Reading Conformational Changes in Proteins with a New Colloidal

Subscriber access provided by WEBSTER UNIV

Biological and Medical Applications of Materials and Interfaces

Reading Conformational Changes in Proteins with a New Colloidal-Based Interfacial Biosensing System Mostak Ahmed, Laura G. Carrascosa, Paul Mainwaring, and Matt Trau ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b18269 • Publication Date (Web): 25 Feb 2019 Downloaded from http://pubs.acs.org on February 27, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Reading Conformational Changes in Proteins with a New Colloidal-Based Interfacial Biosensing System Mostak Ahmed1¥, Laura G. Carrascosa1¥*, Paul Mainwaring1, and Matt Trau1,2* 1

Centre for Personalized Nanomedicine, Australian Institute for Bioengineering and

Nanotechnology (AIBN), The University of Queensland, Brisbane, QLD 4072, Australia 2

School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD

4072, Australia *corresponding authors ¥ joint-first

authors

ACS Paragon Plus Environment

1

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 38

KEYWORDS: protein conformation, protein folding, gold nanoparticles, colorimetric response, tannic acid

ABSTRACT: Many biological events such as mutations or aberrant post-translational modifications can alter protein conformation and/or folding stability and their subsequent biological function, which may trigger the onset of diseases like cancer. Evaluating protein folding is hence crucial for the diagnosis of these diseases. Yet, it is still challenging to detect changes in protein folding, especially if they are subtle, in a simple and highly sensitive manner with the current assays. Herein, we report a new colloidal-based interfacial biosensing approach for qualitative and quantitative profiling of various types of changes in protein folding, from denaturation to variant conformations in native proteins, such as protein activation by underlying mutations or phosphorylation. The approach is based on the direct interfacial interaction of proteins freely available in solution with added tannic acid-capped gold nanoparticles (AuNPs), enabling interrogating protein’s folding in their solubilized form. We found that under the optimized conditions, proteins can modulate the solvation of the colloids according to their folding or conformational status, which can be visualized in a single step, by the naked eye, with minimal protein input requirements (LOD of 1 ng/µL). Protein folding detection was achieved regardless of protein topology and size without using conformation-specific antibodies or mutational analysis, which are the most common assays for sensing malfunctioning proteins. The approach showed excellent sensitivity, superior to Circular Dichroism, for the detection of the very subtle conformational changes in EGFR and ERK proteins induced by activating mutations and phosphorylation, enabling their detection even in complex samples derived from lung cancer cells, which contained up to 95% excess of their wild-type forms. Broader clinical translation was showed via monitoring the action of conformation restoring drugs, such as Tyrosine Kinase


2

Page 3 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Inhibitors on EGFR conformation and its downstream protein network, using ERK protein as a surrogate.

1. INTRODUCTION Proteins naturally switch among different conformations to regulate their functions in different biological contexts and respond to cellular needs.1 The switch among these conformations, referred to as native conformation variants, is often mediated by the addition of post-translational modifications (PTMs), which locally change chemical and physical properties of the protein, and the overall energy landscape to induce the required conformational change. For example, phosphorylation of serine, threonine or tyrosine amino acids in proteins cause a change in the conformation and function of proteins because of the electrostatic perturbation induced by the phosphate group at these sites.2 In normal conditions, this process is reversible allowing proteins to regulate nearly every aspect of cell life through the choice of various conformations.3 However, events such as mutations can disrupt the reversibility of this process by altering the protein sequence and inducing key conformational changes that favor the irreversible phosphorylated conformation variant, resulting in pathological functions and the onset of diseases like diabetes, cancer and Parkinson’s and Alzheimer’s diseases.4-6 The discovery of this mechanism as an important driver of cancer has been key to the design of conformation-rescue drugs, such as tyrosine kinase inhibitors (TKIs), over the last decade. TKIs target these abnormally phosphorylated proteins and restore their former phosphorylation levels and function — a therapeutic breakthrough that has become the basis of many recent anti-cancer strategies.7 Our understanding of the processes influencing changes in protein conformation is rapidly growing, but further research is needed to fully understand the biophysical link between protein


3


Page 4 of 38

conformation and disease, and to identify clinically actionable targets that would lead to more effective approaches to treat the associated diseases. To address this need we have focused on methodologies involving the direct interaction of proteins with gold nanoparticles (AuNPs). These interactions govern the overall behavior of the resulting colloidal system and its potential applications,1, 8-15 especially for cancer detection at early stage.16-18 Although many conditions can influence the overall protein adsorption process and behavior of the resulting colloidal system, (e.g., AuNPs properties such as surface chemistry (i.e., type of capping/stabilizing agents), size, material, morphology, curvature, types of proteins, temperature or incubation conditions) protein conformation can also play a key role.8-9, 19-23 In this regard, it was observed that proteins might undergo conformational changes during adsorption on large AuNPs, and this can lead to AuNPs aggregation under certain conditions.23, 27-29 Also, AuNPs previously derivatised with Cytochrome C proteins could vary their solvation properties and undergo aggregation upon addition of a misfolding agent (e.g., pH change). This effect was presumably due to the action of this agent on the conformation of the protein suggesting a potential use of AuNPs for protein conformation analysis.30 However, this approach requires prior covalent binding of the protein to AuNPs surface — a process that can distort the original protein conformation and bias the subsequent protein folding analysis. More recently, misfolded and fully unfolded antibodies were also found to cause AuNPs aggregation, and this phenomenon has been used to develop an approach for evaluating the structure and function of antibodies. This approach did not require prior covalent binding of the protein to AuNPs surface,31 hence representing a promising avenue to interrogate protein folding. Yet, it is still unclear whether it could be extended to proteins other than immunoglobulins, or if it would have enough sensitivity to determine more subtle conformational changes (e.g., native conformation variants), which does not involve protein misfolding. More importantly, it


4

Page 5 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


remains unexplored whether the changes in AuNPs solvation with distinct protein conformations could translate into a robust tool for clinical applications such as cancer detection or therapy monitoring. Herein, we evaluate AuNPs solvation behavior against a collection of proteins with different topologies and sizes, which had various degrees of conformational changes, including misfolded or unfolded conformations (e.g., denatured proteins), and also native variant conformations. Given that AuNPs can be synthetized with tannic acid as a surfactant, and that this molecule has been reported to have unique interactions with proteins via hydrophobic and hydrogen bonds,24-25 we have further explore role of this type of capping agent in tuning the adsorption of various protein conformations. We found that under optimized conditions, involving the interaction of tannic acidcapped AuNPs with proteins in aqueous solutions, protein conformation can effectively modulate protein adsorption behavior on AuNPs surface and control solvation properties of the colloidal system. We exploit this phenomenon to develop a colloidal-based interfacial biosensing system that could accurately detect changes in protein conformation with great simplicity and minimal protein input requirement (LOD 1 ng/µL), showing a clear advantage over other conventional methods such as Circular Dichroism, which require markedly higher protein amounts. The approach successfully detected misfolding in proteins, regardless of their size or topology, and demonstrated broader applicability by also identifying subtle conformational changes, arisen from protein activation by underlying mutations or addition of phosphorylation modifications. Our method showed high sensitivity and specificity in detecting mutation-induced conformational changes in EGFR that leads to its constitutive auto-phosphorylation and subsequent activation of intracellular phosphorylation pathways. This detection was achieved even in heterogeneous samples with up to 95% excess of its wild-type isoform. This high level of sensitivity highlights


5


Page 6 of 38

the potential of this approach to avoid the current need for mutational analysis for detecting the presence of mutated EGFR in cancer. We also demonstrated the use of this approach to monitor the conformational changes involving the activation of intracellular proteins, which are downstream in the EGFR phosphorylation cascade, using ERK as an example. In this case, the approach showed similar sensitivity levels as per mutation-induced conformational changes in EGFR, showing promise in the analysis of protein phosphorylation-associated conformational changes, regardless of their origin. This finding underscores that this strategy could be used for mapping networks of protein-phosphorylation cancer biomarkers in cells. Finally, we show the potential application of this strategy for cancer therapeutics by monitoring the action of TKIs drugs in restoring EGFR and ERK protein phosphorylation levels and conformation in treated cells. Our approach offers significant advantages over conventional methods for reading the conformational landscape of proteins, namely simplicity and naked eye detection while avoiding conformationspecific antibodies, mutational analysis, labelling or other protein processing steps. Hence, we believe this strategy holds great promise for clinical-translation in cancer care.

