Continuum Biomechanics Modeling of Homologue Proteins

Jonathan Chang, Regine Labog, Sweta Ramachandran Abstract Actin and mreB possess homologous traits that interest scientists who believe that the conservation of protein sequencing suggests a common ancestor. After observing their similarities in sequence and structure, we will use COMSOL to discover whether or not their biomechanical properties account for actin's and mreB's differing biological functions. To do so, f-actin and mreB were modeled while taking into account f-actin's 122 degree twist and mreB parallel structure. These models revealed that mreB's maximum displacement was significantly lower than actin's. This exponential displacement of actin is due to the g-actin's angle when the force was applied and the linear displacement of mreB was due to the parallel application of force. By apply forces on the two proteins, we see that the flexibility of actin is necessary for actin which must handle multiple functions in a eukaryotic cell. However, in bacteria where mreB's main function is to provide the structure for bacteria, it requires a more rigid structural component. Our model accounts for the structures of the two proteins which will, in the future, help in determining when the two proteins diverged from their common ancestor. Introduction Actin is a component of the cytoskeletal system allowing cell movement and cellular processes. Actin filaments are called microfilaments of thin filaments that undergo constant rearrangement to create movement. Actin is a globular protein with the ATP binding site at the center of the molecule. G-actin is short for globular actin, a short polypeptide chain made up of 375 amino acids. Gactin combines with other g-actin monomers to create an actin filament. It serves as a site for nucleation and further growth of the actin protofilament. Once ATP is incorporated into the actin filament, it hydrolyzes immediately and the ADP remains in the actin filament until it depolymerizes and an ATP is sent into the nucleation site while the previous ADP exits. Gactin creates filamentous actin (F-actin) when ATP, Mg, and K are present. However, above the critical concentration of G-actin, the molecules polymerize. Below the critical concentration, the actin filaments depolymerize. Actin filaments possess polarity. The positive end of G-actin is opposite the cleft holding the ATP molecule, which is the negative end. Growth and polymerization occurs more rapidly in the positive end. Though the intermolecular interactions of two actin molecules is weak, adding a third actin monomer stabilizes the overall complex. Once the dimer becomes a trimer, the actin molecules adds more monomers and forms a nucleation site. Adding actin filaments or key actin binding proteins elongates the actin molecules to form a long helical polymer. After the growth period, the polymer reaches an equilibrium phase where depolymerization controls the length of the polymer as new monomers are added. mreB Bacillus subtilis mreB is a bacterial, actin-like protein that has been shown to perform essential functions in cellular physiology. It affects cell growth, cell shape, chromosome segregation and polar localization of proteins, and localization as helical filaments under the cell membrane. MreB

performs dynamic, motor-like movements in the cells and extend along helical tracks in seconds. MreB is a bacterial protein considered an actin homologue based on its similarities in tertiary structure and conservation in the active site's peptide sequence. MreB has filaments located under the cellular membrane to control the width of rod shaped bacteria Aside from tubulin, the other major component of the eukaryotic cytoskeleton is F-actin (filamentous actin), a relatively thin protein composed of two strands twisted around each other. Actin works in cell motility, shape determination, phagocytosis, cytokinsesis, and rearragement of surface components. It is 43kDa bi-lobed protein that binds ATP in a cleft between the two lobes. The mreB gene is associated with prokaryotic cell shape determination but not cell envelope synthesis. Research on Bacillus subtilis showed that the large spirals encircling the cytoplasm under the cell membrane suggests that mreB forms filamentous structures in bacteria similar to the eukaryotic actin cytoskeleton. In vitro, purified mreB forms polymers consisting of protofilaments of 51 angstroms which is close to the spacing between the subunits of filamentous actin which is 55 angstroms. The three-dimensional structure of actin and mreB is also very similar. The striking difference between mreB and actin is that the Factin twists around each other whereas mreB protofilaments are straight. Research Design and Methods We used COMSOL to model the actin and MreB based on the values determined through literature research; this includes density, Young's Modulus, Poisson's ratio, and the dimensions of F-actin, as well as the dimensions of its subunits. The values we have determined are as follows: F-actin total diameter 7 nm, length of interest 20 nm, subunit diameter of 5.4nm, Young's Modulus of 44e6 Nm-2, and a Poisson's ratio of 0.3. The length of interest was determined to be the length at which it makes

its designated 166 degree twist, the primary design to incorporate. In COMSOL, we used a 3D, structural, static model. Since we were more interested in comparing the difference in response to loads between actin and MreB, using a transient model was not of interest. With the static model, one end was fixed and the other end was applied a vertical/parallel load. By simplifying the model of F-actin to incorporate half the number of subunits for clarity sake, calculated the precise positions and direction vectors of the subunits was possible while the overall structural design was not sacrificed. Edge gaps between subunits was modeled in both actin and MreB since separation does naturally occur between subunits--the gaps were designed to be as consistent as possible between the two COMSOL models. Two forces that were determined through literature research to be the usual load forces for these proteins was applied to the non-fixed end: 100 pico-Newtons, and 100 micro-Newtons. This led to interesting results wherein displacement along inside edge of both proteins could be determined and outputted as a graph. Results

