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. 2020 Nov 6;5(45):29017–29024. doi: 10.1021/acsomega.0c03416

Time-Dependent Fluorescence Spectroscopy to Quantify Complex Binding Interactions

Samuel P Bernhard 1, Candace K Goodman 1, Erienne G Norton 1, Daniel G Alme 1, C Martin Lawrence 1, Mary J Cloninger 1,*
PMCID: PMC7675582  PMID: 33225133

Abstract

graphic file with name ao0c03416_0008.jpg

Measuring the binding affinity for proteins that can aggregate or undergo complex binding motifs presents a variety of challenges. In this study, fluorescence lifetime measurements using intrinsic tryptophan fluorescence were performed to address these challenges and to quantify the binding of a series of carbohydrates and carbohydrate-functionalized dendrimers to recombinant human galectin-3. Collectively, galectins represent an important target for study; in particular, galectin-3 plays a variety of roles in cancer biology. Galectin-3 binding dissociation constants (KD) were quantified: lactoside (73 ± 4 μM), methyllactoside (54 ± 10 μM), and lactoside-functionalized G(2), G(4), and G(6)-PAMAM dendrimers (120 ± 58 μM, 100 ± 45 μM, and 130 ± 25 μM, respectively). The chosen examples showcase the widespread utility of time-dependent fluorescence spectroscopy for determining binding constants, including interactions for which standard methods have significant limitations.

Introduction

Protein–ligand binding interactions are currently measured by many different methods.1,2 Some of the most common methods include microcalorimetry, fluorescence intensity, absorbance intensity, fluorescence anisotropy, surface plasmon resonance, and the enzyme-linked immunosorbent assay (ELISA). However, many important interactions such as protein–carbohydrate interactions are low-affinity associations that are difficult to measure accurately with commonly available methods. Low protein solubility and protein aggregation at higher concentrations also often complicate measurements of ligand binding.3

Fluorescence is a preferred method for monitoring binding interactions because of its speed and sensitivity.4,5 Binding is often readily detected by measuring the change in fluorescence intensity at the monitored wavelength for side chains such as tryptophan.6,7 When changes in fluorescence intensity can be observed for the existing protein side chains, then the appendage of an additional fluorescence label is unnecessary.8

For systems that undergo very small changes in fluorescence upon binding, fluorescence lifetime (rather than equilibrium) measurements can be invaluable. However, the conventionally longer acquisition times and complex data analysis of fluorescence decay approaches such as time-correlated single photon counting (TCSPC)4 have, in the past, made it more difficult to obtain time-dependent spectra. In contrast, Direct Waveform Recording technology (Fluorescence Innovations) is capable of collecting time-resolved data in a fraction of the time of traditional systems. This technique allows decay curves to be treated as a type of spectrum, resulting in a simplified and accelerated analysis process that works well for fluorescence lifetime measurements with both intrinsic fluorescent groups and appended fluorescent dye labels. Thus, in principle, direct waveform recording makes the fluorescent lifetime technique more practical and accessible for protein binding studies.9 In practice, however, this has yet to be widely adopted for the routine determination of dissociation constants.

The binding of carbohydrates to galectin-3 serves to exemplify the advantages of the fluorescence lifetime methodology for measuring low-affinity binding interactions. Galectin-3 is a β-galactoside binding protein with a shallow binding pocket for carbohydrate binding and an N-terminal domain for protein aggregation.10,11 Binding to carbohydrates plays an important role in cancer progression,12−14 but binding interactions can be difficult to study with galectin-3. Individual association constants for simple mono- and disaccharides with galectin-3 are weak, and the protein aggregates when approaching an oligomeric carbohydrate-functionalized system.15 Because of the inherent difficulty of studying these galectin-3 binding interactions, a wide range of dissociation constants for carbohydrate binding to this protein have been reported. The reported values of the dissociation constants (KD) for the binding of galectin-3 to galactose-β1,4-glucose (lactose), for example, range from 1616 to 860 μM17 and include the values 55,18 90,19 130,20 140,21 and 61822 μM. In addition, the reported KD of methyl-(Gal-β1,4-Glc) (methyllactose) is 224 μM.23 This span in reported KD values is a testament to the challenge of accurately measuring weak associations between proteins that are difficult to study and their low affinity but important ligands.

