Browsing by Subject "Amino Acid Substitution"
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Item Characterization of U2AF26, a Paralog of the Splicing Factor U2AF35(2004-08-19) Shepard, Jeremiah Brian; Lynch, Kristen W.; McKnight, Steven L.The essential splicing factor U2 auxiliary factor (U2AF) mediates 3' splice site recognition during spliceosome assembly. The mammalian U2AF is composed of a large subunit, U2AF65, and a small subunit, U2AF35. U2AF65 recognizes the pyrimidine tract and U2AF35 binds to the AG dinucleotide, both of which are specific 3' splice site sequence motifs. In the present work U2AF26, a paralog of the conventional U2AF35, has been studied. U2AF26 shares 84% primary amino acid identity with U2AF35, suggesting functional homology. However, U2AF26 has two amino acid substitutions in ribonulceoprotein consensus sequence-2 (RNP-2) and significant differences within the RS domain, two regions thought to be important for the function of U2AF35. The goal of this study was to characterize the functional differences between the two small subunits. Western blot analysis revealed that U2AF26 protein expression varies relative to U2AF35 in different mouse tissues. Site-specific crosslinking analysis of sixteen permutations of the nucleotide composition upstream and downstream of the AG indicates that U2AF26 and U2AF35 bind to the UAGG motif with the highest affinity. Interestingly, U2AF26 binds the UAGU motif better than U2AF35. This observation suggests that U2AF26 and U2AF35 have overlapping binding affinities, but that U2AF26 might be capable of recognizing a specific 3' splice site motif better than U2AF35. Initial evidence suggested that U2AF26 is regulated by circadian rhythm. Analysis of U2AF26 over a 24-hour period in the mouse forebrain indicates that expression of the full length transcript does not change significantly, but the alternative splicing of the U2AF26 transcript fluctuates during the day:night cycle. Examination of U2AF26 alternative splicing in other tissues revealed that this splicing event is temporally regulated in the liver, but with a two-peaked pattern of splicing. Further analysis of other alternative splicing events in the liver indicates that the polypyrimidine tract binding (PTB) transcript is regulated in a similar manner. The two-peaked pattern of splicing in the liver suggests that the alternative splicing of U2AF26 and PTB is not regulated by circadian rhythm. However, this is the first time it has been observed that pre-mRNA splicing changes as a function of the day:night cycle.Item Nucleocytoplasmic Localization of MAPKs(2007-08-08) Yazicioglu, Mustafa Naci; Cobb, Melanie H.Mitogen-activated protein kinases (MAPKs) comprise a family of protein-serine/threonine kinases, which participate in signal transduction pathways that control intracellular events. MAPKs are regulated by phosphorylation cascades, which are usually initiated by external stimuli including a variety of ligands. At least two upstream protein kinases are activated in series to lead to activation of a MAPK. The kinase that activates the MAPK is a MAPK kinase (MAP2K or MEK) and the kinase that phosphorylates the MAP2K is a MAP3K or MEK kinase (MEKK). Upon activation, MAPKs may translocate to the nucleus to phosphorylate nuclear targets. Previous findings from our laboratory showed that a constitutively active and nuclear form of the MAPK ERK2 is sufficient for transformation of immortalized fibroblasts (Robinson MJ et al,1998). However the mechanisms of nuclear localization of MAPKs are still not fully understood clearly. Although most nucleocytoplasmic localization events require carrier proteins known as karyopherins (importins and exportins), ERK2 enters the nucleus of permeabilized cells even if these carrier proteins are missing. This is explained by direct binding to proteins in the nuclear pore complex (NPC). Similar to ERK2 targets, NPC proteins also contain Phe-Xxx-Phe (FXF) motifs. My first aim in this project was to examine the roles of ERK2 residues that are crucial for FXF binding on nuclear localization of ERK2. Mutating these ERK2 residues decreased the nuclear import of ERK2 proteins in permeabilized cells. Secondly, the regulation of ERK2 nuclear export was analyzed. It was observed that ERK2 export occurs by two distinct processes; one energy-dependent and the other energy-independent. My final aim was analyzing the activation and nucleocytoplasmic trafficking of other MAPKs, JNK and p38.Item The Structural Properties of Adaptation and Allostery in Proteins: A Case Study in a PDZ Domain(2017-03-08) Raman, Arjun Swaminathan; Rosen, Michael