Chemistry of conjugation to gold nanoparticles affects G-protein activity differently
© Singh et al.; licensee BioMed Central Ltd. 2013
Received: 10 October 2012
Accepted: 6 March 2013
Published: 19 March 2013
Gold nanoparticles (AuNP) are extensively used as biophysical tools in the area of medicine and technology due to their distinct properties. However, vivid understanding of the consequences of biomolecule-nanomaterial interactions is still lacking. In this context, we explore the affect of conjugation of Gαi1 subunit (of heterotrimeric G-proteins) to AuNP and examine its consequences. We consider two bio-conjugation strategies covalent and non-covalent binding.
Affinity of the AuNP to the Gαi1 is 7.58 × 10 12 M-1. AuNP conjugated Gαi1 exhibits altered kinetics of activation, non-covalent bio-conjugates displays retarded kinetics, up to 0.88 fold when GTPγS was used as ligand, of protein activation contrary to covalent conjugates which accelerates it to ~ 5 fold. Conjugation influence intrinsic Gαi1 GTPase function in conflicting modes. Non-covalent conjugation inhibits GTPase function (decrease in activity upto 0.8 fold) whilst covalent conjugation drastically accelerates it (12 fold increase in activity). Altered basal nucleotide uptake in both types of conjugates and GTPase function in non-covalent conjugate are almost comparable except for GTPase property of covalent conjugate. The effect is despite the fact that conjugation does not change global conformation of the protein.
These findings provide clear evidence that nanoparticles, in addition to ‘passive interaction’ with protein (biomolecule), can interact “actively” with biomolecule and modify its function. This concept should be considered while engineering nanoparticle based delivery systems in medicine.
Impressive developments have occurred in nanoscience technology in the past decade, despite which a detailed understanding of nanoparticle (NP) interaction at cellular, sub-cellular and biomolecule level is lagging behind [1–19]. Cedervall et al. have demonstrated that binding and dissociation parameters of protein-nanoparticle complex depend on surface characteristics of nanoparticle as well as physico-chemical properties of the protein . It has been demonstrated that NPs can elevate the rate of protein fibrillation potentially leading to proposals of novel mechanisms for amyloid diseases offering therapeutic opportunities for treatment . Further, imaging studies provide crucial information that nano-conjugation uniformly promotes endocytosis of EGFR, influencing its compartmentalization, and the mechanism of endocytosis . Thus, nano-conjugation cannot be construed as an innocuous tool but may directly alter the cellular processes at the molecular level . Improved understanding of the interactions at nano-bio interface will give answers to questions concerning the effect of conjugation on protein conformation and hence its function.
Majority of the drugs target GPCR, which transduce signal by activating heterotrimeric G-protein which in turn switches on a cascade of downstream signal transduction pathway. The activation status of heterotrimeric G protein regulates the downstream cascade events. Hence, Gαi1 is a very important model protein to investigate the effects of different types of conjugation to nanoparticle. G proteins are ubiquitously expressed and despite the variety in their function and biochemical effects, their structures are very highly conserved. These properties of G proteins, additionally, make them very vital model systems for studying the effects of nanoparticles; an area that is fast gaining importance in biology and medicine.
In the present study, we investigate the effect of the different conjugation strategies on the conformation and function of G proteins. A comparative study is presented, between non-covalently and covalently bound AuNP-Gαi1 conjugates. In the non- covalent conjugate, the rate of basal nucleotide uptake was retarded in a concentration dependent manner of AuNP, whereas in the covalent conjugate, the rate was accelerated. Both types of conjugation influenced the intrinsic Gαi1 GTPase function affecting the kinetics of GTP hydrolysis in opposite modes. Non-covalent conjugation showed inhibitory effect on GTPase function whilst covalent conjugation dramatically accelerated it. We propose that the mode of interaction with nanoparticles modulate the function of the protein in the conjugate, which may alter related cellular physiological pathways. These findings provide strong evidence that nanoparticles can interact “actively” with biomolecules and modify their function.
Bio-conjugation exploiting two different approaches
N-terminal covalent conjugation using EDC chemistry: Site specific conjugation was achieved by forming a peptide bond between N-terminal primary amine of the protein and carboxylic acid groups of negatively charged AuNP utilizing 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide, EDC, chemistry. Retardation of AuNP’s electrophoretic mobility on agarose gel confirmed conjugation (Additional file 1: Figure S1A). Negative control (AuNP in presence of EDC without protein) also exhibited negligibly small amount of retardation in mobility due to the formation of O-acylisourea intermediate between AuNP and EDC. Changes in mobility of Au:Gαi1 complex depends on the concentration of EDC used.
