Date of Award
7-2014
Document Type
Thesis
Degree Name
Master of Science (MS)
Department
Chemistry
Keywords
Leucine zippers, Proteins, Cell interaction
Abstract
Heterogeneous nuclear ribonucleoprotein C is ubiquitous in vertebrates, and exists as two alternatively spliced isoforms, hnRNP C1 and hnRNP C2 (hnRNP C). hnRNP C has been associated with pre-mRNA packaging, pre-mRNA, splicing, mRNA stability, internal ribosome entry site medicated translation, and has even been reported to be an integral component of the telomerase enzyme. Two proteins, hRaly and hRalyl, exhibit a great deal of primary sequence similarity with the C proteins and also conserve structural and functional motifs that have been identified in hnRNP C. A leucine zipper motif has been shown to be the oligomerization domain of hnRNP C and this sequence is conserved in hRaly and hRalyl. To determine if the three proteins are truly separate or whether they form various combinations of homo and hetero-oligomers previous cloning experiments conducted using polycystronic vectors (Peetha, 2013) showed when cloning one gene (either hRaly or hRalyl), Escherichia coli remained virulent whereas when hRaly and hRalyl were cloned in the same polycystronic vector, E. coli cells died. This lethality was attributed to the hypothesis that the heterodimeric structure between hRaly and hRalyl is the physiologically relevant structure. The research presented here tests the efficacy of this hypothesis by using molecular docking studies. These studies were v conducted using FlexPepDock from Rosetta dock. It was shown that hetero-dimers consisting of hnRNP C/hRaly, and hnRNPC/hRalyl were equally and in some cases more stable than their homo-dimer counterparts. To investigate the positional relevance of heptads 1-4 in determining stability a “scrambled” leucine zipper was generated, this sequence contained random heptads from hRaly, hRalyl, and hnRNP C. The resulting structure was only slightly less stable than any of the other dimers. Analysis of all of the structures identified two salt bridges that were common to all of the dimers modeled but was lacking in the mutated sequence. To determine if the decreased stability of the scrambled sequence resulted from the loss of these two intermolecular salt bridges, these were incorporated. The residues forming these bridges were mutated into the sequence. To determine if this were indeed the case these residues were incorporated into the mutated sequence. The resulting structure’s binding energy was increased by 4 kcal/mol, and was not as stable as all of the other modeled structures. The lack of specificity between the different zippers suggests the possibility of compositionally diverse hnRNP C, hRaly, hRalyl proteins in the cell.
Recommended Citation
Yambay-Tilman, Evelyn Rebecca, "Leucine zipper interacts promiscuously? Analysis of leucine zipper specificity in the c-protein family." (2014). Electronic Theses & Dissertations. 124.
https://digitalcommons.pittstate.edu/etd/124
Comments
Born digital thesis, 56 p.