Previously reported methods to quantify galectin-3 binding affinity have employed calorimetry,17,24,25 surface plasmon resonance (SPR),18 NMR,20 fluorescence intensity measurements, and competitive fluorescence polarization.23,26 Some of these methods including calorimetry, NMR, and fluorescence techniques are performed in solution, while SPR experiments and ELISAs rely on surface-bound ligands, receptors, or inhibitors. Isothermal titration microcalorimetry and NMR must be performed at relatively high concentrations of protein (100 μM), while fluorescence measurements including fluorescence polarization and the fluorescence lifetime measurements described here can be performed at much lower concentrations (4.3 μM in this experiment, hundred nanomolar in other time-dependent fluorescence measurements, and even low nanomolar concentrations in fluorescence polarization experiments).26−29 Fluorescence intensity measurements can be quite successful19 but are much more susceptible than the fluorescence lifetime measurements to variations caused by inner filter effects, laser power fluctuations, and concentration discrepancies because of imprecise pipetting which can produce inaccurate results if not considered.

All of these methods have some advantages, and comparisons of the relative association strengths of different ligands when they are determined using the same type of assay are meaningful. However, effects including those mentioned above such as concentration and surface interaction can make it difficult to obtain meaningful absolute values of KD that can be compared across assay types.

Because galectin-3 has weak monovalent binding interactions with simple mono- and disaccharides and readily aggregates in the presence of carbohydrate-functionalized macromolecules, this protein is ideal for showcasing the utility of time-dependent fluorescence spectroscopy to determine even difficult to measure binding constants. Here, fluorescence lifetime binding studies using galectin-3 with lactoside, methyllactoside, and a series of biomimetic multivalent lactose-functionalized glycodendrimers are reported. These values should serve as unbiased solution-based values to which values obtained from other assays could be benchmarked. Of course, the reported fluorescence lifetime methodology also works well for more straightforward ligand–receptor binding interactions that are not complicated by issues such as aggregation, solubility, or low affinity and can therefore be widely utilized.

Results and Discussion

Galectin-3 Binding

The carbohydrate recognition domain (CRD) is a 130 amino acid β-sandwich composed of residues 112–250 (Figure 1).30,31 The S-face of the CRD exhibits a channel capable of binding a range of galactoside-based ligands. Galectin-3 contains three tryptophan residues including a key tryptophan (W181) that mediates a C–H/π interaction in the canonical S-face binding site (Figure 1, PDBID: 3ZSJ(32)). The other two tryptophan residues are located early on the N-terminus (W22 and W26) and do not participate in specific binding interactions.32,33

Figure 1.

Figure 1

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(a) Crystal structure of galectin-3 CRD, shown in green, with lactose in the canonical binding pocket. The environment around the key tryptophan (W181), shown in magenta, is altered by ligand binding. (b) Hydrogen bonds between lactose and H158, R162, N174, E184, and R186 galectin-3 residues, yellow, are shown.

Complex binding events that involve multiple accessible binding sites and potential protein oligomerization, such as those described here, can be modeled by eq 1.

graphic file with name ao0c03416_m001.jpg 1

In this equation, Fb is the fraction of protein present in the bound state, [L] is the concentration of the ligand available in solution, and Bmax is the maximum specific ligand binding extrapolated for an extremely high concentration of the ligand.34,35KD is the dissociation constant, and n is the Hill coefficient. A key feature of this analysis is that observed waveform changes continue with increasing lactose concentration up to a point and then stop, indicating that binding saturation has occurred. Values of Fb plotted for each concentration were the coefficients obtained by fitting each waveform to a linear combination of unbound and saturated galectin-3 waveforms. As described in the Supporting Information, no waveform is explicitly determined for the saturated state; instead, the values associated with a saturated waveform are calculated.

The Hill coefficient, n, acts in this equation to account for complex stoichiometry and cooperativity. When n = 1, this indicates that galectin-3 is binding a single carbohydrate in a noncooperative manner. Values greater than one indicate that galectin-3 is involved in more complex binding interactions that may include nonspecific binding, complex stoichiometries, or positive cooperativity. Hill coefficient values less than one indicate negative cooperativity.36

Under conditions where a single ligand interacts with a single receptor, as is the case for lactose and methyllactose, data can be appropriately modeled by eq 1 but with the Hill coefficient set to n = 1. The multivalent behavior of galectin-3, including its capacity to oligomerize, is widely accepted.37−40 Therefore, allowing the Hill coefficient to be calculated as a noninteger value is also important to approximate the known nature of galectin-3 in multivalent binding scenarios. Data obtained using both fit models are reported as appropriate. Standard deviations are reported to represent the accuracy of the value determined by the curve fit.35