2. EXPERIMENTAL SECTION Details on some methods including cell culture, protein isolation, characterization of the tested proteins, and specificity of our colloidal-based interfacial biosensing approach are included in the Supporting Information.

2.1. Chemicals and Materials. Lung cancer cell lines including NCI-H1666, NCI-H1975, and HCC827 were purchased from ATCC (USA) and the cell culture materials such as growth medium (RPMI 1640), fetal bovine serum and antibiotics were procured from Gibco, Life Technologies


6

Page 7 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(Australia). BSA, MUC1, BGG and HER2 proteins were collected from Bio-Rad (Australia). Pierce Classic Magnetic IP/Co-IP Kit containing protein A/G magnetic beads, lysis buffer, elution buffer, and neutralization buffer were purchased from Thermo Scientific (Australia). Silver staining stuff (Silver stain plus kit was bought from Bio-Rad (Australia). Western blot reagents such as NuPAGE 4-12% Bis-Tris Gel, NuPAGE LDS Sample Buffer (4X), NuPAGE MOPS SDS Running Buffer 20X, NuPAGE transfer Buffer 20X, Nitrocellulose Membrane were obtained from Novex, Life Technologies (Australia), the NuPAGE ladders from New England Biolabs and BioRad (Australia), and the Odyssey Blocking Buffer from LI-COR (USA). Phospho-specific antiEGFR (phosphoY1068) monoclonal antibody (ref. ab32430) was purchased from Abcam (Australia); phospho-specific anti-ERK monoclonal antibody (ref. ab136926) from Abcam (Australia); IRDye 680RD anti-rabbit (ref. 926-68071) and IRDye 800CW anti-mouse (ref. 92632210) secondary antibodies from LI-COR (USA). Gefitinib (ref. 4765) was bought from Cell Signalling Technology (Australia). 2.2. Characterization of AuNPs using Dynamic Light Scattering (DLS). DLS measurements were performed with a ZetaSizer 3000-HA (Malvern Instruments, UK). Samples were diluted 1:20 in H20 to a total volume of 1.0 mL. Measurement runs (5 X 10) were performed, with standard settings including refractive index = 1.331, viscosity = 0.89, and temperature = 25°C. Zeta-potential was also performed with the same instrument. Samples were diluted 1:20 in H20 to a total volume of 250 µL and a number of measurements were 50. Standard settings include viscosity 0.89, dielectric constant 78.5 and temperature 25°C. 2.3. Detection procedure of protein conformation based on protein-AuNPs interactions. Unless otherwise stated, experiments were done by mixing 5 µL of each protein at 6 ng/µL (i.e.,


7


Page 8 of 38

30 ng) with 10 µL of 10 nm tannic acid-capped AuNPs (Nanocomposix, Australia). In the original assay form, where no salt was added, the incubation was run for up to 30 min. In the final assay form, the incubation time was reduced to only 3 min, and protein conformation was revealed by subsequent addition of 1.5 µL of Sodium Saline Citrate (1XSSC), which resulted in a color change in the protein-AuNPs system from reddish to bluish if the protein was denatured or phosphorylated. Colorimetric observations were also quantified by a UV-Vis spectrophotometer (Biolab, Australia). 2.4. Circular Dichroism (CD). CD experiments were performed using the Jasco J-715 CD Spectrometer, UK. Secondary structure evaluation required 300 µL of pure non-phosphorylated and phosphorylated EGFR (500 ng/uL) proteins in 10 mM buffer (a mixture of Glycine.HCl and Tris.HCl). The standard settings were less than 600 V HT voltage, far-UV (200-240 nm) wavelength, 3-5 L/min N2 flow rate, and continuous scanning mode with 50 nm/min scanning speed. Tertiary structure evaluation required 500 µL of pure EGFR (1000 ng/µL) in the same buffer under all the aforementioned settings except near-UV wavelength (250-350 nm). 2.5. SERS experiments. All SERS experiments were performed using a portable IM-52 Raman Microscope with a 785 nm wavelength laser and a laser power of 70 mW for excitation. For this test, 20 µL of 6 ng/µL of each protein (non-phosphorylated EGFR and phosphorylated EGFR) was incubated with 40 µL of 10 nm AuNPs for 30 min and then SERS spectrum was recorded at 20 ms integration time.


8

Page 9 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3.

RESULTS AND DISCUSSION

3.1. Distinguishing misfolded proteins in solution using a colloidal-based interfacial biosensing system. To evaluate the effect of protein misfolding on AuNPs solvation, we chose a simple salt-induced aggregation assay (Figure 1A (Scheme)), wherein 5 µL of solution containing 30 ng of either native or denatured protein (6 ng/µL) was added to 10 µL of highly monodispersed 10 nm tannic acid-capped AuNPs (reddish) and incubated at room temperature for 3 min. This step was followed by the addition of 1.5 µL 1XSSC (sodium saline citrate). In this assay, salt was added to destabilise and aggregate the preformed protein-AuNPs system unless the AuNPs were effectively protected by the adsorbed proteins22, 32 — a process that causes a colour transition from reddish to bluish. The colour changes associated with colloidal aggregation were visible by naked eye and could be further analysed quantitatively by UV-Vis spectrophotometry via measuring the absorbance ratio at 520 nm and 658 nm. Monodispersity of the AuNPs was previously confirmed by Dynamic Light Scattering (DLS) which showed low polydispersity index (PdI: 0.136) along with the narrow hydrodynamic size (z-average: 19.2) and single zeta potential peak at -37.5 mV (Figure S1, Supporting Information). To demonstrate the potential of this strategy for detecting major folding changes across multiple protein types, we selected a cohort of nine native proteins representing various categories of protein topologies (i.e., globular: ERK, BSA, BRAF, mTOR and BGG and transmembrane: EGFR, MUC1, PD-L1 and HER2) and sizes (PD-L1 (34kDa) < ERK (44 KDa) BSA > MUC1. This trend indicates that the extent of colloidal aggregation varied widely among proteins and was not biased towards any protein size or topology. For example, denatured EGFR and ERK proteins showed the largest absorbance ratio changes (ΔAnative EGFR - denatured EGFR = 2.45 and ΔAnative ERK – denatured ERK = 2.26) upon salt addition, despite their vastly different topologies (transmembrane vs intracellular) and markedly different sizes (180 KDa vs 44 KDa). Interestingly, we noted that the approach could detect the subtle conformational changes associated with denaturation of bovine serum albumin (BSA), which is believed to cause only minor impacts on the formation and reshuffling of specific disulphide (SS) bonds. 33 This is because native serum albumin has no beta-sheet in its structure34 and it has been suggested that thermally denatured BSA has probably the same fundamental type of folding of the polypeptide chains as the native one — 55% alpha-helix and 45% random conformation according to X-ray scattering studies.31 It thus seems that this SS-reshuffling may have been sufficient to induce significant changes in protein adsorption, presumably by altering the number of exposed thiol groups to AuNPs (thiols are well-known for their high affinity towards gold). To clarify the role of thiols in inducing significant changes in AuNPs solvation, we checked whether the magnitude of the colloidal aggregation could be biased towards proteins with cysteine-rich domains, involved in protein dimerization via disulphide bond formation. Within our protein cohort, only ERK, EGFR, HER2 and MUC1 had these types of cysteine-rich domains.35-38 Of these, EGFR and MUC1 respectively produced the largest (ΔA= 2.45) and smallest (ΔA= 0.77) absorbance ratio changes upon salt addition. This suggests that, while these domains may have an important role in driving the aggregation of the colloidal solution during protein adsorption, they did not have any significant effect in biasing the overall magnitude of colloidal aggregation.