Figure 1a: COMSOL Diagram of Actin Protein, Front View

Figure 1b: COMSOL Diagram of Actin Protein, Front View, Meshed

Figure 4: COMSOL Diagram of MreB with Force Applied, Boundary View

Figure 2: COMSOL Diagram of MreB Protein, Front View

Figure 5: COMSOL Diagram of MreB with Force Applied, Streamline View

Figure 3: COMSOL Diagram of Actin with Force Applied, Boundary View

Figure 6: COMSOL Diagram of Actin with Displacement Edge Outlined in Red

Figure 7: COMSOL Diagram of Total

Displacement Along Actin Edge, Load of 100 Micro-Newtons

Figure 8: COMSOL Diagram of Total Displacement Along Actin Edge, Load of 100 Pico-Newtons

Figure 11: COMSOL Diagram of Total Displacement Along MreB Edge, Load of 100 Pico-Newtons Maximum Displacement 1.8e-8 meters 4.614e-28 meters

Protein Actin MreB

Figure 12: Maximum Displacements with 100 Pico-Newtons Load

Figure 9: COMSOL Diagram of MreB with Displacement Edge Outlined in Red

Figure 10: COMSOL Diagram of Total Displacement Along MreB Edge, Load of 100 Micro-Newtons

Discussion Maximum displacement was measured and analyzed for both actin and MreB. The displacement curve of actin (Figures 7 and 8) is exponential, which can be explained by the angle of the subunit on which the force is applied due to the helical conformation of the protein. In contrast, the displacement of MreB (Figures 10 and 11) is linear because the uniaxial force is applied in parallel to the major axis of the MreB filaments. Based on our results, it is apparent that the maximum displacement of MreB (Figure 4) is significantly smaller than that of actin (Figure 3). This can be explained by the rotational twist in the F-actin conformation, which makes the protein less rigid. Thus, it can be inferred that these homologue proteins, which have similar amino acid sequences and tertiary structures, play different roles in eukaryotic and prokaryotic cells. Since actin must handle multiple functions in a eukaryotic cell, including mechanical support, cell motility, cargo transport, and cytokinesis, flexibility and an ability to change conformations efficiently may be an essential characteristic for the protein. The larger

displacement that was observed supports this motif. However, the primary function of MreB in bacteria is to provide the organism with a rigid, inter-cellular backbone. Consequently, the smaller displacement observed in MreB upholds the notion that the bacterial protein must be relatively inflexible and stiff. The models of actin and MreB that were constructed represent the fundamental building blocks of the two proteins. Only four subunits of the protein were modeled, and in the future, a larger number of subunits can be modeled to verify that the proteins behave similarly at the subunit level and as a complete protein. Moreover, the interactions between the individual filaments, such as hydrogen bonding and amino acid interactions, were not considered. In order to account for these interactions, the individual amino acids can be modeled to determine if these interactions affect the displacement of the protein as a whole. Once a thorough model of actin is established, it would be interesting to study the elongation of the actin filament and the biomechanics that underlies the propagation of the protein through the cytosol of a cell. Limitations: did not take into account interactions between the actin filaments only modeled 4 subunits of the protein Future Studies: interactions between actin and other proteins elongation of actin References
1. Figge, Rainer M., Arun V. Divakaruni, and James W. Gober. "MreB, the cell shape-determining bacterial actin homologue, co-ordinates cell wall morphogenesis in Caulobacter crescentus."Molecular Microbiology 2004: 1321-332. Blackwell Publishing Ltd. Web. <http://www.biochemistry.ucla.edu/biochem/Faculty/Gob er/PDF/1321.pdf>. Van den Ent, Fusinita, Linda Amos, and Jan Lwe. "Bacterial Ancestry of Actin and Tubulin."Current Opinion in Microbiology 2001: 634-48. Elsevier Science Ltd. Web. 3 Dec. 2009. <http://www2.mrclmb.cam.ac.uk/groups/jyl/PDF/current%20opinion%20mi cro%202001.pdf>. Defeu, Jol, and Peter Graumann. "Bacillus subtilis actinlike protein MreB influences the positioning of the replication machinery and requires membrane proteins

MreC/D and other actin-like proteins for proper localization." BMC Cell Biology. PubMed central, 3 Mar. 2005. Web. 3 Dec. 2009. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC555950/ >.

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