Modeling DNA unzipping in the presence of bound proteins

Slide 1: Modeling DNA unzipping in the presence of DNA binding proteins Farhat Habib, Dr. Ralf Bundschuh Department of Physics, The Ohio State University

Slide 2: Overview  Importance of understanding DNA-protein interactions  Single molecule experimental techniques  The problem statement  Theory and description of the applied model  Results and conclusions  Future directions

Slide 3: DNA  Carries genetic information  Double stranded polymer consisting of monomer units called nucleotides  4 nucleotides labeled A, G, C, and T  Basepairing A≡T, G≡C  Each strand carries complete information of the other

Slide 4: Proteins  Most diverse of macromolecules  Intermediaries in most biological reactions  Composed of smaller units called amino acids Proteins play a vital role in DNA replication, transcription, recombination, repair, and in activating/inhibiting gene expression

Slide 5: Unzipping force analysis of protein association (UFAPA)  Single molecule experiment  Right sensitivity for probing DNA-protein interactions  F ~ 10 – 20 pN  Distances ~ nm

Slide 6: Goals  To investigate the limitations of UFAPA  The minimum binding energy for which the protein can be detected  Minimum distance between two proteins for which they can be resolved

Slide 7: Theory and methods  We describe the protein-DNA system’s thermodynamic behavior or properties in terms of the partition function of the system Model  Break the DNA-protein system into two parts  The double stranded (ds)DNA with (or without) proteins  The single stranded (ss)DNA on which force is being applied

Slide 8: Model  Partition function for dsDNA m   E (i ) N (m)  e i Where E(i) is the stacking energy of the ith basepair  The ssDNA  In the highly stretched regime we will operate the Extended Freely Jointed Chain (EFJC) model is the most accurate one h 2 ml / l W ( R; m)  C [q (h)] b p e  hR 2R

Slide 9: Model (cont.)  The protein  Include protein-DNA interaction by adding the extra free energy due to the presence of the protein at the binding site  Partition function for the entire system Z N ( R )  N (m)W (R; m)(1   (m  m0 )e E prot / k BT ) where m0 is the protein binding site m  To obtain force at a given extension once we have the partition function, we use  f ( R )   k BT log Z N ( R) R

Slide 10: Minimum protein strength 30 kJ/mol  Top plot shows the force- 17 extension curves from a Force (pN) protein of progressively 16 lower binding energy at same position 15 -5 kJ/mol  Average force = 15.3 pN; 1300 1400 1500 1600 1700 1800 Std deviation = 0.7 pN R(nm) GC:AT  At less than 10 kJ/mol the 4 75:25 50:50 peak from the protein is Change in force (pN) 25:75 3 within one standard deviation of the mean 2 1 20 40 60 80 Protein binding energy (kJ/mol)

Slide 11: Minimum resolvable distance between two proteins p) 10 (b n 20 tio ra 30 pa se 40 n ei ot Pr 50 N) 22 20 rce (p 18 16 Fo 14 12 1400 1500 R(nm ) 1600 1700 1800

Slide 12: Averaged minimum resolvable distance ×½ Resolvable distance (basepairs) 40 ×1 30 ×2 20 10 25 50 75 100 Binding energy (kJ/mol)  Minimum resolvable distance for pairs of proteins with 3 different relative binding energies

Slide 13: Conclusions and Future Directions  We investigate the limits of the UFAPA technique by considering the protein-DNA system thermodynamically  Average force for bare DNA was found to be 15.3 pN with a standard deviation of 0.7 pN  Minimum binding energy for a protein to be detected using UFAPA in the absence of FEC of bare DNA is around 10 kJ/mol  Minimum resolvable distance between two proteins can be up to 50 basepairs depending on relative protein strengths and the underlying DNA sequence Future: Consider the kinetics of the process