11


Page 12 of 38

3.2. Detection of mutational- and phosphorylation-induced conformational changes in proteins. To demonstrate the broader applicability of our colloidal-based interfacial biosensing system for detecting more subtle conformational changes, we challenged our approach to the detection of variant conformations in native proteins, such as protein activation by underlying mutations or phosphorylation, using EGFR and ERK proteins as an example. Transmembrane EGFR protein, upon binding its cognate ligand, experiences dimerization and conformational change in its intracellular tyrosine kinase domain. This conformational change is a key to allow the access of ATP and the subsequent phosphorylation of the protein at key sites. 39 These phosphorylated sites also act as docking sites for interacting and activating downstream intracellular proteins (e.g., ERK) via a phospho-transfer reaction. However, in many cancers, including lung cancer, the EGFR gene contains mutations that alter the sequence and conformation of the encoded protein.4, 40 When these changes disrupt the native conformation of its tyrosine kinase domain, the protein may adopt a mutation-induced conformation that favours protein’s constitutive autophosphorylation, irrespective of binding of its external cognate ligand. This, in turn, leads to an overactive protein that promotes tumour growth via further activation of downstream signalling pathways. For testing our approach, we focused on two of these EGFR mutant variants, i.e., EGFRE746-A750 Del

and EGFRT790M, which are frequently seen in lung cancer patients. With analysis of these two

mutant variants, we sought to understand if our methodology could detect the presence of a mutation-induced overactive EGFR protein in cancer cells without performing mutational analysis, which is a current method in the clinical setting.41-42 However, we also wanted to explore if our approach could detect a similar conformational change associated with phosphorylation, wherein a DNA mutation is not involved. To this end, we evaluated ERK protein in cells with


12

Page 13 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


previously active EGFR (in cells ERK is present in the cytoplasm in non-phosphorylated form unless activated by upstream phosphorylated EGFR protein). This test would allow us to understand whether our approach could potentially be used to identify any protein involved in an overactive phosphorylation signalling pathway regardless of the signal triggering the phosphorylation of that protein. Such possibility is key for devising more personalised and effective therapeutic strategies that could identify any of these malfunctioning proteins to improve patient outcomes dramatically. Prior to detection with the AuNPs, EGFR or ERK proteins were isolated from selected lung cancer cell lines containing either native EGFR (from H1666 cells) or mutated EGFR variants (from H1975 and HCC827 cells, where EGFR is permanently phosphorylated) via Immuno-purification (IP). The purity of all proteins of interest was validated by highly sensitive silver staining (Figure S2 A, Supporting Information). Also, the phosphorylation status and isoformity of EGFR and ERK proteins were confirmed by western blot (Figure S2 B, Supporting Information). For achieving high sensitivity towards EGFR and ERK native variant conformations, we optimised the assay incubation conditions to favour a rapid aggregation of the colloidal system only in presence of phosphorylated (including mutation-induced phosphorylated) proteins. To this end, we tested conditions such as protein amount (5-40 ng), incubation time (1-7 min), total solution pH (3-9) and AuNPs size (10-40 nm). All of these conditions played a significant role in tuning the solvation of the colloidal system and the resultant sensitivity and specificity towards phosphorylation (Figure 2). In almost all tested conditions, the AuNPs solution remained reddish for non-phosphorylated EGFR protein, while experienced aggregation along with the colour


13


A

B 4

A b s o r b a n c e 5 2 0 /6 5 8

3

2

1

0

3

2

1

9

8

7

6

5

3

0 4

0 3

0 2

1

0

1

0 4

A b s o r b a n c e 5 2 0 /6 5 8

4

pH

P r o te in a m o u n t (n g )

D

C

4

A b s o r b a n c e 5 2 0 /6 5 8

4

3

2

1

3

2

1

7

5

0 4

0 2

0 1

3

0

0

1

A b s o r b a n c e 5 2 0 /6 5 8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 38

A u N P s s iz e (n m )

T im e (m in )

Figure 2. Optimization of operating parameters in protein-AuNPs (5 µL:10 µL) interactions for non-phosphorylated (black) and phosphorylated (red) EGFR compared to control (green) samples containing the buffer only. Parameters tested are (A) protein amount, (B) total solution pH, (C) AuNPs size, and (D) adsorption time. Each data point represents the average of three separate trials and error bars represent the standard deviation of measurements.

change from reddish to bluish for its phosphorylated isoform. The key difference among these conditions was the sensitivity towards detection of protein conformations, as reflected by the magnitude of their associated colour changes. For example, the increase in the protein amount up to 30 ng (6 ng/µL) provided the largest difference in the absorbance ratio between each protein isoform, indicating that this value was crucial for achieving a considerable sensitivity towards this


14

Page 15 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


conformational change. The sensitivity also increased sharply with pH and showed the most significant difference between conformations at pH 7. In contrast, increasing the incubation time for periods longer than 3 min did not increase the sensitivity or specificity of the assay, indicating that the protein adsorption process is very rapid for both protein isoforms in our system. We also observed that an increase in the size of AuNPs significantly reduced our ability to distinguish between conformations clearly. This is presumably due to larger particles favouring both protein conformations to undergo denaturation during adsorption and their subsequent crosslinking-type aggregation.19, 23, 43-45 Hence, we selected 30 ng of protein incubated with tannic acid-capped AuNPs of 10 nm size at total solution pH 7 for 3 min followed by salt addition, as the ideal conditions for monitoring phosphorylation-induced conformational changes. Prior to optimizing these parameters, we first optimized three possible salt concentrations scenarios, i.e., 0.5X, 1X, and 1.5X sodium saline citrate (1.5 µL each). As shown in Figure S3, the 1XSSC was found to provide the maximum gap between control and target. In addition, the limit of detection (LOD) (i.e., 1 ng/µL) and limit of quantitation (LOQ) (i.e., 2 ng/µL) was showed in Figure S5. The specificity of our approach was thoroughly confirmed by control experiments where the immunoprecipitation (IP) was performed in the absence of EGFR/ERK antibody or input protein (Figure S4, Supporting Information). To further evaluate the sensitivity of our assay, we tested its ability for detecting rare populations of conformation variants in complex samples by detecting phosphorylated EGFR or ERK proteins in a sample containing their non-phosphorylation isoforms in different proportions (0%, 5%, 10%, 25%, 50%, 75%, 90%, and 95%). It was possible to detect


15


B

% EG FR 100 95 90 75 50 25 10 5

0

5 4 3 2 1 0 0

5

10 25 50 75 90 95 100

% ERK

A b s o r b a n c e 5 2 0 /6 5 8

A A b s o r b a n c e 5 2 0 /6 5 8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 38

