Structural and functional characterization of bacterial response and neuroprotective proteins

Competence;NMR;Calbindin;response regulator;mass spectrometry;differential surface modification

Hobbs, Carey Anne ; John Cavanagh, Committee Chair,William Miller, Committee Member,David Bird, Committee Member,Dennis Brown, Committee Member,Kenneth Tomer, Committee Member,Hobbs, Carey Anne ; John Cavanagh ; Committee Chair ; William Miller ; Committee Member ; David Bird ; Committee Member ; Dennis Brown ; Committee Member ; Kenneth Tomer ; Committee Member

University:North Carolina State University

关键词: Competence;    NMR;    Calbindin;    response regulator;    mass spectrometry;    differential surface modification;   

Others  :  https://repository.lib.ncsu.edu/bitstream/handle/1840.16/5423/etd.pdf?sequence=1&isAllowed=y

美国|英语

来源: null

【 摘 要 】

The studies described here involve the structural and functional characterization of two proteins, calbindin-D28k and ComAC.Calbindin-D28k is a calcium binding protein that plays a unique role in eukaryotic cells, acting as both a calcium buffer and sensor.Upon binding calcium, calbindin-D28k transitions from an ordered apo- state into a disordered state as calcium is bound.Once fully loaded with four calcium ions it again takes on an ordered state.The high resolution NMR solution structure of holo- calbindin-D28k has been solved.The structure of the apo- calbindin-D28k, however, remains to be solved.In the absence of a high resolution structure of apo- calbindin-D28k it has not been possible to compare the three-dimensional structure of the two states, making it impossible to analyze how this change affects the function of calbindin-D28k.This study uses differential surface modification analyzed by mass spectrometry to probe the tertiary structural changes that occur upon calcium binding.This comparison provides the first look at the conformational change of calbindin-D28k upon calcium binding.In the second study, the structure and function of the C-terminal DNA binding domain of ComA (ComAC) from Bacillus subtilis was investigated.ComA is a response regulator protein involved in the two-component signal transduction system regulating the development of genetic competence.The protein can be divided into two distinct domains, an N-terminal regulatory domain and a C-terminal effector domain.The C-terminal domain, ComAC, is a DNA binding domain responsible for binding to target operons, including the srfA operon.The transcriptional activation of the srfA operon ultimately controls the expression of the genes responsible for expressing the protein machinery that bind to and take up exogenous DNA. This research determined the high resolution solution structure of ComAC using NMR.This structure was then used in conjunction with SPR, NMR DNA titration studies and molecular modeling to analyze and determine how ComAC interacts with its cognate DNA promoter sequences.