficiencies of the BAP1 cell line or the presence of AcpH, a phosphodiesterase that cleaves the Ppant arm in E. coli 11,12 . The presence of AcpS, a native phosphopantetheinyl transferase in E. coli, likely explains why some holo-ACP also was obtained from the BL21-DE3 cell line. To ensure that ACPs were in a majority holo-form, Ni-bound ACPs were chemoenzymatically treated in vitro with SfpR4-4 13 in the presence of coenzyme A before the elution step during purification. SfpR4-4 is a mutant of the Bacillus subtilis Sfp discovered by high-throughput phage selection that displays a 300-fold increase in catalytic efficiency and broader substrate specificity than the wild-type Sfp 13 . The successful conversion to 100% holo-ACP was confirmed by liquid chromatography mass spectrometry (LCMS) and protein purity evaluated by SDS PAGE (Figs S1-S3). Holo-AcpP was observed to migrate to ~20 kDa on SDS PAGE despite its molecular weight of ~10 kDa (Fig. S3). This is consistent with previous observations and is likely due to the unusual charge distribution of the protein 14 . A second higher molecular weight band at ~40 kDa observed on non-reducing gels represents a dimer that forms via the disulfide bond between the Ppant arm thiols of monomeric ACPs 15 . Holo-ACT ACP migrated to the expected molecular weight of ~10 kDa on SDS PAGE gel and also exhibited dimer formation under non-reducing conditions (Fig. S3). The migration of FabF on SDS PAGE gels was consistent with the monomeric mass (~45 kDa), although it may exist as a dimer under native conditions (see below). Figure 1. The discovery of a colorimetric assay that reports on ACP-KS interactions. While investigating ACP-KS interactions using a previously reported vibrational spectroscopic mechanistic cross-linking approach (blue arrows), an unexpected color change was observed upon mixing the ACP substrate with the KS partner. Upon investigation, it was revealed that the cyanylation reaction had not gone to completion, and thus instead of the ACP-thiocyanate being mixed with KS, the ACP-TNB − complex was inadvertently added. This led to the realization that the facile activation of ACP to ACP-TNB − enables the colorimetric reporting of mechanistically relevant ACP-KS interactions (red arrows).
Investigation of AcpP and ACT ACP oligomeric state and capacity of these proteins to bind to the KS FabF. SV-AUC measures the sedimentation rate of molecules or proteins in solution and can provide information about molecular shapes, oligomeric state and binding affinity. We observed that the sedimentation profiles of AcpP and ACT ACP fitted to a model-free continuous c(s) distribution. AcpP (12.3 kDa) sediments with a broad (9.97-12.8 kDa) major peak centered at s(20,w) = 1.7 S, whereas ACT ACP (11.4 kDa) sediments slightly differently with a major peak at s(20,w) = 1.5 S. This observation suggests that AcpP sediments at an apparently "larger" molecular radius, which is consistent with how the protein behaves in SDS-PAGE and in solution (Fig. S4) 15 . Nonetheless, it appears that both ACPs exist primarily in a monomeric state under reducing conditions. It appears that AcpP forms a tetramer (Fig. S4), but whether this self-association is physiologically relevant remains to be investigated.
Next, we used SV-AUC to study ACP-FabF interactions by looking for significant changes in the single boundary of the sedimentation velocity profile of FabF at varying concentrations of ACP. Prior to measuring the binding affinity, we first determined that FabF sediments mostly as a dimer in solution (Fig. S4), which is consistent with previous crystallography studies 16 . We observed that the FabF sedimentation boundary changes upon addition of AcpP but remains unchanged when ACT ACP is added (Fig. 2). These data suggest that AcpP complexes with FabF whereas ACT ACP does not, which is consistent with previous in vitro cross-linking studies [6][7][8] .
