Tyrosine hydroxylase (TyrH) catalyzes the rate-limiting step in the synthesis of the catecholamine neurotransmitters dopamine, epinephrine, and norepinephrine. Phosphorylation of Ser40 of rat TyrH activates the enzyme by decreasing the affinity for catecholamines. In humans, there are four different TyrH isoforms with varying lengths for the regulatory domain. DOPA and dopamine binding studies were performed on the phosphorylated and unphosphorylated human isoforms. The Kd for DOPA was increased two times upon phosphorylation of hTyrH1, but no change was seen for hTyrH4; the Kd value decreased with the increase in the size of regulatory domain. The small effect on the Kd value for DOPA upon phosphorylation of hTyrH suggests that DOPA does not regulate the activity of hTyrH. Dopamine binds very tightly and upon phosphorylation the affinity for dopamine is decreased. This Kd value decreases with the increase in the length of the regulatory domain. The crystal structures of substrate complexes of the homologous enzyme phenylalanine hydroxylase (PheH) show a large movement of a surface loop (residues 131-155) upon amino acid binding. The corresponding loop residues (175-200) in TyrH play an important role in DOPA formation. This conformational change in TyrH loop was studied with fluorescence anisotropy. Three tryptophan residues in the TyrH, at positions 166, 233, and 372, were mutated to phenylalanine, and Phe184 was mutated to tryptophan. An increase in anisotropy was observed in the presence of phenylalanine and 6-methyl-5-deazatetrahydropterin (6M5DPH4), but the magnitude of the change of anisotropy with 6M5DPH4 was greater than that with phenylalanine. Further characterization of the sole tryptophan in the loop showed a decrease in the amplitude of the local motion only in the presence of 6M5DPH4 alone. The conformational change in wild type TyrH was examined by H/D exchange LC/MS spectroscopy in the presence of the natural ligands. Time-course dependent deuterium incorporation into the loop in the presence of ligands indicated that the pterin alone can induce the conformational change in the loop irrespective of whether iron is reduced or oxidized. From these results, one can conclude that the loop undergoes a conformational change upon pterin binding, making the active site better for amino acid binding.

Calmodulin (CaM) is a highly conserved protein able to bind Ca2+. When Ca2+ is bound the protein can bind and activate further proteins with several individual functions. CaM switches to a more open conformation when Ca2+-bound and is able to do so at a high rate. Little is known about the conformational switches between apo and Ca2+-bound states. A hypothesis suggests that protonation/deprotonation of a histidine side-chain is part of the answer and thus the dynamics of CaM would be pH dependent. This was further investigated in this thesis. Methods to carry out the project included protein expression of isotope labelled CaM-TR2C E140Q, standard protein purification and protein adapted Nuclear Magnetic Resonance (NMR) spectroscopy. The results suggest that CaM-TR2C E140Q is likely to depend on pH and that histidine 107 (H107) may have a central role in the conformational changes observed. At lower pH it was also suggested that CaM-TR2C E140Q obtained a more open conformation with weakened intramolecular interactions and that the tertiary structure of CaM-TR2C E140Q may have been disrupted. / Calmodulin (CaM) är ett, till hög grad konserverat protein med möjlighet att binda in Ca2+. Då Ca2+ är bundet kan proteinet binda och aktivera ytterligare protein med olika enskilda funktioner. CaM byter med hög hastighet till en mer öppen konformation då Ca2+ binder. Lite vetskap finns kring hur konformationsändringarna mellan apo-form och Ca2+-bunden form går till. En hypotes föreslår att protonering/deprotonering av en histidin-sidokedja kan vara en del av svaret och att CaMs dynamik därför bör vara beroende av pH. Detta undersöktes vidare i detta examensarbete. Metoder som användes för att genomföra projektet inkluderar proteinuttryck av isotopinmärkt CaM-TR2C E140Q, standardiserad proteinrening och proteinanpassad kärnmagnetisk resonans (NMR) spektroskopi. Resultaten föreslår att konformationsändringarna av CaM-TR2C E140Q troligen är pH-beroende och att histidin 107 (H107) kan ha en central roll vid dessa ändringar. Vid lägre pH föreslås att CaM-TR2C E140Q antar en mer öppen konformation med försvagade intramolekylära interaktioner och att tertiärstrukturen av CaM-TR2C E140Q kan ha blivit upplöst.