Lactose is an important ligand for study because it is structurally similar to endogenous galectin-3 epitopes, and although it has been explored extensively in the literature, there is significant variation in reported binding constants because of the complexity of the system and the challenge of the measurement (16–860 μM).16−22Figure 2a shows the binding curve and corresponding KD for lactose binding to GST-purified galectin-3, as determined by the fluorescence lifetime experiment. The KD was determined to be 73 ± 4 μM or toward the lower end of the reported range of values. The KD for His-tagged galectin-3 with lactose was 56 ± 8 μM (Figure 2b). As expected, these binding constants are very nearly the same; with error bars, the values are only 5 μM from overlapping. This indicates that the differences in purification protocols, including the addition of the six amino acid residue histidine sequence appended to the N-terminal domain, do not have a significant impact on selective binding into the C-terminal carbohydrate binding domain.

Figure 2.

Figure 2

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(a) Binding curve for lactose and GST-purified galectin-3. Bmax = 1.18. (b) Binding curve for lactose and His-tagged galectin-3. Bmax = 1.26. The concentration of galectin-3 is 4.3 μM. Error bars represent the standard deviation arising from triplicate repetitions of the assay.

PolyLacNAc, LacdiNAc, and LacNAc are the primary endogenous ligands for vertebrate galectins, and the Thomsen–Friedenreich antigen (Galβ1, 3GalNAcα1-O-Ser/Thr) is the putative ligand for interactions such as the cancer cell surface association of Thomsen–Friedenreich-modified Mucin-1 to extracellular galectin-3. As such, these ligands, which are associated with galectin-3-mediated processes, are nonreducing sugars.41−46 The binding interaction between galectin-3 and a nonreducing lactose derivative, methyllactose, was explored in order to compare nonreducing and reducing sugar binding. As demonstrated in Figure 3, binding constants for the galectin-3/methyllactose interaction were determined (GST-purified galectin-3). Using eq 1 with n = 1, a KD value of 54 ± 10 μM was found, which is slightly lower but quite similar to values observed for the reducing lactoside hemiacetal. This indicates that galectin-3 binding assays using reducing sugars are generally useful despite the fact that the native carbohydrate ligands are often nonreducing.

Figure 3.

Figure 3

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Binding curve for methyllactose and GST-purified galectin-3. Bmax = 1.20. The concentration of galectin-3 is 4.3 μM. Error bars represent the standard deviation arising from triplicate repetitions of the assay.

Lactose-functionalized poly(amidoamine) (PAMAM) dendrimers were synthesized according to our previously published procedure47,48 and feature a moderate carbohydrate loading (Figure 4). These multivalent ligands were selected in order to observe and quantify the monovalent binding characteristics present within a multivalent system. For these interactions, it is appropriate to calculate binding based on eq 1 in which the Hill coefficient, n, is not set to 1 in order to accommodate the multivalent nature of binding.

Figure 4.

Figure 4

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Schematic representation of glycodendrimers used in the reported experiments.

Comparatively, the glycodendrimer binding affinities are all equivalent with values of 120 ± 58, 100 ± 45, and 130 ± 25 μM for G(2), G(4), and G(6), respectively. It is important to note that the n terms observed for both the G(2) and the G(6) glycodendrimers were less than one (n = 0.63 and 0.66, respectively). These Hill coefficients suggest that more than one galectin-3 binds per glycodendrimer. Upon binding of galectin-3 to a lactoside endgroup on the glycodendrimer surface, the remaining lactosides must overcome steric crowding to bind additional galectin-3 proteins.49,50 Some lactosides will not be able to access galectin-3 binding sites, causing negative cooperativity from the perspective of the lactoside ligand (Figure 5a,c, Table 1). For G(4), the Hill coefficient, n, was determined to be 1.0 ± 0.2 which is consistent with noncooperative binding. This deviation from the other glycodendrimers can be explained through the optimal flexibility and available surface area of G(4), as compared to G(2) and G(6), which allows for steric hindrance of lactoside dendrimer endgroups to play a less influential role in subsequent binding interactions.51−54Table 1 summarizes the reported binding and cooperativity constants.

Figure 5.

Figure 5

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Binding curve for galectin-3 with (a) lactose-functionalized G(2)-PAMAM (Bmax = 1.37), (b) lactose-functionalized G(4)-PAMAM (Bmax = 0.96), and (c) lactose-functionalized G(6)-PAMAM (Bmax = 1.36). The concentration of galectin-3 is 4.3 μM. Error bars represent the standard deviation arising from triplicate repetitions of the assay.