To measure the binding strength between FabF and AcpP, we applied analysis of weighted-average (signal average) sedimentation coefficient (s w ) as a function of AcpP concentration. Partial saturation of the reaction boundary was achieved by titrating 16 μM FabF with up to 230 μM AcpP (Fig. S5). An EPT s(w) fast isotherm was constructed by defining the reaction boundary between 4 and 8 S, and a second isotherm was constructed by integrating the c(s) distribution between 1 and 8 (Fig. S5). The k D value of 6.4 ± 2.0 μM was obtained when both isotherms were fit globally to an A + B ↔ AB model by fixing free FabF at 5.9 S, AcpP at 2.0 S and fitting to a convergent dimer of heterodimers s-value of 6.6 S (Fig. S5). The calculated k D is in close agreement with the k D of 4.1 ± 1.8 μM previously reported using isothermal calorimetry 9 , validating SV-AUC as a method to quantify ACP-FabF interactions in solution.
Conversion of Holo-ACPs to ACP-TNB − . After using SV-AUC to establish that FabF binds to holo-AcpP but not to holo-ACT ACP, we sought to characterize these same interactions using the coupled mechanistic cross-linking reaction/vibrational spectroscopic approach developed in our lab 6 . To install the thiocyanate probe on the terminal thiol of the Ppant arm of holo-AcpP and holo-ACT ACP, we first needed to convert the holo-ACPs to their corresponding ACP-TNB − adducts. Holo-ACPs were treated with excess 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), commonly known as Ellman's reagent 17 , to install the TNB − probe on the terminal thiol end of the 4′-Ppant arm of holo-ACPs. To avoid a second TNB − probe adding to a cysteine residue near the N-terminus of ACT ACP, the C17S ACT ACP construct was utilized. A bright yellow color signifying the release of half of DTNB (TNB 2− ion) into solution was observed when the holo-ACPs reacted with DTNB. The ACP-TNB − products were purified via size exclusion chromatography (SEC) with a PD10 column and subsequently characterized by LCMS (Figs S1 and S2). Comparison of the holo-ACP to ACP-TNB − circular dichroism (CD) spectra revealed that the installation of the TNB 2− molecule to the Ppant arm did not perturb the overall helical structure of the protein (Fig. S6).

AcpP-TNB − forms a mechanistically relevant cross-link with the active site cysteine of FabF.
In the course of generating and using the AcpP-SCN product for mechanistic crosslinking with FabF 6 , it was noted that a reaction evolved a bright yellow color upon mixing, reminiscent of the color change when TNB 2− is released. This observation led us to hypothesize that the unexpected color change was driven by the KS active site thiolate acting as a nucleophile and attacking the thiol-bound TNB − adduct, as outlined in Fig. 1. Upon analysis www.nature.com/scientificreports www.nature.com/scientificreports/ of the starting material by LCMS, we confirmed that in this case the reaction of the ACP-TNB − with sodium cyanide had not gone to completion and thus the AcpP-TNB − adduct had been mixed with FabF. Recognizing the potential fortuitous discovery of a facile method to obtain a mechanistically relevant KS-ACP complex, we further investigated this unexpected result.
We obtained pure AcpP-TNB − and mixed up to three molar equivalents of this protein with FabF. Analysis of the potential ACP-KS cross-link via non-reducing SDS PAGE indicated indeed a larger molecular weight complex was formed of ~65 kDa (Fig. 3A), which is the same size as the previously reported AcpP-FabF complex 6 . The mixing of FabF with increasing amounts (0-3 molar equivalents) of AcpP-TNB − resulted in a decrease in the intensity of the bands corresponding to the standalone proteins and increase in the putative cross-linked heterodimer. Addition of the reducing agent betamercaptoethanol (BME) led to the dissociation of this complex into two bands of ~20 and 45 kDa (Fig. 3B), consistent with the higher molecular weight complex being linked by the redox-sensitive disulfide bond. We speculate that the faint band at ~75 kDa observed under non-reducing conditions could represent two AcpPs, the second docking onto FabF in a non-mechanistically relevant manner, although further investigation is required to confirm the identity of this band. Analysis of the reaction components and products by SEC further confirmed that the ACP-TNB − and FabF combined to form a higher molecular weight complex (11 min) thus releasing a small molecule product with a λ max at 412 nm (27 min; Fig. 4), which is consistent with the reaction scheme outlined in Fig. 1.