Protein homo-oligomerization is the process through which identical peptides bind together to form higher order complexes. Self-interactions in many cases are constitutive and stable, used as building blocks for biological structures, such as rings, filaments and membranes. Further, homo-oligomerization can also be a regulatory process that influences the proteins' function such as change in transcriptional activities for transcription factors. Innovative methods to measure oligomerization in live cells are needed in order to understand regulation and function of homooligomerization in the native cellular context. This thesis examines the case of the tumor suppressor p53, whose homo-tetramerization greatly influences its activity as a transcription factor. We develop methods to quantify p53's self-interaction in individual living cells and follow it in time after DNA damage. The two methods we developed have complementary qualities and different applications. We first use fluorescent correlation spectroscopy to study the molecular events occurring in the first three hours of the p53 in response to double strand breaks. We find that in the absence of stress p53 is present in a mixture of, monomers, dimers and tetramers. When damage is sensed, oligomerization is rapidly induced and nearly all p53 is found bound in tetramers. We combine our data with a mathematical framework to propose the existence of a dedicated mechanism triggering p53 oligomerization independently of protein stabilization. Next, we use bimolecular fluorescent complementation to probe for tetramerization in the longer timescales of p53's response to ultraviolet radiation. In this context we find that even though the rate of p53 accumulation increases with the dose of radiation, p53 tetramers are formed at a steady rate. We hence propose the existence of an inhibitory mechanism that prevents the oligomerization reaction from following a linear input-output relation. We identify ARC, a known cofactor of p53, as part of this inhibitory mechanism. Downregulation of ARC restore the linear relation between to total and tetrameric p53. Finally, in both experimental setups higher oligomerization lead to an increase in p53 activity, underscoring the connection between regulation of oligomerization and the transcriptional activity of p53 in cancer cells. Collectively, this work emphasizes the importance of precise measurements to investigate the regulation and function of higher order complexes and provides generally applicable methods to quantify homo-oligomerization in live single cells.

Presented in this dissertation are studies of protein dynamics and protein/protein interactions using solution phase hydrogen/deuterium exchange in combination with mass spectrometry (HXMS). In addition, gas phase fragmentation behaviors of deuterated peptides are investigated, with the purpose of increasing resolution of the HXMS. In the area of single protein dynamics, two protein systems are studied. Studies on the cytochrome c2 from Rhodobacter capsulatus indicate its domain stability to be similar to that of the horse heart cytochrome c. Further comparison of the exchange kinetics of the cytochrome c2 in its reduced and oxidized state reveals that the so-called hinge region is destabilized upon oxidation. We also applied a similar approach to investigate the conformational changes of photoactive yellow protein when it is transiently converted from the resting state to the signaling state. The central β-sheet of the protein is shown to be destabilized upon photoisomerization of the double bond in the chromophore. Another equally important question when it comes to understanding how proteins work is the interactions between proteins. To this end, two protein complexes are subjected to studies by solution phase hydrogen deuterium exchange and mass spectrometry. In the case of LexA/RecA interaction, both proteins show decreases in their extents of exchange upon complex formation. The potential binding site in LexA was further mapped to the same region that the protein uses to cleave itself upon interacting with RecA. In the sHSP/MDH system, hydrogen/deuterium exchange experiments revealed regions within sHSP-bound MDH that were significantly protected against exchange under heat denaturing condition, indicative of a partially unfolded state. Hydrogen/deuterium exchange therefore provides a way of probing low resolution protein structure within protein complexes that have a high level of heterogeneity. Finally, the feasibility of increasing resolution of HXMS by gas phase peptide fragmentation is investigated by using a peptide with three prolines near the C-terminus. Our data show that deuterium migration indeed occurs during the collision activated dissociation process. Caution is required when interpreting the MS/MS spectra as a way of pinpointing the exact deuterium distribution within peptides.

