| 1. |
- de Marothy, Tuuli Minttu Virkki, 1984-
(author)
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Marginally hydrophobic transmembrane α-helices shaping membrane protein folding
- 2014
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Doctoral thesis (other academic/artistic)abstract
- Most membrane proteins are inserted into the membrane co-translationally utilizing the translocon, which allows a sufficiently long and hydrophobic stretch of amino acids to partition into the membrane. However, X-ray structures of membrane proteins have revealed that some transmembrane helices (TMHs) are surprisingly hydrophilic. These marginally hydrophobic transmembrane helices (mTMH) are not recognized as TMHs by the translocon in the absence of local sequence context.We have studied three native mTMHs, which were previously shown to depend on a subsequent TMH for membrane insertion. Their recognition was not due to specific interactions. Instead, the presence of basic amino acids in their cytoplasmic loop allowed membrane insertion of one of them. In the other two, basic residues are not sufficient unless followed by another, hydrophobic TMH. Post-insertional repositioning are another way to bring hydrophilic residues into the membrane. We show how four long TMHs with hydrophilic residues seen in X-ray structures, are initially inserted as much shorter membrane-embedded segments. Tilting is thus induced after membrane-insertion, probably through tertiary packing interactions within the protein.Aquaporin 1 illustrates how a mTMH can shape membrane protein folding and how repositioning can be important in post-insertional folding. It initially adopts a four-helical intermediate, where mTMH2 and TMH4 are not inserted into the membrane. Consequently, TMH3 is inserted in an inverted orientation. The final conformation with six TMHs is formed by TMH2 and 4 entering the membrane and TMH3 rotating 180°. Based on experimental and computational results, we propose a mechanism for the initial step in the folding of AQP1: A shift of TMH3 out from membrane core allows the preceding regions to enter the membrane, which provides flexibility for TMH3 to re-insert in its correct orientation.
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| 2. |
- Hedin, Linnea E, 1981-
(author)
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Intra- and intermolecular interactions in proteins : Studies of marginally hydrophobic transmembrane alpha-helices and protein-protein interactions.
- 2010
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Doctoral thesis (other academic/artistic)abstract
- Most of the processes in a living cell are carried out by proteins. Depending on the needs of the cell, different proteins will interact and form the molecular machines demanded for the moment. A subset of proteins called integral membrane proteins are responsible for the interchange of matter and information across the biological membrane, the lipid bilayer enveloping and defining the cell. Most of these proteins are co-translationally integrated into the membrane by the Sec translocation machinery. This thesis addresses two questions that have emerged during the last decade. The first concerns membrane proteins: a number of α-helices have been observed to span the membrane in the obtained three-dimensional structures even though these helices are predicted not to be hydrophobic enough to be recognized by the translocon for integration. We show for a number of these marginally hydrophobic protein segments that they indeed do not insert well outside of their native context, but that their local sequence context can improve the level of integration mediated by the translocon. We also find that many of these helices are overlapped by more hydrophobic segments. We propose, supported by experimental results, that the latter are initially integrated into the membrane, followed by post-translational structural rearrangements. Finally, we investigate whether the integration of the marginally hydrophobic TMHs of the lactose permease of Escherichia coli is facilitated by the formation of hairpin structures. However our combined efforts of computational simulations and experimental investigations find no evidence for this. The second question addressed in this thesis is that of the interpretation of the large datasets on which proteins that interact with each other in a cell. We have analyzed the results from several large-scale investigations concerning protein interactions in yeast and draw conclusions regarding the biases, strengths and weaknesses of these datasets and the methods used to obtain them.
