Size Dependence of Gold Nanoparticles – How Site-Specific Conjugation to Dihydrofolate Reductase Alters Characteristics Público
Ragupathi, Ashwin (Spring 2018)
Published
Abstract
Using the enzyme Dihydrofolate reductase (DHFR) and 5 nm, 15 nm and 30 nm AuNPs, both the effect of site-specific binding and the effect of AuNP size on the activity of the enzyme can be investigated. In order to allow AuNPs to conjugate to different locations on DHFR, the two intrinsic cysteines were replaced with non-thiol amino acids, and a single cysteine or His-Tag was placed into desired attachment sites.
The characterization of the free enzymes and conjugates, as well as the stability and preparation of both was investigated. The free mutants and wild type DHFR were prepared using TEV cleavage, and demonstrated a similar enzyme activity of about 30 turnovers s-1 and activation energies of approximately 75-79 kJ/mol. Once conjugates were formed, Tween 20 was used to stabilize them. SDS-PAGE showed that the DHFR mutants adequately conjugate with AuNPs and a novel fluorescence assay was developed to determine the concentration of protein bound to AuNPs. Ratios of conjugated enzyme to AuNP were also determined. UV/Vis analysis determined free AuNP had a SPR λmax absorbance at 519 nm while conjugation red-shiftted the λmax absorbance to 525 nm. Dynamic Light Scattering was used to determine that a monolayer of DHFR forms around the AuNP. Also, the activity of the conjugates mostly ranged from about 0.4 to 1.5 turnovers s-1. Varying the sizes of the AuNP did not have much of an effect on the activity when compared to that of other conjugates. Also, these findings demonstrate that conjugation to AuNP greatly decreases the activity of DHFR, and that AuNP attachment affects the kinetics and dynamics of DHFR.
Table of Contents
Part I: Introduction to Gold Nanoparticles and Dihydrofolate Reductase 1
1.1 Introduction 1
1.1.1 Goals and Aims 1
1.1.2 The Gold Nanoparticle 2
1.1.3 What is DHFR? 3
1.1.4 Mutants of DHFR 5
Part II: Preparation and Characterization of DHFR Mutants 8
2.1 Purpose 8
2.2 Materials and Methods 8
2.2.1 TEV Cleavage 8
2.2.2 Activity of Free Mutants 10
2.2.3 Arrhenius Information 11
2.3 Results and Discussion 11
2.3.1 TEV Cleavage 11
2.3.2 Activity of Free Mutants 12
2.3.3 Arrhenius Information 13
Part III: Conjugation and Stability 15
3.1 Purpose 15
3.2 Materials and Methods 15
3.2.1 Synthesis of 15 nm AuNP 15
3.2.2 Conjugation 16
3.2.3 Tween 20 and Free Protein 17
3.2.4 Tween 20 and Conjugates 17
3.3 Results and Discussion 17
3.3.1 Synthesis of 15 nm AuNP 17
3.3.2 Tween 20 and Free Protein 18
3.3.3 Tween 20 and Conjugates 19
Part IV: DHFR-AuNP Conjugate Characterization 22
4.1 Purpose 22
4.2 Material and Methods 22
4.2.1 UV/Vis Verification 22
4.2.2 SDS-Page Gel 22
4.2.3 Dynamic Light Scattering 22
4.2.4 Concentration Determination 23
4.2.5 Conjugate Activity 23
4.2.6 DHFR to AuNP conjugate ratios 23
4.3 Results and Discussion 24
4.3.1 UV/Vis Verification 24
4.3.2 SDS-Page Gel 25
4.3.3 Dynamic Light Scattering 27
4.3.4 Concentration Determination 29
4.3.5 Conjugate Activity 30
4.3.6 DHFR to AuNP conjugate Ratios 31
Part V: Conclusions 33
Part IV: References 35
About this Honors Thesis
| School | |
|---|---|
| Department | |
| Degree | |
| Submission | |
| Language |
|
| Research Field | |
| Palabra Clave | |
| Committee Chair / Thesis Advisor | |
| Committee Members |
Primary PDF
| Thumbnail | Title | Date Uploaded | Actions |
|---|---|---|---|
|
|
Size Dependence of Gold Nanoparticles – How Site-Specific Conjugation to Dihydrofolate Reductase Alters Characteristics () | 2018-04-09 10:57:31 -0400 |
|
Supplemental Files
| Thumbnail | Title | Date Uploaded | Actions |
|---|