5

100 95 90 75 50 25 10 5

0

4 3 2 1 0 0

5

% P -E G F R

10 25 50 75 90 95 100

% P -E R K

Figure 3. Sensitivity and specificity of the assay for detecting protein phosphorylation under the optimized conditions in pure and heterogeneous samples (5-95%). (A) Absorbance values for native (non-phosphorylated) EGFR isolated from H1666 cells (black), mutated (phosphorylated) EGFR isolated from HCC827 cells (orange), and both mixed in different proportions (grey). (B) Absorbance values for inactive (non-phosphorylated) ERK (blue), active (phosphorylated) ERK (purple) and both mixed in different proportions (grey). ERK’s effect on AuNPs solvation directly correlated with the conformation status of upstream EGFR activation status. Each data point represents the average of three separate trials and error bars represent the standard deviation of measurements. (P-EGFR: phosphorylated EGFR and P-ERK: phosphorylated ERK).

phosphorylated EGFR in excess of up to 95% non-phosphorylated EGFR indicating an excellent performance (Figure 3A). A similar result was also obtained for downstream ERK protein (Figure 3B). This level of sensitivity and selectivity is highly relevant for clinical applications, as biopsies often contain a mixture of healthy and cancer cells with various levels of malignancy.46 Moreover,


16

Page 17 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the amount of protein required is significantly lower than in most conventional techniques such as X-ray scattering,47 fluorescent-based techniques,48 and several spectroscopies5, 49-50 which also require extensive processing before analysis. Hence, they are rather tedious and sophisticated for clinical use, and may also face challenges in detecting rare populations in complex samples. Mutation-based analysis, including DNA sequencing, albeit laborious and expensive, can circumvent the inconveniences of direct protein analysis by identifying the underlying mutation at the DNA level. However, their clinical use is typically limited to mutations already known to cause a misfolding or an aberrant conformation that severely impacts protein function.4, 40, 51 In this regard, our approach may potentially detect any aberrant conformation affecting protein function without previous knowledge of the underlying mutation. It also has advantages over immunoassays using antibodies against specific abnormal conformations or post-translational modifications (e.g., phospho-sites). Although, this technique has dramatically simplified the detection of pathological protein conformations by directly performing detection on tissue preparations, their market availability is restricted to a narrow range of protein types, and there are additional concerns about their selectivity and reliability.52-54 Hence our approach, not only could be applied to a larger number of proteins, but it also requires less time. 3.3. Role of the tannic acid capping agent in driving the solvation behaviour of AuNPs against protein conformational changes and in increasing assay selectivity. Using a similar salt-induced strategy, it was previously reported31 that citrate-capped AuNPs incubated with misfolded or denatured antibodies resulted in colloidal aggregation, whereas native (perfectly folded) antibodies were able to positively stabilize the system. Those data indicated that misfolded and denatured antibodies, unlike their native ones, were unable to sufficiently interact and adsorb


17


Page 18 of 38

onto AuNPs. Hence this protein-AuNPs system was very unstable and collapsed quickly upon salt addition. In our approach, AuNPs aggregation with the denatured proteins would occur via an alternative mechanism triggered by our choice of tannic acid as the capping agent for AuNPs instead of widely used citrate ions. Tannic acid has a unique ability to interact with and bind to proteins strongly ― an interaction that involves hydrophobic and hydrogen bonds without contribution from covalent or ionic bonds.24-26, 55 Because of this interaction, this agent plays a crucial role in our system, by initially attracting proteins to the AuNPs surface for subsequent adsorption onto AuNPs surface. Since the tannic acid-protein interaction is mostly hydrophobic and the denatured proteins are more likely to expose their previously hidden hydrophobic residues, we hypothesized that the tannic acid capping agent would interact with and bind to denatured proteins more favourably than their native ones. Once the protein-AuNPs complex is formed, the complex containing denatured proteins would have greater tendency to interact with other denatured proteins-AuNPs complexes via protein-protein hydrophobic interactions, which would lead to a crosslinking-type aggregation of the colloidal system.23 This mechanism would also explain why tannic acid-capped AuNPs are ideal for detecting not only major folding changes, such as denaturation, but also other subtle conformational changes, such as variant conformations in native proteins. Many of the changes seen in native conformation variants, e.g., changes in the position of loops, frequently involve the exposure of previously hidden hydrophobic amino acids or the exposure of amino acids with a relatively high affinity towards gold. For example, phosphorylation-induced EGFR activation involves conformational changes around three hydrophobic residues located in the juxtamembrane segment.56 In our recent reports, we also proved that phosphorylated proteins have a higher affinity towards flat gold


18

Page 19 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


surfaces than their non-phosphorylated counterparts due to the phosphorylated conformation exposing more favourably residues with large gold-affinity.57-58

Figure 4. (A) UV-Vis spectra for up to 30 min incubation of native or denatured EGFR with (left) tannic-capped AuNPs and (right) citrate-capped AuNPs. (B) UV-Vis spectra for up to 30 min

incubation of non-phosphorylated or phosphorylated EGFR with (left) tannic-capped AuNPs and (right panel) citrate-capped AuNPs. Orange colour is for both types of AuNPs in A and B. The redshifts observed under certain conditions are noted as Dl. The protein denaturation condition was 95°C for 5 min. Other conditions were 5 µL of 6 ng/µL (i.e., 30 ng) protein, 10 µL of 10 nm AuNPs, and total solution pH 7. Each dotted rectangular box containes zoomed part for the red shfit of the absorbance peak(s) (i.e., wavelength difference, Δλ in the X-axis) of the main figure.


19


Page 20 of 38

To experimentally prove the unique role of tannic acid in enabling this mechanism, we first performed incubation of EGFR native and denatured forms with either tannic acid-capped or citrate-capped AuNPs of 10 nm size. We allowed them to interact for up to 30 min without adding any salt (conditions: 5 µL of 30 ng protein, 10 µL of AuNPs, and total solution pH 7). In case of tannic acid-capped AuNPs, protein addition resulted in almost no visible colour change of their original reddish colour irrespective of proteinʼs folding status. We only noted a small but measurable change in UV-Vis peak at 520 nm. The denatured EGFR protein caused a ~3-fold decrease in the peak intensity compared to its native form (Figure 4A (left)). This decrease in the peak intensity was accompanied by a broadening in peak width, along with a slight red-shift, which was also larger for denatured EGFR protein in contrast with its native one (5 nm vs 3 nm). This outcome suggests that the added proteins, particularly the denatured ones, were somehow destabilising the colloidal system and thus leading to a minimal aggregation. In contrast, when we performed the same experiment but using 10 nm citrate-capped AuNPs (in-house synthesized) instead of tannic acid-capped ones, we observed that EGFR native protein stabilised the colloidal solution, as evidenced by an increase in the peak intensity (Figure 4A (right)), as opposed to the decrease seen for tannic acid-capped AuNPs. This finding goes in line with the expected mechanism for citrate-capped AuNPs, as previously reported for correctly folded antibodies. We further noted that addition of the denatured EGFR protein caused a minimal decrease in the peak intensity, but far lower in magnitude than for the tannic acid-capped AuNPs system. This observation is also in agreement with the previous report of misfolded antibodies having less ability to interact effectively with and bind to citrate-capped AuNPs.31 Altogether, this data clearly proves that native and denatured proteins interact very differently with citrate-capped AuNPs than tannic acid-capped AuNPs.