K.; Ranganathan, Rama; Rice, Luke M.Complex systems are present at all scales of biology spanning amino acid interactions in proteins to inter-species interactions in ecosystems. Despite their ubiquity, an understanding of how to study complex systems methodically is lacking. Fundamentally, this is because a procedure to discover the appropriate parametrization of such systems into relevant parts is absent. The Ranganathan Lab developed an evolutionary approach, Statistical Coupling Analysis (SCA), to understand how amino acid interactions within proteins give rise to basic protein characteristics such as fold and function. The result of this approach was a structural decomposition of proteins into units of coevolving residues termed protein 'sectors'. Is the transformation from amino acids to protein sectors a useful and relevant representation of the essence of proteins? Previous work has demonstrated that specifying co-evolution is sufficient to design synthetic natural-like proteins and encodes the information needed to execute a primary function. As evolved biological systems however, proteins must also also adapt quickly to new functions. In addition, a fundamental characteristic of many proteins is the ability to transmit information over a significant distance in the protein structure, a phenomenon known as allostery. Where in the protein do these characteristics lie? Here, we address the sequence origins and structural properties of a) the adaptive capacity of proteins and b) allosteric communication within proteins. Understanding how proteins can adapt quickly to new function is an outstanding question in biology, marked by failed attempts at engineering new or altered function guided by structural approaches focused on the importance of active site or binding pocket positions. It is often the case that mutations located distal to the active site harbor adaptive potential, a non-obvious observation given the crystal structure of a protein. To more fundamentally understand the adaptive process in proteins, here we examine a two-step mutational path to new specificity in a model protein PSD95pdz3 where one intermediate, a Glycine (G) to Threonine (T) mutation at position 330, removed from the binding site of the protein, maintains native function while simultaneously adapting the protein to an alternate function (termed a 'conditionally neutral' mutation) whereas the other intermediate, a Histidine (H) to Alanine (A) mutation at position 372, promotes a direct specificity switch, abrogating native function. Through a stochastic population dynamics simulation, we find the conditionally neutral intermediate promotes adaptation over a wide range of mutation rates and rates of environmental fluctuation while the direct specificity switching mutation facilitates adaptation only within a special regime of population dynamics parameters. We comprehensively identify the spatial distribution of all adaptive mutations in PSD95pdz3, finding that direct specificity switching mutations are found exclusively at sector positions directly at the binding site whereas conditionally neutral mutations are generally found distal to the binding site but connected to it through the protein sector. Crystal structures reveal how a mutation at position 330 creates plasticity at the binding site to create a dual function protein. Overall, these results illustrate the importance of a spatially distributed network of coupled residues for adapting in a fluctuating environment. Revealing paths of allostery in protein structures has been a central goal of structural biology. In PSD95pdz3, we find that the G330T mutation causes local backbone remodeling and structurally affects solely a distant conserved helix. Detecting structurally coupled residues in the protein reveals a network of connected residues spanning the 330 position to the distant helix propagating through the core of the protein sector. As a check of this allosteric path, we are able to abrogate the allosteric transmission through a mutation in the middle of the protein sector. Both the ability to adapt to new function and the property of allostery are found almost exclusively within the protein sector. The work highlighted here in addition to previous studies in the lab thus appear to suggest that the sector is a good descriptor and model for understanding how proteins work. It will therefore be important for future studies to determine if other methods of protein design such as Direct Coupling Analysis and Rosetta provide equivalently sufficient descriptions of proteins and what additional information, if any, these approaches reveal.