This work describes a method of the secondary structure prediction of proteins based on cellular automaton (CA) model - CASSP. Optimal model and CA transition rule parameters are acquired by evolutionary algorithm. Prediction model uses only statistical characteristics of amino acids, so its prediction is fast. Achieved results was compared with results of other tools for this purpose. Prediction cooperation with a existing tool PSIPRED was also tested. It didn't succeed to beat this existing tool, but partial improvement was achieved in prediction of only alpha-helix secondary structure motif, what can be helful if we need the best prediction of alpha-helices. It was developed also a web interface of designed system.

Some proteins require presence of their specific ligand, cofactor or prosthetic group for their activity. Binding of this specific molecule can cause conformational changes which permit to perform their function. In some occasions the identification of conformational changes could be really challenging task. In this thesis we describe the novel approach for monitoring structural changes in proteins using chemical cross-linking and high resolution mass spectrometry and its application on model calmodulin system. It is demonstrated that analysis using isotope-labelled cross-linking agents enabled us to get insight into the structural rearrangement caused by presence or absence of the protein ligand. However, it is shown that the method has potential drawback due to limited enzymatic proteolysis. The novel approach that also makes it possible to quantify the changes in protein structure was used together with other methods for characterization of the neutral trehalase Nth1 in complex with Bmh1 protein (yeast isoform of protein 14-3-3). The results revealed that Bmh1 induce structural rearrangement of Nth1 molecule with changes within the EF- hand like motif which is essential for the activation process.

The inhibition of HIV-1 protease plays an important role in combating HIV. Nine HIV-1 protease inhibitors have been succesfully marketed for the treatment since 1995. However, their efficiencies decrease due to the resistance development. More potent compounds with novel structural motifs and mechanisms of action are therefore still needed. Several inhibitory compounds have been reported to bind to the protease at the loci different from the active site. Interestingly, darunavir, which is the last approved inhibitor with supposedly competitive mode of action, was also suggested to bind to the flap region of the protease. Two studies discussed this alternative binding mode based on the X-ray structural and kinetic analysis, respectively. Nevertheless, it is questionable, if such a mechanism is relevant also in physiological conditions or if it is only an artifact of crystallization. Another study provided a strong evidence for the alternative binding of darunavir to highly mutated HIV-1 protease. Based on thermodynamic analysis, it was shown that two molecules of darunavir bind to the protease dimer. Surprisingly, this observation was not confirmed by the X-ray structure analysis since the inhibitor was bound only within the active site. However, this protease variant was employed in further...

The most common structural platform in biology, the βα-repeat classes of proteins, are represented by the (βα)8TIM barrel topology and the α/β/α sandwich, CheY-like topology. Previous studies on the folding mechanisms of several members of these proteins have suggested that the initial event during refolding involves the formation of a kinetically trapped species that at least partially unfolds before the native conformation can be accessed. The simple topologies of these proteins are thought to permit access to locally folded regions that may coalesce in non-native ways to form stable interactions leading to misfolded intermediates. In a pair of TIM barrel proteins, αTS and sIGPS, it has been shown that the core of the off-pathway folding intermediates is comprised of locally connected clusters of isoleucine, leucine and valine (ILV) residues. These clusters of Branched Aliphatic Side Chains (BASiC) have the unique ability to very effectively prevent the penetration of water to the underlying hydrogen bond networks. This property retards hydrogen exchange with solvent, strengthening main chain hydrogen bonds and linking tertiary and secondary structure in a cooperative network of interactions. This property would also promote the rapid formation of collapsed species during refolding. From this viewpoint, the locally connected topology and the appropriate distribution of ILV residues in the sequence can modulate the energy landscapes of TIM barrel proteins. Another sequence determinant of protein stability that can significantly alter the structure and stability of TIM barrels is the long-range main chain-side chain hydrogen bond. Three of these interactions have been shown to form the molecular underpinnings for the cooperative access to the native state in αTS. Global analysis results presented in Chapter II and Chapter III, suggest that the off-pathway mechanism is common to three proteins of the CheY-like topology, namely CheY, NT-NtrC and Spo0F. These results are corroborated by Gō-simulations that are able to identify the minimal structure of kinetically trapped species during the refolding of CheY and Spo0F. The extent of transient, premature structure appears to correlate with the number of ILV side chains involved in a large sequence-local cluster that is formed between the central β-sheet and helices α2, α3 and α4. The failure of Gō-simulations to detect off-pathway species during the refolding of NT-NtrC may reflect the smaller number of ILV side chains in its corresponding hydrophobic cluster. In Chapter IV, comparison of the location of large ILV clusters with the hydrogen exchange protected regions in 19 proteins, suggest that clusters of BASiC residues are the primarily determinants of the stability cores of globular proteins. Although the location of the ILV clusters is sufficient to determine a majority of the protected amides in a protein structure, the extent of protection is over predicted by the ILV cluster method. The survey of 71 TIM barrel proteins presented in Chapter V, suggests that a specific type of long-range main chain-side chain hydrogen bond, termed “βα hairpin clamp” is a common feature in the βα-repeat proteins. The location and sequence patterns observed demonstrate an evolutionary signature of the βαβ modules that are the building blocks of several βα-repeat protein families. In summary, the work presented in this thesis recognizes the role of sequence in modulating the folding free energy landscapes of proteins. The formation of off-pathway folding intermediates in three CheY-like proteins and the differences in the proposed extent of structure formed in off-pathway intermediates of these three proteins, suggest that both topology and sequence play important and concerted roles in the folding of proteins. Locally connected ILV can clusters lead to off-pathway traps, whereas the formation of the productive folding path requires the development of long-range nativelike topological features to form the native state. The ability of ILV clusters to link secondary and tertiary structure formation enables them to be at the core of this cooperative folding process. Very good correlations between the locations of ILV clusters and both strong protection against exchange and the positions of folding nuclei for a variety of proteins reported in the literature support the generality of the BASiC hypothesis. Finally, the discovery of a novel pattern of H-bond interactions in the TIM barrel architecture, between the amide hydrogen of a core ILV residue with a polar side chain, bracketing βαβ modules, suggests a means for establishing cooperativity between different types of side chain interactions towards formation of the native structure. See Additional Files for copies of the source code for the global analysis program and the cluster analysis program.

