|Abstract or Summary
- Cells represent microcosms of spatial and temporal structural organization, with the achievement of internal spatial organization relying upon a collection of macromolecular motor complexes to transport and localize components throughout the cell. Cytoplasmic dynein is one such motor complex, and is the principal ATP-dependent motor for retrograde transport along microtubules in the cell. The large (~1.2 MDa) cytoplasmic dynein complex is comprised of multiple protein subunits, including two copies of the intermediate chain (IC), the N-terminal half ('N-IC') of which, is central to the dynein cargo attachment sub-domain. N-IC is a prototypical example of the intrinsically disordered protein (IDP) class, serving as a primarily disordered polybivalent molecular scaffold for its numerous binding partners (including regulators of the dynein motor complex), itself often becoming more ordered upon binding interaction. This dissertation presents studies aimed at the biophysical characterization of N-IC itself and also its interactions with several of its binding partners to elucidate structure-dynamics-function relationships, and to gain insights into how these binding interactions might be regulated, as these protein-protein interactions can ultimately determine the sub-cellular targeting and function of dynein within the cell.Chapter 1 opens with a brief introduction to IDPs, as they are a relatively new class of proteins whose recognition and presence in the reported scientific literature have grown exponentially in the past decade-and-a-half. The prevalence of intrinsically disordered proteins and protein regions in the proteome, the peculiarities in their binding interactions with partners, and their functionality in the absence of fixed, three-dimensional structure are outlined. This sub-section is followed by a thorough review of cytoplasmic dynein motor functions in the cell and its protein subunit composition, with particular emphasis placed upon the intermediate chain--the central protein of this thesis. A thorough review is also given for regulatory complexes of the dynein motor including dynactin, ZW10 RZZ, and NudE EL. Chapter 2 presents a review of the premier biophysical technique for the study and characterization of IDPs--solution-state protein NMR spectroscopy--and enumeration of particular considerations that must be taken into account (stemming largely from the conformational dynamics and motional freedom of these polypeptide chains) in the interpretation of data garnered from these techniques when applied to IDPs or unfolded proteins.Chapters 3 and 4 present original research work on the characterization of the N-terminal 143 residues (IC:1-143) of the Drosophila melanogaster dynein intermediate chain and its interactions with binding partners. The work presented in Chapter 3 demonstrates that, although predominately disordered, IC:1-143 deviates from random coil behavior, particularly in the form of two regions of α-helical structure near the N-terminus. Furthermore, these helical segments were determined to exist in two non-contiguous segments of IC that interact with the regulator dynactin p150[superscript Glued] protein, and the results of this study provided insights into the biophysical basis by which the IC–p150[superscript Glued] interaction (and thus the association between dynein and dynactin) might be regulated. In Chapter 4 a more detailed conformational and dynamical examination was performed on IC:1-143 using NMR residual dipolar couplings (RDCs) and paramagnetic relaxation enhancement (PRE) experiments, revealing unprecedented detail concerning further deviations of this protein from random coil behavior. The Tctex1 and LC8 light chains binding regions in IC exhibit enhanced polyproline II conformational sampling (relative to a random coil description), and the IC:1-143 protein exhibits further deviations from random coil behavior in the form of significant transient tertiary structure, shedding light on how the association state of IC with dynactin p150[superscript Glued] vs. NudE might be controlled in Drosophila melanogaster when both regulatory proteins are simultaneously present.Chapter 5 presents a summary of the key findings of the work presented in this dissertation, as well as an assessment of outstanding questions in this field and proposed work to help fill these gaps in knowledge. Overall, the results presented in this dissertation provide detailed descriptions of the structure and dynamics of the N-terminal half of N-IC (which constitutes a 'hotbed' for binding activity), revealing both subtle and pronounced deviations from random coil behavior in the form of secondary structure of varying degrees, as well as transient tertiary structure, all of which underlie interactions of IC with its binding partners, and also provide insights into the biophysical bases for regulation of these binding interactions.
- Cells represent microcosms of spatial and temporal structural organization