Non-covalent conjugation: In non-covalent conjugation AuNP capping ligand plays an important role in the bio-conjugate. In the present study, AuNP is capped with DHLA which gives an overall negative charge on its surface. The protein may interact with AuNP in a number of orientations, or many AuNP’s could be attached to a given molecule of protein. Non-covalently bound protein-NP complex was also retarded in electrophoretic mobility, compared to AuNP itself (Additional file 1: Figure S1B), confirming their conjugation. To explore further, whether the conjugation was cysteine mediated, Gαi1 sulfhydryl groups were modified using Iodoacetamide. Conjugation to AuNP was observed even with cysteine modified Gαi1 (Additional file 1: Figure S1B), as evidenced by retardation in its electrophoretic mobility. Mobility of Gαi1-AuNP and cysteine-modified-Gαi1-AuNP were similar, ruling out cysteine mediated interaction between AuNP and Gαi1. To further demonstrate that non-specifically conjugated Gαi1 has free sulphydryl groups, N-(3-pyrene) maleimide (NPM) was used to check formation of fluorescent adducts with free thiol groups (Additional file 2: S2). Cysteine-modified Gαi1-AuNP, upon treatment with NPM displayed fluorescence spectrum with peaks at 377 nm, 397 nm and 418 nm similar to Gαi1-NPM adduct, though, with a lesser intensity. These results confirm the presence of free sulphydryl groups of Gαi1 even after non specific conjugation with AuNPs.
Quenching of Gαi1 Tryptophan fluorescence by AuNP
Rate of activation is differentially affected by the nature of conjugation
Fluoroaluminates activate Gαi1-GDP by mimicking the γ-phosphate of GTP in its binding site. Time dependent fluorescence changes from Gαi1 upon activation by AlF4- binding was monitored for non-covalent and covalent AuNP conjugated Gαi1 and activation rates were calculated.
Effect of AuNP on basal rate constants of Gαi1-AlF4- binding
Non-Covalent AuNP- Gαi1 [AuNP] nM
k app (sec -1 )
Covalent AuNP- Gαi1
(ii) Covalent conjugation: Conversely, covalent conjugation at the N-termini of the protein caused enhancement in the rate of AlF4- mediated activation, 3.2 fold in comparison to unconjugated protein (Figure 2B, Table 1).
Both non-covalently and covalently bound AuNP did not perturb the characteristic feature of Gαi1 to bind GDP nucleotide and its behaviour to undergo activation-dependent changes induced by transition state mimetic, AlF4-. Non-covalent and covalent conjugation, modulated kinetics of AlF4- induced activation of Gαi1 in contrasting manner. The rate of activation by AlF4- is much faster in case of covalently conjugated protein and the peak fluorescence of active protein was comparable with respect to unconjugated protein (Figure 2).
Conjugation does not affect the secondary structure of the protein
Far-UV Circular Dichroism (CD) spectra were recorded to monitor secondary structural features of the protein. Non-covalent or covalent conjugation with AuNP did not cause changes in secondary structure of the protein suggesting the global structure of complex of Au:Gαi1(GDP) to be intact (Additional file 3: Figure S3). These findings clearly indicate that the conjugation of AuNP changes the activity of Gαi1 without affecting the conformation of the protein.
Mode of interaction between Gαi1 and AuNP alter the kinetics of basal GTPγS binding
Effect of AuNP conjugation on Gα i1 basal GTPγS uptake
Non-Covalent AuNP- Gαi1[AuNP] nM
k app (sec-1)
Covalent AuNP- Gαi1
AuNP conjugation modulates Gαi1 intrinsic GTPase activity
We used an extrinsic fluorescent probe, N' – Methylanthraniloyl (mant)-GTP (mGTP) in order to quantitatively study effect of AuNP conjugation on binding and release of nucleotide (GTP/GDP) and monitor Gαi1 activation. Fluorescence resonance energy transfer (FRET) was monitored as a function of time by exciting the intrinsic tryptophan fluorescence at 295 nm and measuring the mGTP fluorescence at 448 nm. 200 nM Gαi1 was titrated with several concentration of mGTP (100 nM to 800 nM). mGTP fluorescence increased upon addition to Gαi1 and then decayed at a slower rate, confirming mGTP hydrolysis to mGDP. Further, addition of 10 μM GTPγS decreased the mGDP fluoresecence rapidly. To obtain the corrected mGTP uptake and hydrolysis, fluorescence remaining after GTPγS addition was subtracted.