20

Page 21 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


To further prove the role of the tannic acid capping agent also in the detection of native variant conformations, we performed the same experiment as above for non-phosphorylated (i.e., native) and phosphorylated EGFR isoforms with both types of nanoparticles (Figure 4 B (left and right)). For tannic acid-capped AuNPs, we observed the same spectral features as per our previous experiments with native and denatured proteins, i.e., almost no visible colour change of their original reddish colour irrespective of proteinʼs conformation, however a small but measurable change in UV-Vis peak at 520 nm. The phosphorylated EGFR protein caused a ~2-fold decrease in the peak intensity compared to its non-phosphorylated form (Figure 4B (left)). This decrease in the peak intensity was accompanied by a slight red-shift, which was also slightly larger for phosphorylated EGFR protein in contrast with its native one (3.5 nm vs 3 nm). This data indicates that phosphorylated protein, compared to its non-phosphorylated isoform, is more likely to interact positively with the tannic acid capping agent and promote AuNPs aggregation, as we previously hypothesised. The above features were not observed in the citrate-capped AuNPs system. Here, the differences in the spectra between both isoforms were minimal. As previously, the non-phosphorylated (i.e., native) EGFR protein positively stabilized the system, as evidenced by a slight increase in the peak intensity at 520 nm. On the other hand, the phosphorylated EGFR protein caused a slight decrease in the peak intensity at 520 nm. However, this decrease was ~3.6 times lower than per the tannic acid-capped colloidal system, indicating that the phosphorylated variant conformation has a significantly lower impact in the AuNPs solvation than the tannic acid-capped system, and hence, that the citrate system has lower sensitivity to distinguish between these isoforms. The distinct solvation behaviour of these two colloidal systems with native variant isoforms confirms our


21


Page 22 of 38

previous hypothesis that each colloidal system interacts differently with proteins, and follow a different mechanism. To further confirm the role of tannic acid in enabling greater sensitivity to visualise native variant conformations, we performed our previous salt-induced aggregation assay under the optimised conditions using 10 nm citrate-capped AuNPs instead of 10 nm tannic acid-capped AuNPs (Figure S7, Supporting Information). Although it was still possible to see protein conformational changes with this system, we observed significantly lower sensitivity and specificity for citrate-capped than tannic acid-capped AuNPs. Hence, although variant conformations could potentially be realised in other AuNPs systems via a different mechanism, the tannic acid cap of our AuNPs appears to have a vital role in modulating how AuNPs behave against various protein conformations. It is important to note that certain conditions can favour aggregation of protein-AuNPs system irrespective of protein folding status.23 For example, large nanoparticles have low surface curvature enabling proteins to interact with the surface via multiple points — a process that may result in a slight denaturation during interactions that leads to a colloidal aggregation.19, 43-45 This process typically occurs unless the AuNPs are previously protected with particular surface coatings such as polyethylene glycol (PEG).23 In our case, we have minimised the chance of this slight denaturation process by using small-size nanoparticles of only 10 nm (as shown experimentally in Figure 2C). Hence, the small colloidal destabilization seen in the presence of native proteins could be attributed to the presence of tannic acid on the AuNPs surface. Indeed, the extraordinary ability of the tannic acid to attract proteins towards the AuNPs surface has already been associated with the formation of an enhanced corona and a broader tendency of the colloidal system towards aggregation.59


22

Page 23 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Altogether, our data suggest that (i) the addition of salt seems highly effective in accelerating the aggregation process of colloidal systems containing denatured proteins or native conformation variants, but not in the systems containing native proteins, and that (ii) the tannic acid-mediated colloidal-based interfacial biosensing approach, can distinguish protein misfolding and subtle conformational changes (such as those involved in native variant conformations) with extreme simplicity, specificity and accuracy regardless of protein size, topology or presence of cysteinerich domains. 3.4. Exploring

protein-AuNPs

interactions

and

phosphorylation-induced

protein

conformational changes with Surface Enhanced Raman Spectroscopy (SERS) and Circular Dichroism (CD) respectively We used SERS to gain further insight into the protein-AuNPs interaction mechanism and elucidate how the different conformations, associated to the phosphorylated and non-phosphorylated EGFR proteins, interact with our tannic acid-capped AuNPs (conditions: 20 µL of 6 ng/µL of each isoform was incubated with 40 µL of 10 nm AuNPs for 30 min). This technique allows determining the key amino acid residues involved in protein adsorption for each isoform by observing the changes in their enhanced Raman signals. The Raman spectra in Figure 5A indicates that the overall SERS peak for phosphorylated EGFR is more intense than that for its non-phosphorylated isoform, suggesting higher adsorption of phosphorylated EGFR protein. This finding goes in line with our previous report of phosphorylated EGFR and ERK proteins having larger affinities to interact with flat gold surface.58 It also suggests that the tannic acid from our AuNPs is more interactive with phosphorylated proteins. This preferred interaction could be due to hydrophobic interactions with previously hidden hydrophobic residues, such as those involving the phosphorylation of the residues from the juxtamembrane segment, which are essential for EGFR


23


Page 24 of 38

Figure 5. (A) SERS spectra of protein-AuNPs assembly in solution. (Condition: 20 µL of 6 ng/µL

of each protein (EGFR and P-EGFR) was adsorbed onto 40 µL of 10 nm AuNPs for 30 min). (B) Circular Dichroism (CD) study of secondary and tertiary conformations of phosphorylated and non-phosphorylated EGFR proteins in solution. Secondary structure evaluation required 300 µL of pure non-phosphorylated and phosphorylated EGFR (500 ng/uL) in 10 mM buffer (a mixture of Glycine.HCl and Tris.HCl). Tertiary structure evaluation required 500 µL of pure nonphosphorylated and phosphorylated EGFR (1000 ng/µL) in the same buffer.


24

Page 25 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


activation.56-58 Indeed, the peaks associated with tryptophan — a hydrophobic residue, were greatly increased in the phosphorylated protein-AuNPs system spectra. We also note that some peaks (particularly for thiols and amines, which are known to have a higher gold affinity) are specific to the spectra involving the phosphorylated isoform, indicating that phosphorylationinduced conformational changes might allow these specific groups to become more exposed towards the AuNPs gold surface, compared to the non-phosphorylated EGFR protein. There are many other features, which are unique in the spectra of the phosphorylated protein-AuNPs system, such as the increase in peak intensity at around 930 cm-1, which is commonly assigned to the C-C stretch of α-helical structure. This data is an indication of specific conformational arrangements occurring during the interaction and adsorption onto the AuNPs. This finding suggests that the phosphorylated protein is more prone to experience folding changes upon adsorption onto the gold surface than the non- phosphorylated version, and further explains its larger tendency towards interacting with other proteins-AuNPS wherein the protein has undergone a similar unfolding process to cause a cross-linking aggregation of the colloidal system. To confirm that the majority of these changes are associated to the distinct conformation of each EGFR isoform, and thus, are not the result of a denaturing process induced by the AuNPs, we explored the key conformational features of each protein isoform before interacting with the AuNPs using Circular Dichroism (CD). This experiment includes aqueous solutions containing either phosphorylated or non-phosphorylated EGFR isoform in the absence of the tannic-capped AuNPs. Data clearly suggests that each isoform is different in terms of their secondary and tertiary conformations. Both conformations show a double minimum at 208 and 222 nm related to the αhelical structure, and a single minimum at 217 nm indicative of β-sheet structure.60-61 However, as shown in Figure 5B (left), the phosphorylated EGFR has relatively larger content of α-helix