Table 1. Dissociation Constants for Galectin-3 with Monosaccharides and Glycodendrimers.

compound KD (μM) Hill coefficient (n)
lactosea 73 ± 4 1c
lactoseb 56 ± 8 1c
methyllactosea 54 ± 10 1c
lactose funct. G(2)a 120 ± 58 0.63 ± 0.08
lactose funct. G(4)a 100 ± 45 1.0 ± 0.2
lactose funct. G(6)a 130 ± 25 0.66 ± 0.04
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a

GST-purified galectin-3.

b

His-tagged galectin-3.

c

Calculation performed with n = 1.

As demonstrated in Figure 6a, the net intensity of the intrinsic galectin-3 fluorescence is not significantly changed by binding of lactose. Steady-state fluorescence measurements report the average number of emitted photons over a relatively large period of time (milliseconds or seconds) wherein the fluorescing system has achieved an equilibrium. In Figure 6a, steady-state measurements were taken following excitation with 295 nm light, with the fluorescence emission observed from 300 to 700 nm. The similarity in energy between the excitation and emission wavelengths results in only a small segment of the intrinsic galectin-3 fluorescence emission peak (>330 nm) being obtainable without saturation. The lack of a significant change in the fluorescence emission spectrum upon the addition of increasing concentrations of lactose and the limited useful range of the emission spectra showcase the need for fluorescence lifetime measurements.

Figure 6.

Figure 6

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(a) Emission wavelength intensities resulting from excitation at 295 nm. (b) Changes in the fluorescence waveform caused by increasing concentrations of lactose.

Plotting the normalized waveform differences for galectin-3 fluorescence upon binding of lactose further demonstrates that fluorescence intensities are insufficient for quantification of galectin-3 binding (Figure 6b). Specifically, an increase in intensity in the 17–25 ns time regime and an equivalent decrease in intensity from 25 to 60 nm suggests that the rate of fluorescence emission is changed by binding, but the overall intensity is not. It is important to note that the differences shown in Figure 6b are very small relative to the amplitude of the corresponding waveforms, which are shown in the Supporting Information.

Despite the limited range of emission spectra, binding constants were quantified for the steady-state method. Using eq 1 with n = 1, a KD of 167 ± 18 μM was determined. The limited sensitivity of the steady-state measurements and the corresponding large error bars at low concentrations of lactose likely cause the discrepancy in binding constants obtained using steady-state experiments as compared to values obtained using fluorescence lifetime measurements. For both steady-state and lifetime protocols, control experiments were carried out to ensure that fluorescence was not altered by PBS additions, ruling out concentration effects, or by ligand additions independent of galectin-3, ruling out the introduction of unintended fluorophores.

Conclusions

The use of time-dependent fluorescence spectroscopy as a quantitative method for determining dissociation constants of difficult to study receptor–ligand interactions is demonstrated. Binding studies with low-affinity monomeric ligands and proteins that are prone to oligomerization are readily obtainable even when only small changes in the fluorescence emission spectra occur upon ligand binding. Improved accuracy was achieved relative to steady-state fluorescence binding studies, and the fluorescence lifetime method of measuring binding constants can provide benchmark values for assessment of other techniques such as surface-based and steady-state studies.

We showcased the utility of this fluorescence lifetime binding methodology by studying carbohydrate binding to galectin-3. Galectin-3 oligomerizes at higher concentrations and in the presence of multivalent ligands, and the monomeric ligand–receptor binding interaction is relatively weak. For these reasons, binding studies are challenging, and a wide range of binding constants has been reported for the interaction of lactose with galectin-3. Time-dependent fluorescence spectroscopy allows accurate determination of solution binding constants for the binding of lactose by galectin-3 (56–73 μM, depending on the exact nature of galectin-3 that was used). Comparisons of galectin-3/lactose binding constants to those of methyllactose (because native galectin-3 binds to nonreducing sugars) are reported. In addition, the binding constants for the ligands on a multivalent glycopolymer binding to galectin-3 were reported and were observed to be impacted by receptor crowding effects. Fluorescence lifetime measurements of binding interactions were demonstrated to work well for a challenging, low-affinity system and are be expected to be even more facile for higher affinity, easier to handle ligand–receptor associations.