The FYVE domain is a lipid binding domain found in approximately 27 different mammalian proteins. It specifically interacts with the lipid, PI(3)P, which is enriched on early endosomes. Consequently, many FYVE domain-containing proteins localize to the endosome, however the ability of FYVE domains to target to endosomal membranes is variable, despite high sequence conservation. Here we describe the structural requirements necessary for endosomal localization and liposome avidity. As FYVE domains are lipid binding domain, many FYVE domain-containing proteins have been implicated in membrane trafficking. We performed an RNAi screen of FYVE domain-containing proteins to identify general regulators of endocytosis in Caenorhabditis elegans. In this screen, we identified the EEA1, a known regulator of endocytosis and two novel genes: WDF2 and KIAA1643. Initial characterization of WDF2 suggest that its function is conserved in humans. Of all the FYVE domain-containing proteins, we have been particularly interested in SARA (Smad Anchor for Receptor Activation); a protein implicated in the TGFβ signaling pathway. This protein contains a binding domain for the TGFβ mediated transcription factor, Smad2/3, and a FYVE domain. It was the presence of the FYVE domain, an endosomal targeting signal, in SARA that lead us to hypothesize that endocytosis might be a necessary step in TGFβ signaling. SARA localizes to the early endosome; the TGFβ receptors also internalize into these endosome. When this internalization is prohibited, there is correlative decrease in Smad2/3 phosphorylation, Smad2 nuclear translocation and TGFβ mediated transcription. Overexpression of a dominant negative SARA construct and SARA siRNA oligonucleotides inhibit TGFβ signaling. We conclude that TGFβ receptor signaling to Smad2/3 occurs on the endosome and this signaling requires SARA. Receptor mediated endocytosis has been classically thought of as an important mechanism for attenuating signaling pathways. We have redefined the role of endocytosis to include the necessary and positive regulation of specific signaling pathways. We have also extended our insights into the biological role of the endosome as a compartment specialized for the assembly and propagation of specific extracellular signals.

The high resolution structure of protein molecules and protein-protein complexes is important to investigate their functions. Today, large 3D or 2D crystals are required to obtain protein structures by X-ray crystallography or conventional Electron Microscopy, respectively. However, production of such crystals of good quality is a solely empirical procedure, which relies on screening numerous crystallization conditions. At the same time, multilayered protein crystals are often a by-product of attempts to grow 3D or 2D crystals and could be obtained more easily. So far, multilayered protein crystals have not been used in electron microscopy for structure determination, as the interpretation of an electron diffraction pattern is rather complicated. In this thesis we present the first protein structure bovine liver catalase at 4 Å resolution solved using electron diffraction data from multilayered crystals. 55 diffraction patterns (17 tilt series) were recorded and used for the reconstruction. The tilt geometry of each individual diffraction pattern was determined by a least-squares algorithm or Laue zone analysis to perform spot indexing. The phase problem was solved by molecular replacement. The influence of the missing data cone on the self-rotation function and interpretation of reconstructed map is discussed.