To determine if the putative cross-linked product tethers the mechanistically relevant sites of ACP and FabF, the ~65 kDa band on the non-reducing SDS PAGE gel (Fig. 3A) was further interrogated by tandem proteolysis mass spectrometry. Analysis of these data revealed a peak at m/z = 7256 Da consistent with the trypsin-digested AcpP fragment containing the reactive site serine bound via the Ppant arm to the trypsin-digested fragment of www.nature.com/scientificreports www.nature.com/scientificreports/ the FabF containing the active site cysteine (Fig. S7). We also observed a decrease in counts of the unmodified, trypsin-digested fragments of AcpP containing the serine point of Ppant arm attachment and FabF containing the active site Cys163, consistent with the covalent modification of these fragments. Considering that FabF contains three additional cysteines 8 , these results highlight the regioselectivity of the ACP-FabF cross-linking reaction for the mechanistically relevant product.
The reaction of AcpP-TNB − with FabF can be monitored by UV-vis because while the AcpP-TNB − adduct is colorless, the TNB 2− ion released upon AcpP-TNB − binding to FabF absorbs at 412 nm (ε = 14150 M −1 cm −1 at 25 °C) [18][19][20] . Given that one mole of TNB 2− should be released per mole of AcpP-FabF cross-link formed, the amount of ACP-FabF produced can be readily quantified using the Beer-Lambert law. As shown in Fig. 3C, an increase in A 412 is observed as the 0-1 molar equivalents of ACP-TNB − are added to FabF, and a plateau is observed upon addition of excess ACP-TNB − . Notably, the plateau observed at A 412 = 0.4 corresponds to ~28 μM of TNB 2− released, which is consistent with the 25 μM FabF added to solution forming a cross-link with ACP at a 1:1 molar ratio. Thus, UV-vis can be used to quantitatively monitor the progress of the reaction between ACP-TNB − and FabF to form ACP-FabF.  280 nm) shows that when AcpP-TNB − is mixed with FabF (top), a higher molecular weight complex is formed (11 min) and the small molecule TNB 2− is released (26 min). Upon mixing FabF with ACT ACP-TNB − (bottom), neither the higher molecular product nor TNB 2− is observed. ACPs alone were loaded in holo-form, which display a lower A 280 than the corresponding ACP-TNB − adducts. www.nature.com/scientificreports www.nature.com/scientificreports/ ACT ACP-TNB − does not form a mechanistically relevant cross-link with the active site cysteine of FabF. We next investigated whether the cross-linking of the ACP-TNB − adduct to KS was selective to those proteins that bind in vitro in their holo form. It has previously been shown by us 6 and others 21 that ACT ACP does not bind to FabF, a result that we verified in this work using SV-AUC as outlined above. Upon mixing ACT ACP-TNB − with FabF, we did not observe a color change, which is consistent with a lack of cross-link formation. The inability of ACT ACP-TNB − to form a cross-link with FabF is supported by the lack of a higher molecular weight complex observed under non-reducing conditions (Fig. S8), a lack of increase in A 412 observed upon mixing ACT ACP-TNB − with FabF (Fig. S8c), and SEC (Fig. 4). A faint band is observed above the FabF band in the SDS PAGE gel under non-reducing conditions, which could represent non-specific, low-level binding of the ACT ACP to FabF. Nonetheless, these data support the model that the cross-linking reaction between ACP-TNB − and FabF is selective for functional interactions and can be applied to screen for other hybrid ACP-KS partnerships.
