Many assays rely upon immobilizing proteins onto gold surfaces. Therefore, simple, stable and specific methods for protein immobilization are needed for the development of applications which rely on the oriented attachment of proteins. Current methods of immobilization utilize non-covalent adsorption (physisorption), or covalent adsorption (chemisorption) to attach proteins to surfaces. Physisorption is not a robust method of attachment and does not permit control of a protein's orientation on the surface, which is critical for preserving the protein's activity. In covalent methods, the proteins are often attached through native residues resulting in proteins which are not oriented and which may have reduced activities.
We have used the tetracysteine (TC) motif (C-C-P-G-C-C) to develop a direct, stable, genetically encodable method for the oriented chemisorption of proteins to gold nanoparticles (Au NPs) while simultaneously suppressing protein physisorption. Mutants of ubiquitin (Ub), enhanced green fluorescent protein (eGFP), recombinant protein G (protein G') and the haloalkane dehalogenase, LinB, containing the tetracysteine motif were produced and displayed stronger adsorption to the NPs than the native proteins. The effect of size on the ability of proteins to stabilize the NPs was investigated on Au NPs of various sizes (14, 18, 28 and 39 nm). Gel electrophoresis, fluorescence spectroscopy and immobilized metal affinity chromatography experiments demonstrate that the TC motif controlled the orientation of the protein on the Au surface, and enhanced the binding of protein-NP conjugates to immobilized targets. The small Ub tetracysteine mutant stabilized several sizes of Au NPs while the eGFP tetracysteine mutant had the strongest chemisorption to the 18 nm NPs. The results suggest that the size of the NPs has a definite effect on the binding of proteins. Small, stable proteins such as Ub tend to have more predictable behavior on a range of NP sizes than larger proteins, however this effect depends on the size and nature of the proteins and not all particle sizes will be compatible with all proteins.
Mutants of recombinant protein G (protein G'), an immunoglobulin G (IgG) binding protein, containing the tetracysteine motif were generated to investigate the effect of orientation on preserving the activity of protein G'. Gel electrophoresis experiments suggest that the TC protein G' mutant is chemisorbed to the surface of Au NPs of various sizes. Fluorescence experiments demonstrate that TC protein G' is oriented and maintains its activity on the surface of the Au NPs, and that physisorbed proteins are poorly oriented and are less active on the surface of the NPs. These results demonstrate that the short, genetically engineered tetracysteine motif provides a stable method for the attachment of proteins, and allows the proteins to be chemisorbed, oriented and active on the surface of Au NPs. The control of orientation is key in many applications including sensor devices, and is useful in the process of creating multifunctional NPs which have active oriented proteins or enzymes on the surface.
|Advisor:||Metallo, Steven J.|
|Commitee:||Moasser, Bahram, Paranjape, Makarand, Swift, Jennifer A.|
|School Location:||United States -- District of Columbia|
|Source:||DAI-B 73/02, Dissertation Abstracts International|
|Subjects:||Biochemistry, Inorganic chemistry, Nanotechnology|
|Keywords:||Chemisorbed proteins, Genetically encodable oriented adsorption, Gold nanoparticles, Oriented protein adsorption, Oriented proteins, Tetracysteine motif|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be