Phosphate is an essential nutrient for all forms of life. Escherichia coli has a PhoR/PhoB two component regulatory system that controls the expression of various genes whose products allow the cell to thrive in low phosphate environments. The signaling mechanism of the PhoR/PhoB system has been studied and the phosphorylation cascade that controls gene expression is well understood. What is still unknown is how PhoR senses the phosphate level of the environment. The PstS, PstC, PstA, PstB, and PhoU proteins play a role in this signal sensing. This work confirms the hypothesis that the PstSCAB complex senses the environmental phosphate and that phosphate signal is passed through PhoU to PhoR. Further, this work characterizes residues important for interaction on PhoU and PhoR and identifies a structural model for interaction. This model points to a potential mechanism for PhoU mediated signaling to PhoR. We tested this model with direct coupling analysis and obtained further confirmation. Further use of these techniques may elucidate more of the interactions necessary for proper phosphate signaling.

Strukturbestämning av proteiner är viktigt för att kunna förstå deras funktion och en snabbt utvecklande metod inom fältet är kryoelektronmikroskopi. Storleksbegränsningar förhindrar en bredare applikation av metoden eftersom små proteiner har för låg signal i förhållande till bakgrund för att kunna visualiseras som enstaka partiklar i elektronmiksoskopibilder. Hypotesen för projektet är att det är möjligt att avbilda väldigt små proteiner och kringgå den konventionella storleksbegränsningen genom att använda ett bärarprotein ((Putrescine Aminotransferase; YgjG) som kopplas till en affibody (Zwt) genom “helical fusion” och sedan binda ett litet målprotein till denna större struktur. Komplexet ska ge en tillräcklig storlek, symmetri och rigiditet för en lyckad elektronmikroskopi av bärare tillsammans med det lilla icke kovalent bundna målproteinet. För att karaktärisera den föreslagna bäraren, genomförs stabilitetstester genom CD, verifiering av inbindning av målproteinet i SPR, renhetsundersökning med SEC och slutligen kryoelektronmikroskopi för att testa konceptet. Det lilla målproteinet som kommer att avbildas i konceptstudien är en annan affibody (Z963), som i så fall skulle vara det minsta proteinet som någonsin har lösts med kryogenelektronmikroskopi. Resultaten visar att den undersökta tetramera-bäraren är väldigt stabil (Tm~ 85 oC) och kan tolerera en affibody-fusion med bibehållen bindning av multipla säten. Proteinet kan uttryckas rekombinant och renas till högt utbyte och bildar tetramerer även med fuserad affibody. De slutgiltiga resultaten från den kryoelektronmikroskopiska analysen inväntas fortfarande, men lovande griddar har skapats och en partikelselektion har gett klara 2-D klasser som också framhäver att det lilla målproteinet har bundit. Sammanfattningsvis har biofysikalisk karaktärisering indikerat att YgjG är en lovande bas för ett “imaging scaffold” och att preliminära enstaka-partikel kryoelektronmikroskopi analyser visar att den föreslagna strategin att undersöka små målproteiner är möjlig. / Determining structures of proteins is important to understand protein functions, and a rapidly evolving technique in this field is cryogen electron microscopy. However, size limitations are preventing wider applications of the technique because small proteins have poor signal to noise ratios and are not possible to distinguish in single-particle images. The hypothesis of this project is that it is possible to image very small proteins, bypassing the conventional size limitations of single-particle cryo-EM, by utilizing a carrier protein-scaffold (Putrescine Aminotransferase; YgjG) connected through helical fusion to an affibody (Zwt) that can bind to a small protein of interest. The complex provides a sufficient size, symmetry, and rigidity for successful electron microscopy also of the non-covalently bound small protein of interest. To characterise the proposed scaffold, thermal stability through CD, binding of target protein in SPR, purity through SEC and experiments towards proof-of-concept in cryo-EM will be performed. The small protein of interest to be imaged in the proof-of-concept setup is another affibody, called Z963, that would be the smallest protein ever solved with cryo-EM. The results show that the investigated tetrameric protein scaffold is a highly stable protein (Tm~85oC) that can tolerate affibody fusion with retained binding function of multiple sites. The protein can be recombinantly expressed and purified in high yield and forms tetramers also when fused to affibody. The cryo-EM results are still pending, but promising grids have been created and in an initial particle selection clear 2-D classes that also reveal the small bound protein of interest have been generated. To conclude, biophysical characterization indicates that YgjG is a promising base structure for an imaging scaffold and preliminary single-particle cryo-EM analyses show that the proposed strategy to investigate structures of small proteins of interest is feasible.

No description available.

No description available.