ACP-TNB − can be used to screen for small molecule inhibitors of ACP-KS interactions. We reasoned that because the colored TNB 2− molecule is selectively released upon binding between the AcpP Ppant arm and KS active site cysteine, the methodological advance presented above could be applied to screen for small molecule inhibitors of the ACP-KS binding event, a protein-protein interaction that is essential in the construction of microbial fatty acids. To test this hypothesis, we pre-incubated FabF with cerulenin, which serves as a weak inhibitor of the active site Cys163 residue on FabF 22-24 , prior to mixing in equimolar AcpP-TNB − . We observed that increasing concentrations of cerulenin led to a decrease in the ACP-KS complex band on non-reducing SDS PAGE and less of an increase in A 412 (Fig. 5). Consistent with the model that FabF can adopt multiple conformers, one of which is cerulenin-insensitive 25 , we do not observe 100% inhibition, even at concentrations in 10-fold excess. We observe a 2 μM +/− 0.5 IC 50 value, which is consistent with the range of values reported in the literature 23,24 . These data show that the ACP-TNBbased colorimetric assay additionally can be used to screen for small molecule inhibitors of ACP-KS interactions.

Discussion
Microorganisms utilize fatty acid and polyketide synthases to construct primary and secondary metabolites that serve important roles in the ability of the organism to survive and thrive. A central protein-protein interaction occurs during biosynthesis of polyketides and fatty acids between the ACP and KS 1 . These two proteins come together to facilitate two types of reactions: (1) chain transfer, which involves the active site thiolate of the KS acting as a nucleophile in a nucleophilic acyl substitution reaction of the nascent fatty acid or polyketide covalently tethered to the thiol of the ACP Ppant arm via a thioester; and (2) chain elongation, which involves a decarboxylative Claisen-like condensation of malonyl-based extender units tethered to the ACP Ppant arm via a thioester with an acyl group bound to the active site of KS via a thioester. While the ability to study mechanistically relevant ACP-KS interactions is essential to efforts to create new antibiotics through combinatorial biosynthesis and small molecule inhibitor design, progress has been stymied by the lack of methodology to easily obtain and detect ACP-KS interactions.
Incorporating a reactive electrophilic warhead, such as an epoxide or Michael acceptor, into the Ppant arm activates the ACP to form a covalent cross-link with the active site cysteine of a cognate KS in both FASs and type II PKSs 1,7-9 . Similarly, we showed previously that the modification of an ACP Ppant arm to a thiocyanate (ACP-SCN) activates the Ppant arm to form a cross-linked complex with a cognate KS, a methodology that provides easy access to the ACP-KS complex and a handle to report on functional engagement through the release of CN − , which can be monitored by IR spectroscopy 6 . To obtain the relevant ACP-SCN, the holo-ACP is first reacted with Ellman's reagent (DNTB) to make the mixed disulfide ACP-TNB − , which is subsequently reacted with NaCN. In the context of an undergraduate research experience, we discovered that ACP-TNB − serves to activate the ACP Ppant arm to form a complex with the thiolate active site of a cognate KS in a mechanistically relevant manner similar to what has been shown by other means by our lab and others 1,6,7,9,26 . Ppant-Cys cross-linked ACP-and peptidyl carrier protein (PCP)-protein complexes have been leveraged previously both in vivo 27 and in vitro to obtain important information about the structure and function of ACPs and PCPs 6,28 . The discovery presented in this work is a notable advancement from previous work because of the extreme ease of preparation of the activated ACP Ppant arm (a one-pot reaction of holo-ACP with DNTB) as well as the ability to monitor productive engagement in real time via a visible release of TNB 2− , which has a strong absorbance at 412 nm.
Since its introduction in 1959, DTNB (aka Ellman's reagent) has been extensively used as an efficient and inexpensive method for the quantification of protein sulfhydryls and enzyme activity 18,29,30 . While DTNB has been applied to quantify the ratio of apo-to holo-ACPs in solution 31,32 , the application of DTNB to activate the Ppant arm of ACPs to obtain and visualize ACP interactions with proteins harboring an active site thiol represents an innovative use of this reagent. The resulting assay is an improvement from other methods to assess ACP-KS interactions because it is simpler, quicker, cheaper, and less toxic. It is also notable that this discovery emerged from a CURE experience involving undergraduate students engaged in exploring real research questions and learning from unexpected outcomes in the context of STEM education 33,34 .