Experimental Section

General Methods and Reagents

General reagents were purchased from Sigma-Aldrich and Fischer chemical companies, and β-d-lactose (80% β) was purchased from Acros Organics. These were used without any further purification. Methods to synthesize methyllactose are described in the Supporting Information. Glycodendrimers were prepared according to the previously published procedure.47,48 Dialysis was carried out using Spectra/Por 6-regenerated cellulose tubing obtained from Spectrum Labs (Repligen Corp., Waltham, MA). 1H and 13C NMR spectra were recorded on a Bruker DPX 300 (300 and 75 MHz for 1H and 13C, respectively), a Bruker DRX 500 (500 and 126 MHz for 1H and 13C, respectively), and an AVANCE III Bruker 600 (600 and 151 MHz for 1H and 13C, respectively). Mass spectrometry was carried out on a Bruker Autoflex MALDI-TOF using the indole acrylic acid (IAA) matrix. Dynamic light scattering (DLS) data were collected with a Möbius DLS instrument from Wyatt Technologies (Santa Barbara, CA), proteins were in PBS (1× PBS diluted from the Cold Spring Harbor 10× PBS protocol). Fluorescence lifetime measurements were taken with a UV NovaFluor fluorescence lifetime spectrophotometer from Fluorescence Innovations.

GST-Tagged Galectin-3

Glutathione S-transferase (GST)-tagged galectin-3 was expressed in Escherichia coli transformed with the pGEX-6p-Galectin-3 vector, purified, and the GST tag removed as previously described.55 Prior to use, filtration through 0.02 μm filters (Whatman Anotop 25) and analysis with Wyatt Mobius DLS were carried out to ensure that galectin-3 oligomers were not present.15 The concentration of galectin-3 was determined spectrophotometrically (280 nm, PBS reference) with a molar extinction coefficient of 35,870 M–1 cm–1.

His-Tagged Galectin-3

Cloning

Human galectin-3 was amplified by PCR using the pGEX-6p-Galectin-3 vector as a template. The forward and reverse primers, respectively, were 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAGATAGAACCATGCATCACCATCACCATCACGCAGACAATTTTTCGCTCCAT-3′ and 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTATTATATCATGGTATATGAAGCAC-3′. The cloning strategy56 introduced attB sites and a Shine-Dalgarno sequence, and effectively replaced the N-terminal methionine residue with a Met–His6 sequence to give a minimal N-terminal 6× His-tagged construct (Met–His6 fused to residues 2–250 of human galectin-3). The amplified product was inserted into pDONR201 (Invitrogen) by site-specific recombination using the Gateway BP clonase reaction following the manufacturer’s instructions. The sequence-verified construct was then transferred into pDEST14 (Invitrogen), again using Gateway site-directed recombination, yielding the expression vector pDest14-hGal3-1-250-NHis. Theoretical values for the gene product are a mass of 26,975.18 Da and a pI = 8.58, with an extinction coefficient of 35,870 M–1 cm–1.

Expression and Purification of His-Tagged Galectin-3

For protein expression, E. coli BL21 (DE3)-pRIL cells (Stratagene) were transformed with pDEST14-hGal3-1-250-NHis and plated on LB-agar plates containing 100 μg/mL ampicillin and 25 μg/mL chloramphenicol. A single colony was used to inoculate a 5 mL overnight LB starter culture (100 μg/mL ampicillin and 25 μg/mL chloramphenicol), which was subsequently used to inoculate ZYP-5052-rich autoinduction media57 (1:1000). The culture was shaken with rotation at 250 rpm at 37 °C for 18 h. Cells were harvested by centrifugation at 4000g for 30 min, and pellets were stored at −20 °C until needed. For purification, cell pellets were thawed and resuspended in 5 mL lysis buffer (50 mM Tris-Cl pH 8.0, 300 mM NaCl, and 1 mM PMSF) per gram of the cell pellet and lysed by French Press. The lysate was centrifuged at 25,000g for 30 min, and the supernatant was applied to a Ni-NTA affinity column. The column was washed with lysis buffer and eluted with lysis buffer and 300 mM imidazole. Fractions containing galectin-3 were combined and applied to a Superdex S-75 size exclusion column equilibrated with PBS. Galectin-3 was eluted as a monomer. Protein concentrations were determined by Bradford assay58 using the protein assay reagent (Bio-Rad) and BSA as a standard. The purity and molecular weight of galectin-3 were confirmed by SDS-PAGE. Yields were approximately 20 mg of pure galectin-3 per liter of cell culture. Fractions containing pure galectin-3 were combined, and 1 mL aliquots at 0.5 mg/mL were stored at −80 °C.

