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«Kinetic Investigations of Thiolate Protected Gold Nanoparticles: Protein Interactions, Electron Transfer, and Precursor Formation By Brian N. Turner ...»

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Hydrogen peroxide (30% v/v) was purchased from Acros. Peptide synthesis materials (fmoc protected and side-chain protected amino acids, coupling reagents, and resins) were provided generously by the research group of David Wright. 18 MΩ Water was obtained from a U.S. Filter Modulab water system with a 0.2 µm external filter, or from a Barnstead NANOpure Diamond water purification system. Dry nitrogen is provided in house. Monoclonal antibody 1214, 1129, Palivizumab, and human respiratory syncytial virus were provided generously by the Dr. James Crowe, Jr. research group. Work areas that contained HRSV were cleaned thoroughly with bleach and 70% isopropyl alcohol solution.

Peptide Synthesis Most peptides were synthesized by standard solid phase f-moc procedures using an Apex automated peptide synthesizer. The series of peptides used in the QCM experiments were synthesized, purified, and characterized by Malgorzata Broncel. Subsequent peptides that were utilized in the ELISA experiments were synthesized by Joshua Swartz.

The peptides were cleaved from the resin and side-chain-deprotected using Reagent R (90% trifluoroacetic acid, 5% thioanisole, 3% ethanedithiol, 2% anisole). Following extraction in cold diethyl ether with cold centrifugation (0° C), cleaved peptides were further purified using a Waters semi-prep HPLC with a reverse phase column in a continuous gradient of water:acetonitrile following injection in water/acetonitrile/dilute ammonium hydroxide. Samples were collected with a Waters fraction collector, operated manually. (Note: ammonium hydroxide must be used sparingly, 100 mM, to avoid damaging the column or corroding the pumping lines). Following lyophilization, peptides were diluted in 20% acetonitrile and water, occasionally with the addition of ammonium hydroxide to increase solubility, and characterized by MALDI MS on a Voyager MALDI mass spectrometer with α-cyano-4-hydroxycinnamic acid.

Characterization data for peptides used in this study that were not fully characterized by other coworkers are displayed in Table 2.

Table 2: Tabulated characterization data for peptides purified by this author. The data has been relocated to Appendix D.

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Formation of peptide SAMs Clean gold electrode surface was achieved by mixing 3 drops concentrated sulfuric acid to 1 drop 30% hydrogen peroxide (piranha) on the gold electrode three times, each time completely covering the surface and rinsing away with deionized water. The crystal was then rinsed with copious amounts of absolute ethanol and blown dry with cool nitrogen.

Self-assembled monolayers (SAMs) of RSV F A site peptides terminated with cystiene were created on the surface of QCM crystal electrodes by soaking the crystal face in a mixture of 3 parts tiopronin to 1 part peptide (1 mM combined) solution in absolute ethanol with a minimum of deionized water (to aid solubility). The peptide/tiopronin solution was immediately transferred to the QCM crystal in a glass cell clamped on top of the oversized gold electrode. The total time of soaking varied from 3 to 24 hours. The solution was then discarded and the crystal rinsed with copious amounts of absolute ethanol and blown dry with cool nitrogen.

QCM experiment The QCM crystal presenting the SAM on the gold electrode, or just the clean gold presenting crystal was placed in a flow cell interfaced with the Maxtek, Inc. RQCM.

Phosphate buffer solution (25 to 50 mM sodium phosphate with 0 to 150 mM sodium chloride pH adjusted to 7.1) was pumped across the crystal face in a laminar flow until a minimum in frequency and resistance drift is measured. A ten-minute baseline was then collected. Following collection of a baseline, a solution of bovine serum albumin (BSA) (1 mg/mL) was switched with the buffer solution and pumped 5 to 10 minutes beyond when a decrease in frequency was observed. The buffer flow time assures that there are no available gold sites which could participate in non-specific binding. Buffer was then pumped in order to purge the system of BSA. Then, solutions of antibody in buffer (10g/mL) were flowed for 10 minutes and the frequency response was observed. All solutions were pumped at a flow rate of approximately 40 L/min. The following peptide sequences in Table 3 were evaluated.

Table 3: Peptides designed to be evaluated by the QCM step-wise linear epitope mapping method. Odd numbered sequences were made to correlate QCM experiments with ELISA experiments in an Immulon well plate. Even numbered peptides are designed for the QCM experiment itself, having a cysteine residue for thiol linkage to the gold surface. Spaces allow for easy visualization of the stepwise differences between peptides. The amino acid numbering sequence for HRSV F protein is displayed in the top row. Known MARMs against PZ or related mAbs are indicated in bold organge.