Membrane proteins which comprise approximately a third of all proteins are classified for their roles in specific cell signalling, catalysis of metabolic reactions and transport of ions and molecules. One specific membrane protein, called proteorhodopsin (PR) belongs to the family of microbial rhodopsins and functions as a light-driven proton pump. Its lysine residue (Lys231) on helix G forms a Schiff base (C=N) with retinal, its chromophore which photo-isomerizes from the all-trans to the 13-cis form. Photo-isomerization initiates a photocycle, with distinct intermediates (K, M, N, and O). This study tries to emphasize the importance of interactions occurring between the membrane bilayer and PR by examining the kinetics of its photocycle and structure of the retinal chromophore using time-resolved spectroscopy in the visible range and static Raman spectroscopy. Some of the parameters of the membrane that were found to be important include protein to lipid ratio, bilayer thickness, bilayer fluidity and surface charge. The main conclusion is that PR has a very fast photocycle in negatively charged membranes, but a slower photocycle in positively charged ones, as well as in more rigid, thicker membranes. These slower cycles can originate from 1) suppression of conformational changes by the rigid bilayer or dehydration; 2) lack of available protons due to surface charge and 3) impeded isomerization.

Optical modulation has shown the selective and sensitive signal improvement in high background systems in cell imaging; however, cell applications are still limited due to biocompatibility and delivery issues. Fluorescent proteins have a variety of optically accessible states that make them ideal candidates for investigation of modulatability. Combining the optical modulation technique with the biocompatibility of fluorescent proteins is a major advance. This work focuses on evaluation fluorescent proteins and their optical states for modulation, as well demonstrations of cellular imaging. Herein, we evaluate a green fluorescent protein with interesting photophysical properties favorable for optical modulation. Positive for optical modulation, further investigation of the state dictating modulation reveals the presence of a slow component on the order of milliseconds. To better understand the mechanism responsible modulation, blue fluorescent proteins are created to modify the chromophore environment. Extraction of photophysics confirm the alteration timescales of the modulated state. Motivated by the ability to improve imaging and decode hidden dynamics, demodulation of these proteins demonstrates the selective recovery of signal in the presence of high cellular background. The continued investigation of several other fluorescent proteins identifies modulatable proteins across the visible wavelength region. Additionally, solvent environmental factors show varying timescales which, when combined with mutagenesis, suggest a cis/trans isomerization coupled with a proton transfer. This information of the properties dictating optical modulation allows for the engineering of improved modulatable proteins to study cellular dynamics.

Protein structure is not fixed in time, and conformational transitions are the keyto many biological interactions such as enzymatic reactions or signal transduction.Protein dynamics and secondary structure propensities describe these conformationaltransitions, defining how protein structure is likely to evolve in time. Unfortunatelythis kind of information is extremely hard to obtain and the required experimentsare expensive and time consuming.A backbone dynamics and secondary structure propensity predictor that worksfrom sequence only, called DynaMine, has recently been developed. DynaMine addsa new dimension to protein sequences that can, for instance, be exploited to identifyprotein regions involved in specific biological tasks and in protein classification. Thisthesis shows how these predictions can be used to infer emergent properties of pro-teins and how they can highlight hidden evolutionary relationships between remotehomologs.The thesis is divided in 4 parts, corresponding to four different topics in which dy-namics and secondary structure propensities are reported to play a crucial role: thefirst part describes a new pairwise algorithm that uses secondary structure propen-sities and dynamics to align remote homologous proteins. The following three partsdeal with dynamics-related protein emergent behaviours: protein disorder, beta-aggregation and DNA-binding capability in archaea. The results show how backbonedynamics and secondary structure propensities can help improving the prediction ofall the aforementioned subjects. With regard to the identification of DNA-bindingproteins and the prediction of beta-aggregation, experimental validation is also pro-vided and discussed. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

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