Size Dependence of Gold Nanoparticles – How Site-Specific Conjugation to Dihydrofolate Reductase Alters Characteristics 公开

Ragupathi, Ashwin (Spring 2018)

Permanent URL: https://etd.library.emory.edu/concern/etds/3r074t94h?locale=zh
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

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