Residues that bind to motavizumab are indicated in bold blue.

ID Peptide Sequence RSV F 255 260 265 270 275 | | | | |

A) NSELLSLINDMPITNDQKKLMSNN (not evaluated)

1) LSLINDMPITNDQKKLMS

2) CLSLINDMPITNDQKKLMS

3) SLINDMPITNDQKKLMSN

4) CSLINDMPITNDQKKLMSN

5) LINDMPITNDQKKLMSNN

6) CLINDMPITNDQKKLMSNN

7) INDMPITNDQKKLMSNNV

8) CINDMPITNDQKKLMSNNV 9) NDMPITNDQKKLMSNNVQ 10) CNDMPITNDQKKLMSNNVQ ELISA Stepwise linear epitope mapping of the full length RSV F Antigenic Site A Linear fragments and full length linear peptides of antigenic site A were evaluated for their ability to bind Palivizumab in a peptide ELISA assay using maleimide coated well plates. The design of the peptides has been improved by amination of the C terminus, and acylation of the N terminus to better represent the fact that the antigenic site A is an internal sequence. Serine-glycine-serine-glycine spacers were used in favor of PEG-6 for improved synthetic yield, solubility, and ease of purification. The following synthetic peptides were evaluated by the assay (Table 4 lists peptides used for the ELISA assay and





Table 3 lists peptides used in the QCM assay):

Table 4: Peptides evaluated against palivizumab in an ELISA assay. Spaces allow for easy visualization of the stepwise differences between peptides. The amino acid numbering sequence for HRSV F protein is displayed in the top row. “Linker” indicates the inert CSGSG sequence added for peptide immobilization and projection. Known MARMs against PZ or related mAbs are indicated in bold organge. Residues that bind to motavizumab are indicated in bold blue.

ID Peptide Sequence

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Peptide QCM step-wise linear epitope mapping Self-assembled monolayers of synthetic peptide epitopes 2, 4, 6, 8, and 10 showed small (barely greater than three times the noise) amounts of mass loading when mAb 1214 (20 g/mL) in PBS was flowed across the SAM. The small amount of mass loading was not observed when the electrode was blocked with BSA prior to antibody addition, suggesting that mAb 1214 binds to the gold electrode surface. Experiments demonstrating mAb 1214 binding to the clean gold surface of the QCM crystal confirmed this observation. There were significant amounts of mass loading observed when peptide epitopes were mixed with tiopronin prior to addition; however, this was later discovered to be non-specific binding to tiopronin. 11-mercapto-1-undecanol and 6-mercapto-1hexanol, as alternatives to tiopronin as a diluting agent, also exhibited significant amounts of binding to mAb 1214.

It was concluded that neither Synagis nor mAb 1214 binds self-assembled monolayers of synthetic peptide epitopes 2, 4, 6, 8, nor 10 on gold in a high ionic strength (200 mM salt) environment. Additionally, mAb 1214 and PZ fail to bind to synthetic peptide epitopes 1, 3, 5, 7, or 9 on Immulon. Possibly, mAb 1214 will not bind to the conformation adopted by the peptide on Immulon well plate, whereas it might bind to the same peptide in a different context.

Synagis was found to bind synthetic peptide epitope monolayers of 2, 8, and 10 on gold in a low ionic strength (50 mM salt) environment (4 and 6 were not evaluated due to a lack of remaining material) (Figures10-12).

Figure 10: QCM graph illustrating the binding of BSA to bare gold and PZ to a mixed SAM of peptide 2 and tiopronin.

Figure 11: QCM graph illustrating the binding of BSA to bare gold and PZ to a mixed SAM of peptide 8 and tiopronin.

Figure 12: QCM graph illustrating the binding of BSA to bare gold and PZ to a mixed SAM of peptide 10 and tiopronin.

All three of these epitopes demonstrated a reversible binding event with Synagis, which differs from the irreversible binding of synagis with the diluting agent, tiopronin. Of particular interest, Synagis bound in a highly reversible fashion to peptide 8 (Figure 11).

Reversible binding can be associated with biological activity. At the very least, it distinguishes itself from the other peptides. To be sure that the binding is specific, a new diluting agent that does not bind Synagis must be evaluated and mixed with the peptide epitopes.

