ERAP1 is an aminopeptidase that is a component of antigen processing. To distinguish the role of ERAP1 from homologs ERAP2 and IRAP, I identified three specific ERAP1 inhibitors via a high-throughput screen. These compounds inhibit hydrolysis of a decamer peptide, and some inhibit ERAP1 in a cellular assay. These inhibitors enable dissection of ERAP1 mechanism. ERAP1 has been crystallized in two conformations: open and closed. I collected SAXS data on ERAP1 in the presence of various inhibitors. ERAP1 adopts an open conformation in solution, but some inhibitors stabilize the closed form. Compound 3 docks to a distal pocket 28Å from the active site zinc, while DG013 and DG014 bind to the active site. This distal pocket is an allosteric activation site, and allostery is mediated by stabilizing the closed state. I also identified an intermediate step in substrate binding where helix 4a becomes ordered while ERAP1 maintains an open conformation. Helix 4a then rotates and engages substrate when ERAP1 closes. The nonsynonymous SNP rs30187 at position 528 (Lys/Arg) subtly alters ERAP1 activity in vitro and correlates with disease incidence. Position 528 forms a conformation-dependent electrostatic interaction with Glu913 in the closed structure. The energetic contribution of this interaction is stronger for Lys528 than Arg528. Inhibitors that induce closing are more potent for Lys528 than Arg528. I propose a model where either helix 4a stabilization or allosteric site occupancy shift the conformational equilibrium towards a closed state, while substitution at position 528 alters the opening rate.

In this work, we studied three proteins from three different organisms by EPR spectroscopy: NpHtrII is part of the phototaxis system found in halophilic archaea, ChR2 is a cation-selective channel isolated from a green alga, and HsaCCA is an enzyme involved in the protein biosynthesis of humans. The goal was to identify characteristic conformations and dynamics in each of the studied proteins that were linked to their specific functions. The scientific disciplines employed in this work include biochemistry (site-directed spin-labeling), bioinformatics (data analysis, molecular modeling), informatics (device control, software development), molecular biology (mutagenesis, transformation, heterologous protein expression, protein purification, protein characterization), and physics (EPR spectroscopy, optical spectroscopy, experimental assembly). The experimental results show a strong interdependence between protein structure, conformers, dynamics, and function. Hydrogen bonds, although being a transient electrostatic attraction between polar molecules, are the key molecular interactions required for the conservation of protein functionality: Hydrogen bond networks in NpSRII and ChR2 stabilize the helix bundles, and hydrogen bond networks in HsaCCA mediate interdomain flexibility. However, the resulting structural alterations observed in our proteins manifest on a much larger scale: We have detected changes in the protein backbone mobility of the HAMP2 domain in NpHtrII after signaling. We have discovered the TMH B movement in ChR2 accompanying channel opening. And we have documented a substrate-dependent motion of the head domain in HsaCCA during catalysis.

Proteins are composed of a unique sequence of amino acids, whose order guides a protein to adopt its particular fold and perform a specific function. It has been shown that a protein's 3-dimensional structure is embedded within its primary sequence. The problem that remains elusive to biochemists is how a protein's primary sequence directs the folding to adopt such a specific conformation. In an attempt to gain a better understanding of protein folding, my research tests a novel model of protein packing using protein design. The model defines the knob-socket construct as the fundamental unit of packing within protein structure. The knob-socket model characterizes packing specificity in terms of amino acid preferences for sockets in different environments: sockets filled with a knob are involved in inter-helical interactions and free sockets are involved in intra-helical interactions. Equipped with this knowledge, I sought to design a unique protein, Ksα1.1, completely de novo. The sequence was selected to induce helix formation with a predefined tertiary packing interface. Circular dichroism showed that Ksα1.1 formed α-helical secondary structure as intended. The nuclear magnetic resonance studies demonstrated formation of a high order oligomer with increased protein concentration. These results and analysis prove that the knob-socket model is a predictive model for all α-helical protein packing. More importantly, the knob-socket model introduces a new protein design method that can potentially hold a solution to the folding problem.

Undergraduate cellists from diverse pedagogical backgrounds with various levels of technical proficiency often struggle to adapt to the demanding performance standards of the university environment. Frequently, these challenges are caused by technical concepts that have been incorrectly learned or unaddressed in the cellist's previous educational experience. Designed for university cello professors and undergraduate cello students, this study is a remedial and enhancement technical method for developing efficient performance habits; it presents a synthesis of cello performing ideologies, comprehensive technical analysis, physical conformation solutions, original sequential exercises, practice models, and recommendations for supporting literature. The method provides a structured path to expressive mastery and artistic freedom in performing advanced cello repertoire.

A new district is going to be built in the center of Västerås. The area is an old industrial area and the buildings in this area are considered as an cultural heritage environment. When Västerås Stad is planning for development in the central parts of Västerås there are a number of rules to adhere to. For example, Västerås Stad wants the new buildings to incorporate the existing buildings. They want, that an, urban environment is created and that buildings that are already built is telling a story and that history should be preserved. The design program, authored by the City of Västerås and the other stakeholders, has in Kopparlunden Syd, created a clear change in functions and buildings. The district has a clear vision of the relationship between the residents and the city's desired functions. The construction of a vibrant urban environment should be seen as fulfilled. This essay will look how Västerås Stads guidelines for cultural heritage is used in this new district and it will also look at how the design plan for the area will work.Kopparlunden Syd is the first stage of three developments and in case the other two stages follow the same line, Västerås, will receive a much-needed addition of a district with several functions as well as an architectural mix between old and new. If the design program is taking care of other interests is more questionable. The cultural heritage risks being lost among all the new buildings and functions for relaxation and leisure activities are minimal. The district has a tendency to become a transport route through or to once home. It is clear that rules are built in if you look at the design program when it comes to the functions of the buildings. Relationship between the cultural heritage and the vision is fulfilled when it comes to rules to relate to. But the cultural heritage is more than just rules to be followed. The legacy is history. The heritage should be highlight and that the district should not distort the cultural heritage environment.

No description available.

No description available.

No description available.

No description available.

No description available.