טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentPerach Michal
SubjectCorrelations between Protein Coding, Folding, and Function:
The Effect of Silent Mutations and Dynamics
of Multi-Component Machineries
DepartmentDepartment of Medicine
Supervisor Professor Oded Lewinson


Abstract

It is broadly-recognized that protein coding, folding, structure and dynamics are intricately interconnected to ultimately lead to the optimally protein function. Nevertheless, despite great progress in the field, several outstanding questions remain to be fully addressed. In my thesis I have addressed two such fundamental questions: In the first chapter, I investigated how biased use of synonymous codons affects protein folding, expression, and function. In the second chapter, I established a super resolution microscopy platform to investigate the expression, function, and dynamics of complex protein machineries in live bacterial cells.


Chapter 1 - The effects of silent mutations on protein expression and stability

Due to the genetic code redundancy some mutations in the DNA sequence have no effect on protein sequence. Such “synonymous mutations” were originally believed to have no biological effect, but were later found to be involved in human disease and influence protein expression. The link between synonymous mutations and protein expression and folding is poorly understood. It has been suggested that strategically located “slow codons” stall the ribosome and allow sequential folding of proteins during co-translational folding. Such a model may explain why in some cases replacing slow codons with faster ones leads to protein misfolding. However, research in the field led to contradicting results.

We investigated the link between codon usage and protein folding by combining computations and experiments: First, we used bioinformatics tools to analyze gene families from E. coli and B. subtilis and identified evolutionarily conserved slow codons. Then, we chose 8 genes for experimental testing and replaced the slow codons with fast ones. The effects of these replacements on protein expression, folding, and function were investigated using three independent experimental approaches. In 3 out of the 8 genes tested, slow codons replacement had detrimental effects.

These results demonstrate slow codons can play a role in dictating translation attenuation events that are essential for correct protein folding.


Chapter 2- Using super resolution microscopy to characterize the vitamin B12 transport system in vivo

Super resolution localization microscopy is a 21st-century innovation that combines laser optics and dynamic fluorescent markers, to allow imaging of biological samples in vivo at a resolution exceeding the diffraction limit of light.

My goal was to implement super-resolution fluorescent microscopy to image the process of the multi-component vitamin B12 transport machinery in live bacterial cells.

Import of Vitamin B12 into E. coli cells requires the concerted action of seven components: outer-membrane transporter BtuB, the trans-periplasmic energy coupling complex TonB-ExbB-ExbD, inner membrane transporter BtuCD and the periplasmic substrate binding protein BtuF. This transport system has been extensively investigated in vitro, elucidating many aspects of the structure and function of its components. Nevertheless, fundamental information regarding the in vivo concerted function of the system is still missing, including the copy numbers of the proteins involved, their localization and co-localization, and the effect of the substrate on these parameters.  Using super-resolution microscopy in live bacterial cells we can potentially provide the first-ever in vivo concerted description of a multi-component transport system.