As these experiments were performed a number of years ago and abandoned in order to pursue other research directions, it would be appropriate to reflect on the results and conclusions with a fresh perspective in order to aid a future student reading this dissertation for guidance. In retrospect, these studies could have been improved with a number of modifications. Additionally, peptides discussed in the following section could be evaluated with the QCM technique in order to corroborate the results below. Most importantly, the full length sequence of peptide A, in the following section, should be evaluated first with varying amounts of the diluting agent, whether it is tiopronin or a more inert molecule towards PZ. The amount of diluting agent used in these experiments (3:1 molar ratio) was likely too low, not allowing enough space between peptides to avoid steric hindrance to the approach of PZ. Another approach would be to biotinylate the linked terminus of the peptide and conjugate the biotinylated peptide to a layer of streptavidin. The alternative immobilization strategy may allow for more space around each peptide (governed by the surface area and packing density of streptavidin), avoiding the steric problems without the need to vary the quantity of diluting agent. The experiments should be performed at the high ionic strength only, and possibly with the addition of a surfactant such as Tween 20 to cut down on non-specific binding. Low ionic strength binding is near irrelevant in the scope of mimicking a real biological event.

The reversible nature of some of the different peptides (2,8, and 10), while interesting, is difficult to fully explain. One possible explanation is that the peptides are in such a conformation as to cast an alpha helical “umbrella” over the tiopronin layer below, which is known to bind to the antibodies discussed, allowing the antibody to reversibly bind the peptide instead of irreversibly and non-specifically sticking to the tiopronin layer.

Another important problem with these studies was that the peptides were not amidated and acetylated at the C and N termini, not accurately reflecting the fact that antigenic site A is an internal section of the protein.

Peptide ELISA step-wise linear epitope mapping Peptides were evaluated in an ELISA assay at various concentrations of peptide, primary antibody (PZ), and labeled secondary antibody and with different wash solutions and development conditions until optimum conditions were obtained for the sequence A.

These optimum conditions were determined to be 50 µg/mL peptide in PBS with 10mM EDTA, 5 µg/mL PZ in wash buffer, goat-anti-human diluted 1:5,000 in wash buffer, and 10 minutes of developing time with TMB substrate followed by quenching with 1N H2SO4. The results of this assay are presented in Figure 13.

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0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05

–  –  –

Figure 13: Results of the optimized peptide ELISA. The peptide IDs are listed in Table

4. Multiple null/alternate hypothesis pairs were evaluated from the ELISA data using Student’s t-tests at a 95% confidence interval, assuming unequal variances, and a twotailed distribution. The results are displayed in Table 5. Analysis of the true hypotheses allowed conclusions about which parts of the full peptide sequence A are important to the binding of PZ to HRSV F to be drawn.

Student’s t-tests were performed on the data in order help make arguments about which peptides or groups of peptides binds more or less strongly to PZ than others, displayed in Table 5.

Table 5: Student’s t-tests for peptide ELISA of whether a given null hypothesis or alternate hypothesis is supported. H0 is supported if tstat ≤ tcritical given a two tail distribution at a 95% confidence interval assuming unequal variances. Here x refers to the mean absorbance for the set of peptides indicated by its/their ID #(s) from Table 4. 0 means that H0 is supported or A means that HA is suggested to be true with reasonable confidence. HA is assumed to be the opposite case of H0. For instance, for x5 ≤ x(7-15), H0 means that peptide 5 binds PZ less or equally as efficient as peptides 7 through 15, and A indicates that HA is supported, which would be taken to be that peptide 5 binds PZ more efficiently than the pooled results of peptides 7 through 15.

–  –  –

The first significant finding of these tests is that four out of six peptides (2, 3, 5, and 6) are more active towards PZ than peptides oriented in the same direction but missing amino acids earlier in the chain (peptides 7 through 15). Furthermore, the pooled results of peptides 1-6 are significantly more active towards PZ than 7-15. The implication of this hypothesis is that HRSV F amino acids 254-268 represent a “hot spot” for binding of HRSV F to PZ. The conclusion is reasonable to expect given that there are six hydrogen bond donors (two serine, three asparagine, and one threonine residues) and two negatively charged residues at physiological pH (one each glutamic acid and aspartic acid). There are, however, a higher proportion of these residues in the region represented by peptides 7-15: two positively charged lysine residues, two asparagine residues, and one additional serine residue. Furthermore, escape mutations are present in either region.



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