Fluorescence Lifetime Studies

Fluorescence was monitored using a UV NovaFluor fluorescence lifetime spectrophotometer equipped with a high power (>20 μJ) 532 nm passively Q-switched laser (Teem photonics). This serves as a pump for a pyrromethane dye (600 μM in ethanol) to create a 560–610 nm lasing output, which is then passed through a doubling crystal and the subsequent bandpass filter (Semrock) to achieve 285–310 nm excitation (1 μJ/pulse). Samples were excited at 295 nm to selectively excite tryptophan residues within the protein. Emitted light was selected using a monochromator, and fluorescence decay curves were recorded using direct waveform recording technology.9

The concentration of galectin-3 was adjusted by either dilution with PBS or using a protein spin concentrator (Pierce, 10 kDa MWCO) to afford an appropriate concentration (approx. 24 μM, A280 = 0.81) stock solution for experimentation. Galectin-3 samples were prepared by combining the appropriate volumes of galectin-3 stock (24 μM) and PBS to achieve a 200 μL solution at a galectin-3 concentration of 4.3 μM in three wells of a 96-well quartz microplate (Hellma, 730.009-QG). Fluorescence lifetime waveforms were measured in triplicate for galectin-3 solutions at a 0 μM ligand concentration and following each ligand addition. Sugar or glycodendrimer stock solutions were made up and introduced to the protein solution in a stepwise manner. GST-purified galectin-3 was used with lactose, methyllactose, and glycodendrimer experiments, while the His-tagged galectin-3 was used with lactose experiments. Specifically, from a 10.0 mM lactose solution in PBS, small aliquots (4 × 0.5 μL) were introduced, followed by increasingly larger volumes (3 × 1 μL, 3 × 2 μL, and 2 × 5 μL). These sequential additions resulted in lactose concentrations of 25, 50, 74, 99, 148, 196, 244, 338, 431, and 521 μM. For a single experiment, analysis was completed in three wells. Galectin-3 fluorescence waveforms were obtained in triplicate for individual wells and averaged for each concentration. Values of Fb (eq 1) for each concentration were the coefficients obtained by fitting each waveform to a linear combination of unbound ([L] = 0 μM) and saturated galectin-3 waveforms. Fb values, reported as the y-coordinates in ligand binding curves, are the average of individual well coefficients for replicates within an experiment, and the corresponding error bars are the standard deviation. For the purposes of demonstrating experimental reproducibility, lactose binding interactions have been determined using both GST-purified and His-tagged galectin-3. Moreover, this procedure afforded the same KD values across multiple stock solutions, including those made with galectin-3 from different rounds of expression and purification. The experiment can be performed using the serial dilutions described above or using the desired concentrations in multiple wells.

Steady-State Fluorescence

Fluorescence was monitored using a BioTek Synergy H1 microplate reader. Galectin-3 excitation was achieved at 295 nm, and emission was monitored from 300 to 700 nm.

Galectin-3 samples were prepared by combining the appropriate volumes of galectin-3 stock (24 μM) and PBS to achieve a 200 μL solution at a galectin-3 concentration of 4.3 μM in three wells of a 96-well quartz microplate (Hellma, 730.009-QG). Fluorescence emission spectra were measured for galectin-3 solutions at a 0 μM ligand concentration and following each ligand addition. From a 10.0 mM lactose solution in PBS, small aliquots (4 × 0.5 μL) were introduced, followed by increasingly larger volumes (3 × 1 μL, 3 × 2 μL, and 2 × 5 μL). These sequential additions resulted in lactose concentrations of 25, 50, 74, 99, 148, 196, 244, 338, 431, 521, 741, and 950 μM.

Acknowledgments

The authors acknowledge financial support from NIGMS 62444 and from the Tamara Joy Henderson Fund. E.G.N. acknowledges financial support from NIGMS P20GM103474. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c03416.

  • Synthesis procedures and NMR spectra for methyllactose; additional figures for steady-state fluorescence binding experiments; and full description of binding constant calculations (PDF)

  • Data with embedded formulas; template for calculating binding data (XLSX)

  • Bmax values and values used for binding calculations (XLSX)

Author Contributions

The manuscript was written through contributions of all authors.

The authors declare no competing financial interest.

Supplementary Material

ao0c03416_si_001.pdf (838.1KB, pdf)
ao0c03416_si_002.xlsx (9.8MB, xlsx)
ao0c03416_si_003.xlsx (19.3KB, xlsx)

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