The crosslinking methodology presented in this work can be expanded to investigate the interactions of other pantetheine-based moieties, such as coenzyme A and the PCPs of non-ribosomal peptide synthetases. Future efforts will focus on leveraging this methodology to obtain crystal structures of ACP-and PCP-protein complexes to identify the molecular recognition features that guide functional ACP and PCP interactions, as well as demonstrating the high throughput applications and generalizability of the colorimetric assay. There remains unmet need for a facile and high throughput method to evaluate ACP-and PCP-protein interactions from the perspectives of both the rapid screening of functional hybrid protein partnerships as well as small molecule inhibitors of FASs. We envision that the methodological advancement presented herein will help fill this gap and be useful in www.nature.com/scientificreports www.nature.com/scientificreports/ the assembly of synthetic pathways leading to new "unnatural natural" products as well as in the discovery and development of novel antibiotics.
Prior to applying the eluant onto a poly-prep (Bio Rad) column, 200 μL of expressed ACPs equilibrated resin was removed for analysis by LC-MS to evaluate the ratio of apo-vs holo-ACP in solution. In brief, the 200 μL sample was centrifuged at 12,470 g for 1 minute using a microcentrifuge to separate the supernatant, which contained lysis buffer with other proteins, from the resin-bound ACP. After washing the resin twice with 200 μL wash buffer (50 mM sodium phosphate, pH 7.6, 300 mM NaCl, and 30 mM imidazole), proteins were eluted with elution buffer (50 mM sodium phosphate, pH 7.6, 100 mM NaCl, and 300 mM imidazole). The eluted sample (10 μL) was mixed with 90 μL of ddH 2 O and used for LC-MS (see details below).
When necessary, ACPs were converted to 100% holo using an on-resin Sfp reaction protocol developed in our laboratory. Ni-NTA resin harboring the His 6 -tagged ACP was washed with lysis buffer until the A 280 reached baseline and then washed again using wash buffer until the A 280 once again reached zero. Resin was then mixed with 2 mL of an Sfp reaction mixture containing 50 mM sodium phosphate, pH 7.6, 10 mM MgCl 2 , 2.5 mM coenzyme A (CoA), 15 mM fresh dithiothreitol (DTT), and 6 μM Sfp R4-4 phosphopantetheinyl transferase (a generous gift from Jun Lin's Lab at Georgia State University) 13 . The Sfp-catalyzed addition of the Ppant arm onto remaining apo-ACP took place on column upon gentle rotation at room temperature. After reacting for 12-16 hrs, the resin was washed again with washing buffer until the excess coenzyme A was removed from the reaction and A 260 reached baseline. The Ni-bound holo-ACP sample was then eluted in 1-mL fractions with 10 mL of elution buffer (50 mM sodium phosphate, 100 mM NaCl, 300 mM imidazole, pH 7.6). Protein content was assessed by A 280 readings (ε 280 nm AcpP = 1,490 M −1 cm −1 , ε 280 nm ACT ACP C17S mutant = 4,470 M −1 cm −1 ) using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific) and methods described in Pace et al.
Fast Protein Liquid Chromatography (FPLC). AcpP-TNB − , C17S ACT ACP-TNB − , and FabF (diluted to ~1 mg/ mL for FabF and ~0.5 mg/mL for holo-ACPs in 500 μL in sodium phosphate buffer, pH 7.6) were subjected to gel filtration using an ÄKTA pure chromatography system (GE Healthcare Life Sciences) equipped with a Superdex 75 Increase 10/300 GL column (GE Healthcare Life Sciences) with a molecular weight range of 3000-70000 Da. Cross-linked samples were run at 1:2 FabF:ACP-TNB − ratios (25:50 uM). Elution was carried out in 1 mL fractions with 1.50 column volumes (CV) of the same buffer at a flow rate of 0.8 mL/min. Chromatograms were analyzed using the associated UNICORN software package and plotted in Origin (v.8.6.0).