Effect of AuNP conjugation on Gα i1 basal mGTP uptake
Non-Covalent AuNP- Gαi1[AuNP] nM
k app (sec-1)
Effect of AuNP conjugation on Gα i1 GTPase activity
Non-Covalent AuNP- Gαi1[AuNP] nM
k app (sec-1)
Covalent AuNP- Gαi1
In the present study, we address the dependence on the chemistry of conjugation towards alteration in the kinetics of activation of Gαi1. There have been contradicting observations regarding the benefit of using a nanoparticle in medicine and biochemistry [25–27]. In our view, understanding the physico-chemical basis of how an engineered nanoparticle modulates biological processes requires the study of nanoparticle-biomolecule binding and its effects on biomolecule functionality. Recent studies have emphasized that properties like size, shape, surface modification, and charge of nanoparticles can profoundly affect the interaction between NPs and biomolecules.
Guanine nucleotide binding proteins (G-protein) play a vital role in the physiology of a cell. Structure-function relationship of both, the monomeric and the heterotrimeric G proteins are well understood, their crystal structures helping elucidate their mode of action and the biochemical function. Heterotrimeric G-proteins are activated by agonist-stimulated G Protein-Coupled Receptors (GPCRs) that catalyze the exchange of GTP for GDP on G protein α-subunits and relay extracellular signal to intracellular signalling pathways . We chose to use G proteins as model proteins to better understand the effect of AuNP binding to proteins and the biological effect they elucidate.
Here, we exploit two modes of conjugation between Gαi1 and AuNP, covalent and non-covalent. Effect of AuNP conjugation to Gαi1 was examined by monitoring steady-state Trp-fluorescence from the protein. AuNP interacts with Gαi1 with a binding constant (Kb) of 7.58 × 10 12 M-1. Strong tryptophan fluorescence quenching of Gαi1 was observed with increasing concentration of AuNP. Fluorescence quenching could be explained by efficient energy transfer between AuNP and Gαi1 tryptophan residues. No shift in emission wavelength was noticed, suggesting no change in polarity around tryptophan residues on addition of AuNPs.
Cysteine modifications did not alter the AuNP conjugation and, vice versa, conjugation did not lead to the unavailability of the thiol groups of cysteine for the modifying reagents, therefore leading us to conclude that the 10 cysteine residues (of Gαi1) do not interact with AuNP via thiol-Au linkage chemistry. Our finding is in agreement with a previous study which demonstrates that cysteine residue at the end of a C-terminus of protein was much more reactive toward a gold cluster than a cysteine residue introduced in middle region of protein . This study concludes that non-specific AuNP interaction is not protein-sulphydryl mediated even though cysteine residues are present on Gαi1 surface.
A number of studies have shown that nanoparticle protein conjugates undergo conformational changes and result in unfolding of protein [30–32]. For biochemical applications of NP-protein conjugates, it is crucial that labelling does not modify the protein structure. Interestingly, both types of conjugates of Gαi1 retain their secondary structure as evident from far UV-CD spectra profile for Gαi1 and the conjugates (Additional file 3: Figure S3).
We next investigated whether the bioconjugated Gαi1 was functional and active. We report here functional activity of both the covalent and non-covalent AuNP conjugated Gαi1. Time dependent fluorescence measurement using intrinsic tryptophan and extrinsic MANT moiety fluorescence with hydrolyzable and non-hydrolyzable nucleotides were assayed. Detailed kinetics based functional studies for both non-covalent and covalent AuNP-Gαi1 conjugates have provided important insights: (i) reduced rate of activation by AlF4-, GTPγS and mant-GTP were observed as a consequence of non-covalent interaction of AuNP. (ii) N-terminal covalent probing led to enhanced rate of nucleotide uptake “activity” of Gαi1.
In summary, we here report two different bioconjugation strategies, non-covalent and covalent attachment, of Gαi1 to 6 nm DHLA capped AuNP. No effect of change in protein conformation was observed despite the presence of negatively charged capping ligand, DHLA. Non-covalent bioconjugation caused decrease in “activity” of Gαi1 in terms of decelerated rate of nucleotide exchange and inhibited GTPase activity. N-terminal covalent probing of AuNP modulate the active state of Gαi1 state, as displayed by enhanced rate of nucleotide exchange and stimulated GTPase function. These results (extraordinary increase in the Gαi1 GTPase property) have ramification in understanding the probable molecular basis of gold to cure many diseases when used either in powder form (in ayurvedic treatment) or as colloidal gold in modern medicine (e.g., in arthritis).
All the chemicals used were purchased from Sigma-Aldrich, USA.