25


Page 26 of 38

(55.15% vs 53.72%) and ß-sheet (12.35% vs 7.05%) than the non-phosphorylated isoform. As for their tertiary structure, Tyrosine (Tyr) and Tryptophan (Trp) of phosphorylated EGFR display stronger peaks at 275-285 nm and 290-305 nm respectively in comparison with its nonphosphorylated isoform (Figure 5B (right)). We also observed an increase in bands between 255 and 270 nm associated with phenylalanine (Phe) for the phosphorylated EGFR protein. This increase could be due to an additional contribution of cysteine’s disulphide bond (S-S) at around 260 nm. A further increase in the intensity associated with disulfide bonds is observed from 320 nm to 340 nm. This larger contribution of disulfide bonds may suggest that conformational changes associated to phosphorylation have a particular impact in the disulphide bonds mediated by cysteine sites and may be a reason behind the observed larger adsorption for this conformation (e.g., thiol groups on the phosphorylated EGFR protein could be more exposed to AuNPs compared to its non-phosphorylated isoform). 3.5. Time-point monitoring of drug-responses to the cancer cells using our colloidal-based interfacial biosensing system We finally explored the clinical application of our approach for the control and managing of cancer therapy. Monitoring the effectivity of anticancer drugs is vital for lung cancer patients survival, as they often develop drug resistance within a few months of starting their therapy and this needs timely adjustment of administered drugs.4, 62-63 Hence, we wanted to test if our strategy would also enable monitoring the responses of lung cancer cells to Tyrosine Kinase Inhibitors (TKI) such as Gefitinib. This drug restores the former normal conformation and function of aberrantly phosphorylated proteins by blocking ATP binding.64 We performed our assay using drug sensitive (HCC827) and insensitive (H1975) cell lines grown for 24 hours in a culture media containing various doses of Gefitinib. Both cell lines display a mutated EGFR, but only the EGFR from


26

Page 27 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6: Colorimetric detection of drug sensitivity in lung cancer cells expressing drug-sensitive and -insensitive (A) EGFR and (B) ERK proteins. Each data point represents the average of three separate trials, and error bars represent the standard deviation of measurements. (C) Validation of the colorimetric data, excluding for H1666 cell line, using western blot. ACS Paragon Plus Environment

27


Page 28 of 38

HCC827 (EGFRHCC827) is sensitive to Gefitinib. Increasing drug doses from 0.4 µM to 10 µM resulted in larger aggregation trend of the extracted EGFRHCC827 but not EGFRH1975 (Figure 6A), suggesting that only the EGFRH1975 remained phosphorylated and insensitive to this drug. For the largest drug dose tested, EGFRHCC827 reached similar adsorption levels as the non-phosphorylated EGFR derived from H1666 (EGFRH1666), indicating that the optimum concentration for reversing its former auto-phosphorylation was reached. Similar trends were observed for ERK, which showed analogous responses to Gefitinib as EGFR (Figure 6B). These UV-Vis absorbance data were validated by western blots (Figure 6C). Moreover, drug sensitivity behaviour could also be monitored in heterogeneous samples containing EGFR (or ERK) from drug-treated HCC827 cells in excess of wild-type EGFR (or ERK) from H1666 cells (Figure S8, Supporting Information). These data prove that our EGFR and ERK adsorption profiles are unambiguously linked to their conformation-phosphorylation status and sensitivities to TKIs. It further showcases the large clinical translation potential of our approach, with prospective application not only to cancer diagnosis but to therapeutics as well.

4.

CONCLUSIONS

In conclusion, our data clearly demonstrate that our approach has excellent sensitivity towards protein conformations and could potentially be applied in the clinics for detection of subtle protein conformational changes such as protein phosphorylation-induced conformations and for therapy monitoring. For this system, detection provided five significant improvements over existing technologies such as (i) avoiding the general paradigm of requiring phospho-/conformationspecific antibody, (ii) enabling rapid, naked-eye, label-free and cost-effective read-out, iii) requiring no AuNPs surface modifications, iv) no mutational analysis to identify mutation-induced


28

Page 29 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


variants, and v) allowing drug response monitoring. Compared to other conventional methods, such as CD, our protein-AuNPs approach is markedly more rapid and sensitive. It only requires 5 µL of protein at 6 ng/µL, compared to 300 µL of protein at 500 ng/µL (for secondary structure evaluation) and 500 µL of protein at 1000 ng/µL (for tertiary structure evaluation) that were needed for our CD experiments. ASSOCIATED CONTENT Supporting Information Dynamic Light Scattering (DLS) analysis of AuNPs including size distribution, polydispersity (PdI) and zeta potential measurements; silver staining and western blot for characterizing proteins of interest; control experiments for showing the non-specificity during protein purification; and performance of lab-synthesized citrate-capped AuNPs in sensing protein conformations. AUTHOR INFORMATION Corresponding Author *Email: [email protected] (M. T.) *Email: [email protected] (L.G. C.) Notes The authors declare no competing financial interest. ACKNOWLEDGEMENT We thank Kevin Koo and Abu Ibn Ali Sina for insightful suggestions to improve the quality of this manuscript. We also acknowledge funding received by our laboratory from ARCDP


29


Page 30 of 38

(DP140104006) and National Breast Cancer Foundation of Australia (CG-08-07 and CG-12-07) grants awarded to MT. Although not directly funding the research work in this paper, these grants have significantly contributed to the environment to stimulate the research described here.

REFERENCES (1) Grant, B. J.; Gorfe, A. A.; McCammon, J. A. Large Conformational Changes in Proteins: Signaling and Other Functions. Curr. Opin. Struct. Biol. 2010, 20 (2), 142-147. (2) Groban, E. S.; Narayanan, A.; Jacobson, M. P. Conformational Changes in Protein Loops and Helices Induced by Post-Translational Phosphorylation. PLoS computational biology 2006, 2 (4), e32. (3) Huse, M.; Kuriyan, J. The Conformational Plasticity of Protein Kinases. Cell 2002, 109 (3), 275-282. (4) Sharma, S. V.; Bell, D. W.; Settleman, J.; Haber, D. A. Epidermal Growth Factor Receptor Mutations in Lung Cancer. Nature Reviews Cancer 2007, 7 (3), 169. (5) Thomas, P. J.; Qu, B.-H.; Pedersen, P. L. Defective Protein Folding as a Basis of Human Disease. Trends Biochem. Sci. 1995, 20 (11), 456-459. (6) Yeow, K.-M.; Su, I.-H.; Pan, K.-T.; Tsay, P.-K.; Lui, K.-W.; Cheung, Y.-C.; Chou, A. S.-B. Risk Factors of Pneumothorax and Bleeding: Multivariate Analysis of 660 CT-Guided Coaxial Cutting Needle Lung Biopsies. Chest 2004, 126 (3), 748-754. (7) Cohen, P. Protein Kinases—the Major Drug Targets of the Twenty-First Century? Nature reviews Drug discovery 2002, 1 (4), 309.


