New methodologies for the synthesis of proteins and construction of biomolecular assemblies

Protein chemical synthesis has increasely been used for protein structure-function study. Our laboratory has been engaged in the development of new methodologies for protein synthesis and for the preparation of complex biomolecular structural studies. As a general goal of my thesis project, I mainly...

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Main Author: Hou, Wen
Other Authors: Liu Chuan Fa
Format: Theses and Dissertations
Language:English
Published: 2013
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Online Access:http://hdl.handle.net/10356/54710
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Institution: Nanyang Technological University
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spelling sg-ntu-dr.10356-547102023-02-28T23:51:05Z New methodologies for the synthesis of proteins and construction of biomolecular assemblies Hou, Wen Liu Chuan Fa School of Biological Sciences DRNTU::Science::Biological sciences Protein chemical synthesis has increasely been used for protein structure-function study. Our laboratory has been engaged in the development of new methodologies for protein synthesis and for the preparation of complex biomolecular structural studies. As a general goal of my thesis project, I mainly aimed to make a contribution to the field of protein chemistry. Specifically, in this project we expanded the application scope of native chemical ligation by developing a new method for peptide thioester synthesis by Fmoc SPPS. Furthermore, we also developed two new methods for sequential peptide ligation to synthesize larger protein. At last, we made use of the hybridization property of PNA for preparing dimeric macromolecules and for specific formation of unsymmetric disulfide bonds. In chapter 2, a very simple but efficient method for the synthesis of precursors of thioester was described. The thioester precursors can be directly used for native chemical ligation. N to S acyl transfer in a normal Xaa-Cys peptide amide bond is difficult, because it involves the energetically unfavored cis amide conformation and trans-cis amide isomerization requires significant activation energy. So we designed a tertiary amide of the structure Peptide-CO-N(CH2CH2SH)2, namely N, N-bis(2-mercaptoethyl)-amide or BMEA, to bypass this problem as it is always poised to undergo intramolecular thiolysis for thioester formation however it flips about the C-N bond. We show that a C-terminal BMEA peptide could be used directly for ligation with a cysteinyl peptide at weakly acidic pH. These BMEA peptides were easily prepared with standard Fmoc solid-phase synthesis protocols, thus giving a very convenient access to the thioester components for native chemical ligation. This method was demonstrated not only in the synthesis of small model peptides but also in the synthesis of a histone H3 protein. In chapter 3, to synthesize larger peptide or protein, two alternative methods for the sequential peptide ligation of several segments from N to C direction were developed. First method is based on combined use of thiol capture ligation and native chemical ligation. Using this method, a full active protein, single chain monellin (MNEI), was successfully synthesized by three segments via two chemical ligations from N to C direction. The second method makes use of different reaction kinetics between the BMEA peptide ligation developed in chapter 2 and typical native chemical ligation. A peptide containing 46 amino acids was demonstrated by this method which is so simple that no additional protecting groups are needed. In chapter 4, a peptide dimerization and specific disulfide bond formation strategy directed by PNA hybridization was demonstrated. The peptides were linked to a pair of complementary PNAs by native chemical ligation and PNA base pairing brought the two peptides into close proximity for homodimerization or interchain disulfide bond formation. This work points to the potential of PNA as the template in directing molecular assembling and biochemical reactions. ​Doctor of Philosophy (SBS) 2013-07-24T06:09:18Z 2013-07-24T06:09:18Z 2011 2011 Thesis http://hdl.handle.net/10356/54710 en 154 p. application/pdf
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic DRNTU::Science::Biological sciences
spellingShingle DRNTU::Science::Biological sciences
Hou, Wen
New methodologies for the synthesis of proteins and construction of biomolecular assemblies
description Protein chemical synthesis has increasely been used for protein structure-function study. Our laboratory has been engaged in the development of new methodologies for protein synthesis and for the preparation of complex biomolecular structural studies. As a general goal of my thesis project, I mainly aimed to make a contribution to the field of protein chemistry. Specifically, in this project we expanded the application scope of native chemical ligation by developing a new method for peptide thioester synthesis by Fmoc SPPS. Furthermore, we also developed two new methods for sequential peptide ligation to synthesize larger protein. At last, we made use of the hybridization property of PNA for preparing dimeric macromolecules and for specific formation of unsymmetric disulfide bonds. In chapter 2, a very simple but efficient method for the synthesis of precursors of thioester was described. The thioester precursors can be directly used for native chemical ligation. N to S acyl transfer in a normal Xaa-Cys peptide amide bond is difficult, because it involves the energetically unfavored cis amide conformation and trans-cis amide isomerization requires significant activation energy. So we designed a tertiary amide of the structure Peptide-CO-N(CH2CH2SH)2, namely N, N-bis(2-mercaptoethyl)-amide or BMEA, to bypass this problem as it is always poised to undergo intramolecular thiolysis for thioester formation however it flips about the C-N bond. We show that a C-terminal BMEA peptide could be used directly for ligation with a cysteinyl peptide at weakly acidic pH. These BMEA peptides were easily prepared with standard Fmoc solid-phase synthesis protocols, thus giving a very convenient access to the thioester components for native chemical ligation. This method was demonstrated not only in the synthesis of small model peptides but also in the synthesis of a histone H3 protein. In chapter 3, to synthesize larger peptide or protein, two alternative methods for the sequential peptide ligation of several segments from N to C direction were developed. First method is based on combined use of thiol capture ligation and native chemical ligation. Using this method, a full active protein, single chain monellin (MNEI), was successfully synthesized by three segments via two chemical ligations from N to C direction. The second method makes use of different reaction kinetics between the BMEA peptide ligation developed in chapter 2 and typical native chemical ligation. A peptide containing 46 amino acids was demonstrated by this method which is so simple that no additional protecting groups are needed. In chapter 4, a peptide dimerization and specific disulfide bond formation strategy directed by PNA hybridization was demonstrated. The peptides were linked to a pair of complementary PNAs by native chemical ligation and PNA base pairing brought the two peptides into close proximity for homodimerization or interchain disulfide bond formation. This work points to the potential of PNA as the template in directing molecular assembling and biochemical reactions.
author2 Liu Chuan Fa
author_facet Liu Chuan Fa
Hou, Wen
format Theses and Dissertations
author Hou, Wen
author_sort Hou, Wen
title New methodologies for the synthesis of proteins and construction of biomolecular assemblies
title_short New methodologies for the synthesis of proteins and construction of biomolecular assemblies
title_full New methodologies for the synthesis of proteins and construction of biomolecular assemblies
title_fullStr New methodologies for the synthesis of proteins and construction of biomolecular assemblies
title_full_unstemmed New methodologies for the synthesis of proteins and construction of biomolecular assemblies
title_sort new methodologies for the synthesis of proteins and construction of biomolecular assemblies
publishDate 2013
url http://hdl.handle.net/10356/54710
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