Cerulenin inhibition experiment. A 7.6 mM cerulenin (Cayman Chemicals) stock solution was prepared in 1 mL of anhydrous ethanol. A total of 30 μL sample was prepared keeping FabF and AcpP-TNB − at 1:1 ratio (25 μM:25 μM) with varying cerulenin concentrations (0, 1, 10, 30, 100, 250 μM). The amount of ethanol in the sample was kept constant at 3% (v/v). FabF was incubated with cerulenin for 2 hours at room temperature with gentle shaking. For high cerulenin concentration (100 and 250 μM), protein aggregation was observed. After 2 hours, AcpP-TNB − was added to the 25 μM FabF and samples were incubated at room temperature for an additional 20 minutes prior to measuring the absorbance. Samples were mixed with equal volume of 2X sample buffer and loaded onto a 4-20% gradient gel. Gels were run at 120 V for about an hour until the front dye reached the bottom. Gels were washed with ddH 2 O for 5 minutes then stained with GelCode Blue Safe Protein Stain (ThermoFisher Scientific) for 15 minutes. Gel was washed overnight with ddH 2 O prior to being imaged. Absorbance data were plotted in Origin (v.8.6.0).
Tandem Proteolysis Mass Spectrometry. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis was performed by the Proteomics and Metabolomics Facility at the Wistar Institute using a Q Exactive Plus mass spectrometer (ThermoFisher Scientific) coupled with a Nano-ACQUITY UPLC system (Waters). Samples from non-reducing SDS PAGE gels were digested in-gel with trypsin and injected onto a UPLC Symmetry trap column (180 μm i.d. × 2 cm packed with 5 μm C18 resin; Waters). Tryptic peptides were separated by reversed phase high pressure liquid chromatography on a BEH C18 nanocapillary analytical column (75 μm i.d. × 25 cm, 1.7 μm particle size; Waters) using a 95 min gradient formed by solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). Blank gradients (30 minutes each) were run between sample injections to minimize carryover. Eluted peptides were analyzed by the mass spectrometer set to scan m/z from 400 to 2000 in positive ion mode. The full MS scan was collected at 70,000 resolution followed by data-dependent MS/MS scans at 17,5000 resolution on the 20 most abundant ions exceeding a minimum threshold of 20,000. Peptide match was set as preferred; exclude isotopes option and charge-state screening were enabled to reject singly and unassigned charged ions. Peptide sequences were identified using MaxQuant 1.5.2.8. MS/MS spectra were searched against a custom E. coli UniProt protein database containing the E. coli FabF and AcpP protein sequences using full tryptic specificity with up to two missed cleavages, static carboxamidomethylation (57.02146) of Cys, variable oxidation (15.99491) of Met, and variable mass addition of 339.07797 or 364.07321 on Ser, and variable presence of N-term Met. Consensus identification lists were generated with false discovery rates of 1% at protein, peptide and site levels.
Far UV-Circular Dichroism (CD). CD spectra were collected using an Aviv Circular Dichroism Spectropolarimeter (Model 410). Spectra were collected using a High Precision Quartz SUPRASIL cuvette with 0.1-cm pathlength (Hellma Analytics) at 25 °C using a bandwidth 1 nm, 0.5 nm step size, and 3 seconds averaging time. Prior to the acquisition of spectra, large non-specific aggregates were removed using a 0.2 μm low protein-binding filter with HT Tuffryn membrane (Pall Corporation). For data analysis, protein ellipticity, mdeg, was converted to molar ellipticity, [θ], with units degrees cm 2 dmol −1 . The spectra were smoothed using a smoothing function implemented in the Aviv software, applying a window width of 11 data points, degree 2. Data were plotted in Origin (v. 8.6.0).
Analytical Ultracentrifugation, Sedimentation Velocity experiment (SV-AUC). All experiments were performed using a Beckman model Optima XL-A Analytical Ultracentrifuge (AUC) equipped with an An-60 Ti rotor. Sedimentation