30

Page 31 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(8) Lacerda, S. H. D. P.; Park, J. J.; Meuse, C.; Pristinski, D.; Becker, M. L.; Karim, A.; Douglas, J. F. Interaction of Gold Nanoparticles with Common Human Blood Proteins. ACS nano 2009, 4 (1), 365-379. (9) Walkey, C. D.; Olsen, J. B.; Guo, H.; Emili, A.; Chan, W. C. Nanoparticle Size and Surface Chemistry Determine Serum Protein Adsorption and Macrophage Uptake. J. Am. Chem. Soc. 2012, 134 (4), 2139-2147. (10) Zheng, T.; Cherubin, P.; Cilenti, L.; Teter, K.; Huo, Q. A Simple and Fast Method to Study the Hydrodynamic Size Difference of Protein Disulfide Isomerase in Oxidized and Reduced Form Using Gold Nanoparticles and Dynamic Light Scattering. Analyst 2016, 141 (3), 934-938. (11) Zheng, T.; Pierre-Pierre, N.; Yan, X.; Huo, Q.; Almodovar, A. J.; Valerio, F.; Rivera-Ramirez, I.; Griffith, E.; Decker, D. D.; Chen, S. Gold Nanoparticle-Enabled Blood Test for Early Stage Cancer Detection and Risk Assessment. ACS applied materials & interfaces 2015, 7 (12), 68196827. (12) Zhang, S.; Moustafa, Y.; Huo, Q. Different Interaction Modes of Biomolecules with CitrateCapped Gold Nanoparticles. ACS applied materials & interfaces 2014, 6 (23), 21184-21192. (13) Liu, H.; Pierre-Pierre, N.; Huo, Q. Dynamic Light Scattering for Gold Nanorod Size Characterization and Study of Nanorod–Protein Interactions. Gold bulletin 2012, 45 (4), 187-195. (14) Huo, Q.; Litherland, S. A.; Sullivan, S.; Hallquist, H.; Decker, D. A.; Rivera-Ramirez, I. Developing a Nanoparticle Test for Prostate Cancer Scoring. Journal of translational medicine 2012, 10 (1), 44. (15) Neely, A.; Perry, C.; Varisli, B.; Singh, A. K.; Arbneshi, T.; Senapati, D.; Kalluri, J. R.; Ray, P. C. Ultrasensitive and Highly Selective Detection of Alzheimer’s Disease Biomarker Using


31


Page 32 of 38

Two-Photon Rayleigh Scattering Properties of Gold Nanoparticle. ACS nano 2009, 3 (9), 28342840. (16) Kah, J. C. Y.; Kho, K. W.; Lee, C. G. L.; Richard, C. J. Early Diagnosis of Oral Cancer Based on the Surface Plasmon Resonance of Gold Nanoparticles. International journal of nanomedicine 2007, 2 (4), 785. (17) Medley, C. D.; Smith, J. E.; Tang, Z.; Wu, Y.; Bamrungsap, S.; Tan, W. Gold NanoparticleBased Colorimetric Assay for the Direct Detection of Cancerous Cells. Anal. Chem. 2008, 80 (4), 1067-1072. (18) Liu, X.; Dai, Q.; Austin, L.; Coutts, J.; Knowles, G.; Zou, J.; Chen, H.; Huo, Q. A One-Step Homogeneous Immunoassay for Cancer Biomarker Detection Using Gold Nanoparticle Probes Coupled with Dynamic Light Scattering. J. Am. Chem. Soc. 2008, 130 (9), 2780-2782. (19) Lynch, I.; Dawson, K. A. Protein-nanoparticle interactions. Nano today 2008, 3 (1-2), 40-47. (20) Lundqvist, M.; Stigler, J.; Elia, G.; Lynch, I.; Cedervall, T.; Dawson, K. A. Nanoparticle Size and Surface Properties Determine the Protein Corona with Possible Implications for Biological Impacts. Proceedings of the National Academy of Sciences 2008. (21) Nel, A. E.; Mädler, L.; Velegol, D.; Xia, T.; Hoek, E. M.; Somasundaran, P.; Klaessig, F.; Castranova, V.; Thompson, M. Understanding Biophysicochemical Interactions at the Nano–Bio Interface. Nature materials 2009, 8 (7), 543. (22) Niemeyer, C. M. Nanoparticles, Proteins, and Nucleic Acids: Biotechnology Meets Materials Science. Angewandte Chemie International Edition 2001, 40 (22), 4128-4158. (23) Zhang, D.; Neumann, O.; Wang, H.; Yuwono, V. M.; Barhoumi, A.; Perham, M.; Hartgerink, J. D.; Wittung-Stafshede, P.; Halas, N. J. Gold Nanoparticles Can Induce the Formation of ProteinBased Aggregates at Physiological pH. Nano letters 2009, 9 (2), 666-671.


32

Page 33 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(24) Hagerman, A. E. Chemistry of Tannin-Protein Complexation. In Chemistry and Significance of Condensed Tannins; Springer: 1989; pp 323-333. (25) Siebert, K. J.; Troukhanova, N. V.; Lynn, P. Y. Nature of Polyphenol− Protein Interactions. J. Agric. Food Chem. 1996, 44 (1), 80-85. (26) Hou, W.-C.; Moghadam, B. Y.; Corredor, C.; Westerhoff, P.; Posner, J. D. Distribution of Functionalized Gold Nanoparticles Between Water and Lipid Bilayers as Model Cell Membranes. Environmental science & technology 2012, 46 (3), 1869-1876. (27) Zhou, H.; Aoki, S.; Honma, I.; Hirasawa, M.; Nagamune, T.; Komiyama, H. Conformational Change of Protein Cytochrome B-562 Adsorbed Oncolloidal Gold Particles; Absorption Band Shift. Chemical communications 1997, (6), 605-606. (28) Jiang, X.; Jiang, J.; Jin, Y.; Wang, E.; Dong, S. Effect of Colloidal Gold Size on the Conformational Changes of Adsorbed Cytochrome C: Probing by Circular Dichroism, UV− Visible, and Infrared Spectroscopy. Biomacromolecules 2005, 6 (1), 46-53. (29) Shang, L.; Wang, Y.; Jiang, J.; Dong, S. pH-Dependent Protein Conformational Changes in Albumin: Gold Nanoparticle Bioconjugates: A Spectroscopic Study. Langmuir : the ACS journal of surfaces and colloids 2007, 23 (5), 2714-2721. (30) Chah, S.; Hammond, M. R.; Zare, R. N. Gold Nanoparticles as a Colorimetric Sensor for Protein Conformational Changes. Chem. Biol. 2005, 12 (3), 323-328. (31) Woo, J. R.; Lim, D. K.; Nam, J. M. Minimally Stable Nanoparticle‐Based Colorimetric Assay for Simple, Rapid, and Sensitive Antibody Structure and Activity Evaluation. small 2011, 7 (5), 648-655.


33


Page 34 of 38

(32) Brewer, S. H.; Glomm, W. R.; Johnson, M. C.; Knag, M. K.; Franzen, S. Probing BSA Binding to Citrate-Coated Gold Nanoparticles and Surfaces. Langmuir : the ACS journal of surfaces and colloids 2005, 21 (20), 9303-9307. (33) Rombouts, I.; Lagrain, B.; Scherf, K. A.; Lambrecht, M. A.; Koehler, P.; Delcour, J. A. Formation and Reshuffling of Disulfide Bonds in Bovine Serum Albumin Demonstrated Using Tandem Mass Spectrometry with Collision-Induced and Electron-Transfer Dissociation. Scientific reports 2015, 5, 12210. (34) Carter, D. C.; He, X.-M.; Munson, S. H.; Twigg, P. D.; Gernert, K. M.; Broom, M. B.; Miller, T. Y. Three-Dimensional Structure of Human Serum Albumin. Science 1989, 244 (4909), 11951198. (35) Galli, S.; Jahn, O.; Hitt, R.; Hesse, D.; Opitz, L.; Plessmann, U.; Urlaub, H.; Poderoso, J. J.; Jares-Erijman, E. A.; Jovin, T. M. A New Paradigm for MAPK: Structural Interactions of hERK1 with Mitochondria in HeLa Cells. PloS one 2009, 4 (10), e7541. (36) Abe, Y.; Odaka, M.; Inagaki, F.; Lax, I.; Schlessinger, J.; Kohda, D. Disulfide Bond Structure of Human Epidermal Growth Factor Receptor. J. Biol. Chem. 1998, 273 (18), 11150-11157. (37) Burke, C. L.; Stern, D. F. Activation of Neu (ErbB-2) Mediated by Disulfide Bond-Induced Dimerization Reveals a Receptor Tyrosine Kinase Dimer Interface. Mol. Cell. Biol. 1998, 18 (9), 5371-5379. (38) Raina, D.; Ahmad, R.; Rajabi, H.; Panchamoorthy, G.; Kharbanda, S.; Kufe, D. Targeting Cysteine-Mediated Dimerization of the MUC1-C Oncoprotein in Human Cancer Cells. Int. J. Oncol. 2012, 40 (5), 1643-1649. (39) Gajiwala, K. S. EGFR: Tale of the C‐Terminal Tail. Protein Sci. 2013, 22 (7), 995-999.


