Combining enzymes with synthetic materials is the new frontier of biocatalysis, materials science, and protein engineering. Enzymes are biological macromolecule catalysts with incredible efficiency and specificity that are desirable for use in a variety of different fields. However, commercial applications have been limited by the stability and reusability of un-altered enzymes. An avenue for overcoming the challenges to harnessing enzyme power is to combine enzymes with materials to create an enzymatically-active material that has enhanced stability and activity. Unfortunately, the catalytic activity of the hybrid material is often lower than that of the enzyme alone. The activity of an enzyme is directly dependent on its structure and dynamics. Therefore, a deeper understanding of enzyme structure and dynamics upon incorporation into materials will provide the data necessary to rationally design enzymatically-active materials with the desired features. This dissertation explores the behavior of a model enzyme, T4 Lysozyme, with two different artificial material systems, metal-organic frameworks and polyethylene glycol. The underlying structural rationale for the behavior is probed using a variety of techniques, notably, Electron Spin Paramagnetic Resonance. Herein, the implications of structural alterations on activity and opportunities for exploitation are discussed. T4 Lysozyme is a perfect model for this study because it has a well characterized structure-activity relationship, thus providing a vast literature understanding which can be pulled from to verify and assist with interpretation of data. The structural basis of enzyme activity alteration in artificial materials can be used to rationally design systems with desired characteristics. After successfully demonstrating the tunability of proteins in artificial materials using T4L as a model, human Cu/Zn superoxide dismutase 1 was chosen for continuing studies due to its importance in diseased states. However, the superoxide dismutase mutant chosen is aggregation prone, which makes it difficult to express recombinantly in large amounts. Therefore, an efficient protocol for producing the superoxide dismutase protein was developed to set the stage for future studies.

Abstract Adenosine receptors are heterotrimeric guanine nucleotide-binding (G protein)-coupled receptors (GPCRs) that mediate the effects of the endogenous agonist adenosine. The adenosine A3 receptor (A3R) is the least explored among the four human adenosine receptor subtype members (A1, A2A, A2B and A3) and it is implicated in both neuroprotective and neurodegenerative effects. During the course of this work, the production of the recombinant human A3R in yeast and insect cells was evaluated and heteromerization between the human adenosine A2A receptor (A2AR) and the dopamine D2 receptor (D2R) was studied. A3R with carboxyl-terminal GFP tag was expressed in the yeast Saccharomyces cerevisiae upto 15 mg per litre of culture. Another yeast Pichia pastoris increased the expression up to 108 mg/L of the same receptor when grown in bioreactors. Despite the very high expression levels, purification of A3R from both yeasts was a daunting task, as the aggregation of the receptor could not be averted. In this study, insect cells have been found out to be more suitable host for A3R expression: 10µg of the monomeric A3R could be purified from one liter of insect cell culture. For successful crystallization thermostability of the A3R was to be improved. This work has demonstrated that insertion of T4L, a fusion protein, in the third intracellular loop of A3R increased the thermostability of the receptor by 10°C. As a next step, the combination of point mutations based on alanine-scanning mutagenesis and a fusion protein approach could be useful to stabilize and further crystallize the A3R. This work has demonstrated that the amounts of A3R expressed in insect cells and the final yield of the receptor isolated by affinity purifications, forms a good basis for the beginning of biochemical characterization Receptor heteromerization is a mechanism used by GPCRs to diversify their signaling properties and functions. The human A2AR and D2R heteromers exist in the GABAergic enkephalinergic neurons. The domains responsible for forming intermolecular contacts were purified from Escherichia coli (E. coli). Using biochemical/biophysical techniques such as native-PAGE and mass spectrometry, It was validated that purified carboxyl-terminus of the A2AR and the 3rd intracellular loop of D2R form heterodimers. The investigation of purified calmodulin protein binding to the 3rd intracellular loop of D2R showed that the protein-protein interactions are calcium dependent. / Tiivistelmä Adenosiinireseptorit kuuluvat G-proteiinikytkeiset reseptorit (GPCR:t) proteiiniperheeseen. Adenosiinireseptorit välittävät endogeenisen ligandinsa adenosiinin vaikutuksia solukalvolta solunsisäisiin signaalijärjestelmiin. Adenosiini A3 reseptori (A3R) on adenosiinireseptorien neljästä alatyypistä (A1, A2A, A2B ja A3) vähiten tutkittu. Aikaisempien tutkimusten perusteella A3 reseptori yhdistetään sekä hermosoluja suojaaviin että rappeuttaviin tapahtumiin. Tässä työssä arvioitiin sekä ihmisen rekombinantti-A3R:n tuottumista hiiva- ja hyönteissoluissa että tutkittiin ihmisen adenosiini A2A reseptorin (A2AR) ja dopamiini D2 reseptorin (D2R) heteromerisoitumista. Rekombinantti A3 reseptori- vihreä fluoresoiva proteiini (GFP) fuusioproteiinia tuotettiin Saccharomyces cerevisiae -hiivassa 15 mg litrassa kasvatusliuosta. Pichia pastoris -hiivakanta taas kasvatti saman reseptorin tuottumista aina 108 mg/l saakka, kun tuotto tehtiin bioreaktorissa. Hyvin korkeasta tuottotasosta huolimattaA3R:n puhdistus hiivasta oli ylitsepääsemätön tehtävä, sillä reseptorin saostumista ei voinut välttää. Työssä havaittiin, että hyönteissolut sopivat paremmin A3R:n tuottoon: noin 10 µg monomeerista A3R:a voitiin puhdistaa litran hyönteissoluviljelmästä. Reseptorin stabiilisuuden lisääminen helpottaa reseptorin biokemiallista ja biofysikaalista karakterisointia. Tässä työssä osoitettiin, että T4L-proteiinin lisääminen A3R:n kolmannen solunsisäisen silmukan paikalle lisää reseptorin lämpöstabiilisuutta 10 °C. Jatkotutkimuksissa voitaisiin käyttää alaniiniskannausmutageneesiin perustuvien pistemutaatioiden ja fuusioproteiinin yhdistelmää A3R:n lisästabilointiin ja kiteytykseen. Tämän työn perusteella määrät, joilla A3R tuottuu hyönteissoluissa ja jotka saadaan eristettyä affiniteettipuhdistuksilla, muodostavat hyvän perustan proteiinin biokemialliselle karakterisoinnille. Reseptorin heteromerisoituminen on GPCR:en käyttämä mekanismi signalointiominaisuuksien ja toimintojen monipuolistamiseksi. Ihmisessä A2AR ja D2R heteromeereja on GABAergisissä enkefalinergisissä hermosoluissa. Molekyylien välisiin kontakteihin osallistuvat domeenit puhdistettiin Escherichia coli (E. coli) -bakteerista. Biokemiallisia ja biofysikaalisia tekniikoita kuten natiivi-PAGE:a ja massaspektrometriaa käyttäen vahvistettiin, että puhdistettu A2AR:n karboksiterminaalinen osa ja D2R:n kolmas solunsisäinen silmukka muodostavat heterodimeereja. Myös tutkittaessa puhdistetun kalmoduliini-proteiinin sitoutumista D2R:n kolmanteen solunsisäiseen silmukkaan osoitettiin proteiini-proteiini -vuorovaikutuksen olevan kalsiumista riippuvainen.