Synthesis of Gold nanoparticles (AuNPs)
Size and optical characterization of AuNP
Transmission electron microscopy was used to visualise the shape and to determine size distribution of AuNPs (Additional file 5: Figure S5). TEM images were obtained using JEOL 3010, operating at 300 kV accelerating voltage. The average size distribution was determined by using the image analysis software, Image J. The UV–vis absorption spectra were recorded on a Jasco V-660 UV–vis spectrometer at room temperature with 1 cm path length cuvette. Spectra were obtained with a band width of 1.0 nm and a scan rate of 40 nm/minutes (Additional file 6: Figure S6). Toluene was used as reference.
Expression and purification of Gαi1 protein
The DNA fragment containing the WT rat Gαi1 subunit, cloned into the pET28b expression vector was used to transform BL21 (DE3) cells to express an N-terminal hexa-His-tag-WTGαi1 protein in the presence of kanamycin (100 μg/mL) and purified (Additional file 7: S7). The eluted protein fractions with the maximum protein content were estimated by Lowry’s method  and purity checked by SDS-PAGE. The average yield for WT Gαi1 was 10 mg/L of culture.
Non covalent conjugation: 100 μL, 0.2 μM purified AuNP-DHLA (in 5 mM Hepes-Na, pH 8.0) was incubated with 100 μL, 200 μM Gαi1 at room temperature for 20 minutes. Unconjugated protein and free AuNP were removed by centrifugation (12000 rpm, 20 minutes 4°C) (Additional file 1: Figure S1A).
Covalent bioconjugation: 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC) was used as a cross linking agent between carboxyl groups of AuNP and primany amines of Gαi1. For linkage, 100 μL 3 mM EDC (prepared in double distilled water) was added to 100 μL 0.2 μM AuNP and mixed. 100 μL, 200 μM Gαi1 was added to the mixture containing AuNP and EDC and incubated for 15 minutes at room temperature. Unbound protein and AuNP were removed by centrifugation (15 min, 14000 rpm). Gel electrophoresis was used to confirm conjugation (Additional file 1: Figure S1A).
Steady state fluoresecence
Steady state intrinsic tryptophan fluorescence of Gαi1 in inactive form was recorded on HORIBA Jobin Yvon fluorolog spectrometer with excitation light of 295 nm (excitatoin and emission slit width of 5 nm) at 25°C. In a 3 mL quartz cuvette, 400 nM Gαi1 [in 5 mM Hepes-Na (pH 8.0), 10 mM NaCl, 0.5 mM MgCl2 and 1 μM GDP] was taken and titrated with AuNPs. In all cases blank spectra (buffer containing only AuNP) were subtracted from the protein spectra.
Fluorescence-based kinetic assays
Time-based fluorescence activity measurements were performed on a Jasco FP-6500 Spectrofluorometer at 25°C. In a 3 mL cuvette, 400 nM Gαi1 [in 5 mM Hepes (pH 8.0), 10 mM NaCl, 0.5 mM MgCl2 and 1 μM GDP] was taken. 16 μM GTPγS was added to the protein and the relative increase in intrinsic fluorescence (λex = 295 nm, λem = 340 nm) was measured as a function of time. Similar measurments were performed for AuNP conjugated Gαi1. GTPγS exchange rates were determined as described elsewhere . FRET was monitored by exciting the intrinsic Trp fluorescence at 295 nm and measuring the mant-GTP fluorescence at 448 nm. In all cases, blank spectra containing buffer alone were subtracted from the final spectra.
Circular dichroism (CD) spectroscopy
CD measurements were made on a JASCO model J-715 spectropolarimeter. Far-UV-CD spectra were recorded in 1 cm path length cuvette from 200 to 260 nm; each spectrum was the average of 5 scans. Spectra were recorded with the final protein concentration of 100 μg/mL. Appropriate buffer spectra were recorded and subtracted from the protein spectra.
The apparent rate constants (kapp) reported (Table 1, 2, 3) is the mean of several independent experiments and represent kapp x 10-3 sec-1. Initial 1000 (for non-covalent conjugation) and 200 (covalent conjugation) data points were used to calculate the apparent rate constants reported. In Table 4, the apparent rate constants (kapp) reported is the mean of several independent experiments and represents kapp x-10-4 sec-1. The -fold change in the rate of the AuNP- Gαi1 to that of Gαi1 is calculated as kapp(AuNP- Gαi1)/kapp(Gαi1).
Authors acknowledge funding form the Department of Biotechnology (Govt. of India), New Delhi and IIT Madras for providing facilities. We thank Prof. G. Jayaraman, VIT Univeristy, Vellore, INDIA for allowing us to use CD spectrometer.
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