34

Page 35 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(40) Fransén, K.; Klintenäs, M.; Österström, A.; Dimberg, J.; Monstein, H.-J.; Söderkvist, P. Mutation Analysis of the BRAF, ARAF and RAF-1 Genes in Human Colorectal Adenocarcinomas. Carcinogenesis 2004, 25 (4), 527-533. (41) Ko, B.; Paucar, D.; Halmos, B. EGFR T790M: Revealing the Secrets of a Gatekeeper. Lung Cancer: Targets and Therapy 2017, 8, 147. (42) Pham, T. V.; Beekhof, R.; van Alphen, C.; Knol, J. C.; Henneman, A. A.; Rolfs, F.; Labots, M.; Henneberry, E.; Le Large, T. Y.; de Haas, R. R. INKA, An Integrative Data Analysis Pipeline for Phosphoproteomic Inference of Active Phosphokinases. bioRxiv 2018, 259192. (43) Shang, W.; Nuffer, J. H.; Dordick, J. S.; Siegel, R. W. Unfolding of Ribonuclease a on Silica Nanoparticle Surfaces. Nano letters 2007, 7 (7), 1991-1995. (44) Fei, L.; Perrett, S. Effect of Nanoparticles on Protein Folding and Fibrillogenesis. International journal of molecular sciences 2009, 10 (2), 646-655. (45) Rahman, M.; Laurent, S.; Tawil, N.; Yahia, L.; Mahmoudi, M. Protein-Nanoparticle Interactions; Springer: 2013. (46) Burrell, R. A.; McGranahan, N.; Bartek, J.; Swanton, C. The Causes and Consequences of Genetic Heterogeneity in Cancer Evolution. Nature 2013, 501 (7467), 338. (47) Bernadó, P.; Mylonas, E.; Petoukhov, M. V.; Blackledge, M.; Svergun, D. I. Structural Characterization of Flexible Proteins Using Small-Angle X-Ray Scattering. J. Am. Chem. Soc. 2007, 129 (17), 5656-5664. (48) Royer, C. A. Probing Protein Folding and Conformational Transitions with Fluorescence. Chem. Rev. 2006, 106 (5), 1769-1784. (49) Greenfield, N. J. Methods to Estimate the Conformation of Proteins and Polypeptides from Circular Dichroism Data. Anal. Biochem. 1996, 235 (1), 1-10.


35


Page 36 of 38

(50) Kong, J.; Yu, S. Fourier Transform Infrared Spectroscopic Analysis of Protein Secondary Structures. Acta biochimica et biophysica Sinica 2007, 39 (8), 549-559. (51) Clarke, D.; Martinez, A.; Cox, R. S. Analysis of Cosmetic Results and Complications in Patients with Stage I and II Breast Cancer Treated by Biopsy and Irradiation. International Journal of Radiation Oncology* Biology* Physics 1983, 9 (12), 1807-1813. (52) Bradbury, A. R.; Sidhu, S.; Dübel, S.; McCafferty, J. Beyond Natural Antibodies: the Power of In Vitro Display Technologies. Nat. Biotechnol. 2011, 29 (3), 245. (53) Waters, A. M.; Ozkan-Dagliyan, I.; Vaseva, A. V.; Fer, N.; Strathern, L. A.; Hobbs, G. A.; Tessier-Cloutier, B.; Gillette, W. K.; Bagni, R.; Whiteley, G. R. Evaluation of the Selectivity and Sensitivity of Isoform-and Mutation-Specific RAS Antibodies. Sci. Signal. 2017, 10 (498), eaao3332. (54) Riley, D.; Arndt, U. W. X-Ray Scattering by Some Native and Denatured Proteins in the Solid State. Proc. R. Soc. Lond. B 1953, 141 (902), 93-97. (55) Van Buren, J. P.; Robinson, W. B. Formation of Complexes Between Protein and Tannic Acid. J. Agric. Food Chem. 1969, 17 (4), 772-777. (56) Jura, N.; Endres, N. F.; Engel, K.; Deindl, S.; Das, R.; Lamers, M. H.; Wemmer, D. E.; Zhang, X.; Kuriyan, J. Mechanism for Activation of the EGF Receptor Catalytic Domain by the Juxtamembrane Segment. Cell 2009, 137 (7), 1293-1307. (57) Ahmed, M.; Carrascosa, L. G.; Sina, A. A. I.; Zarate, E. M.; Korbie, D.; Ru, K.-l.; Shiddiky, M. J.; Mainwaring, P.; Trau, M. Detection of Aberrant Protein Phosphorylation in Cancer Using Direct Gold-Protein Affinity Interactions. Biosens. Bioelectron. 2017, 91, 8-14.


36

Page 37 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(58) Ahmed, M.; Carrascosa, L. G.; Wuethrich, A.; Mainwaring, P.; Trau, M. An Exosomal-and Interfacial-Biosensing Based Strategy for Remote Monitoring of Aberrantly Phosphorylated Proteins in Lung Cancer Cells. Biomaterials science 2018, 6 (9), 2336-2341. (59) Untener, E. A.; Comfort, K. K.; Maurer, E. I.; Grabinski, C. M.; Comfort, D. A.; Hussain, S. M. Tannic Acid Coated Gold Nanorods Demonstrate a Distinctive Form of Endosomal Uptake and Unique Distribution Within Cells. ACS applied materials & interfaces 2013, 5 (17), 83668373. (60) Pain, R. Determining the CD Spectrum of a Protein. Current protocols in protein science 2004, 38 (1), 7.6. 1-7.6. 24. (61) Kelly, S. M.; Jess, T. J.; Price, N. C. How to Study Proteins by Circular Dichroism. Biochimica Et Biophysica Acta (BBA)-Proteins And Proteomics 2005, 1751 (2), 119-139. (62) Chen, I.-H.; Xue, L.; Hsu, C.-C.; Paez, J. S. P.; Pan, L.; Andaluz, H.; Wendt, M. K.; Iliuk, A. B.; Zhu, J.-K.; Tao, W. A. Phosphoproteins in Extracellular Vesicles as Candidate Markers for Breast Cancer. Proceedings of the National Academy of Sciences 2017, 201618088. (63) Ware, K.; Hinz, T.; Kleczko, E.; Singleton, K.; Marek, L.; Helfrich, B.; Cummings, C.; Graham, D.; Astling, D.; Tan, A. A Mechanism of Resistance to Gefitinib Mediated by Cellular Reprogramming and the Acquisition of an FGF2-FGFR1 Autocrine Growth Loop. Oncogenesis 2014, 2 (3), e39. (64) Yun, C.-H.; Mengwasser, K. E.; Toms, A. V.; Woo, M. S.; Greulich, H.; Wong, K.-K.; Meyerson, M.; Eck, M. J. The T790M Mutation in EGFR Kinase Causes Drug Resistance by Increasing the Affinity for ATP. Proceedings of the National Academy of Sciences 2008, 105 (6), 2070-2075.


37


Page 38 of 38

TOC (3.6 cm height x 8.4 cm width)


38

Reading Conformational Changes in Proteins with a New Colloidal

Recommend Documents