Name: Wissam Mahmoud Hamdi Abdel-Gawad

Date of Birth: November 7th 1972

Nationality: Egyptian

Previous degree: M.Sc. Biophysics, faculty of science, cairo university

Registration date:   July 2005

Awarding date: December 2007

Supervisors: Prof. Dr. Ali A. A. El-lakkani

Examiners:       [1] Prof. Dr. Alberto Spisni, University of Degli Studi di Parma, faculty of medicine.

                        [2] Prof. Dr. Bodil Norrild, institute of cellular and molecular medicine, University of Copenhagen.

Summary : 

1-    membrane protein crystallization In meso: lipid type-tailoring of the cubic phase

Hydrated monoolein forms the cubic-Pn3m mesophase that has been used for in meso crystallization of membrane proteins. The crystals have subsequently provided high-resolution structures by crystallographic means. It is possible that the hosting cubic phase created by monoolein alone, which itself is not a common membrane component, will limit the range of membrane proteins crystallizable by the in meso method. With a view to expanding the range of applicability of the method, we investigated by x-ray diffraction the degree to which the reference cubic-Pn3m phase formed by hydrated monoolein could be modified by other lipid types. These included phosphatidylcholine (PC), phosphatidylethanolamine, phosphatidylserine, cardiolipin, lyso-PC, a polyethylene glycol-lipid, 2-monoolein, oleamide, and cholesterol. The results show that all nine lipids were accommodated in the cubic phase to some extent without altering phase identity. The positional isomer, 2-monoolein, was tolerated to the highest level. The least well tolerated were the anionic lipids, followed by lyso-PC. The others were accommodated to the extent of 20–25 mol %. Beyond a certain concentration limit, the lipid additives either triggered one or a series of phase transitions or saturated the phase and separated out as crystals, as seen with oleamide and cholesterol. The series of phases observed and their order of appearance were consistent with expectations in terms of interfacial curvature changes. The changes in phase type and microstructure have been rationalized on the basis of lipid molecular shape, interfacial curvature, and chain packing energy. The data should prove useful in the rational design of cubic phase crystallization matrices with different lipid profiles that match the needs of a greater range of membrane proteins.

2- Pre-steady state kinetics studies of the fidelity and mechanism of polymerization catalyzed by truncated human DNA polymerase λ

DNA polymerase (Polλ), a member of X-family DNA polymerase, possesses an N-terminal BRCT domain, a proline rich domain, and a C-terminal polymerase β-like domain (tPolβ). In this paper, we determined a minimal kinetic mechanism and the fidelity of tPolλ using pre-steady-state kinetic analysis of the incorporation of a single nucleotide into a one nucleotide gapped DNA substrate, 21-19/41-mer (primer-primer/template). Our kinetic study revealed an income nucleotide bound to the enzyme-DNA binary complex at a rate constant of 1.55 × 108 M-1 s-1 to form a ground state ternary complex while the nucleotide dissociated from this complex at a rate constant of 300 s-1. Since DNA dissociation from tPolλ (0.8 s-1) was less than 3-fold slower than polymerization, we measured saturation kinetics for all 16 possible nucleotide incorporations under single turnover conditions to eliminate the complications from the multiple turnovers. The fidelity of tPolλ was estimated to be in the range of 10-2-10-4 and was sequence- dependent. Surprisingly, the ground state binding affinity of correct (1.1-2.4 μM) and incorrect nucleotide (1.4-8.4 μM) was very similar while correct nucleotides (3-6 s-1) were incorporated much faster than incorrect nucleotide (00.1-0.2 s-1). Interestingly, the microincorporations of dGTP opposite a template base thymine (0.2 s-1) was more rapid than all other misincorporations, leading to the lowest fidelity (3.2 × 10-2) among all mismatched base pair. Additionally, tPolλ was found to possess weak strand-displacement activity during polymerization. These biochemical properties suggest that Polλ likely fills short-patched DNA gaps in base excision repair pathways and participate in mammalian nonhomologous end-joining pathways to repair double stranded DNA breaks.

3-      RNA secondary structure prediction, including pseudoknot,

 using genetic algorithm

A model genetic algorithm was designed by which randomly chosen structural elements, loop and stem, were passed to the offspring and the terminals closed through base pairing where pseudoknot formed during terminal closing. Contribution to free energy was due to two models, hydrogen bonding model and stacking free energy model. The total free energy of base pairing equals the summation of contributions from the structural pairs in the case of hydrogen bonding model and equals the summation of contributions from the neighboring pairs in the case of stacking energy model. The algorithm selected the 20 individuals of the lowest free energy among the 20 parents and 20 offspring. Finally, the lowest free energy individual was displayed in a coded format and the code is decoded and manually drawn.

 

 

 
 
 
 

 

  
 
   
   
   
 

 
   
   
   
   
   

 

 

  
   
   
   
   
   
   
   
   
   
   
   
 

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