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| 3. |
- Hennerdal, Aron, 1981-
(author)
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Application of membrane protein topology prediction
- 2011
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Doctoral thesis (other academic/artistic)abstract
- Membrane proteins often have essential functions in the cell and many are important drug targets, yet only a small fraction of available protein structures are of membrane proteins. Experimental techniques for elucidating membrane protein structures have proven laborious and expensive, opening the field for comparatively inexpensive computational modeling. Topology prediction addresses a sub-problem of structure prediction for α-helical membrane proteins by modeling which parts of the peptide chain are in, and which parts are on either side, of the membrane. This work describes an algorithm for combining the results of several topology prediction methods to increase prediction accuracy and to quantify prediction reliability, and a faster implementation of the algorithm applicable to large-scale genome data. Further, topology prediction is applied, together with other sequence-based methods, to detect duplications in membrane proteins in whole genomes. We find more duplications in the genomes of yeast and E. coli than in human, possibly due to the abundance of nonduplicated GPCRs in human. A gene duplication and subsequent fusion event constitute a likely origin for duplicated proteins, yet only for one superfamily, the AcrB Multidrug Efflux Pump, do we find the duplicated unit in its nonduplicated form. This apparent scarcity of nonduplicated forms is confirmed when extending the study to the whole human genome. Finally, a benchmark study of topology prediction on several comparably large datasets is described. We confirm previous results showing that methods utilizing homology information top the ranking of topology prediction methods. We also see that the separation of membrane proteins from non-membrane proteins has a partially different set of requirements than topology prediction of membrane proteins, and we suggest a pipeline using different methods for these two tasks.
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| 4. |
- Illergård, Kristoffer, 1980-
(author)
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On the effects of structure and function on protein evolution
- 2010
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Doctoral thesis (other academic/artistic)abstract
- Many proteins can be described as working machines that make sure that everything functions in the cell. Their specific molecular functions are largely dependent on their three-dimensional structures, which in turn are mainly predetermined by their linear sequences of amino acid residues. Therefore, there is a relation between the sequence, structure and function of a protein, in which knowledge about the structure is crucial for understanding the functions. The structure is generally difficult to determine experimentally, but should in principle be possible to predict from the sequence by computational methods. The instructions of how to build the linear proteins sequences are copied during cell division and are passed on to successive generations. Although the copying process is a very efficient and accurate system, it does not function correctly on every occasion. Sometimes errors, or mutations can result from the process. These mutations gradually accumulate over time, so that the sequences and thereby also the structures and functions of proteins evolve overtime. This thesis is based on four papers concerning the relationship between function, structure and sequence and how it changes during the evolution of proteins. Paper I shows that the structural change is linearly related to sequence change and that structures are 3 to 10 times more conserved than sequences. In Paper II and Paper III we investigated non-helical structures and polar residues, respectively, positioned in the nonpolar membrane core environment of α-helical membrane proteins. Both types were found to be evolutionary conserved and functionally important. Paper IV includes the development of a method to predict the residues in α-helical membrane proteins that after folding become exposed to the solvent environment.
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| 5. |
- Larsson, Per, 1978-
(author)
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Prediction, modeling, and refinement of protein structure
- 2010
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Doctoral thesis (other academic/artistic)abstract
- Accurate predictions of protein structure are important for understanding many processes in cells. The interactions that govern protein folding and structure are complex, and still far from completely understood. However, progress is being made in many areas. Here, efforts to improve the overall quality of protein structure models are described. From a pure evolutionary perspective, in which proteins are viewed in the light of gradually accumulated mutations on the sequence level, it is shown how information from multiple sources helps to create more accurate models. A very simple but surprisingly accurate method for assigning confidence measures for protein structures is also tested. In contrast to models based on evolution, physics based methods view protein structures as the result of physical interactions between atoms. Newly implemented methods are described that both increase the time-scales accessible for molecular dynamics simulations almost 10-fold, and that to some extent might be able to refine protein structures. Finally, I compare the efficiency and properties of different techniques for protein structure refinement.
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| 6. |
- Moruz, Luminita, 1982-
(author)
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Chromatographic retention time prediction and its applications in mass spectrometry-based proteomics
- 2013
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Doctoral thesis (other academic/artistic)abstract
- Mass spectrometry-based methods are among the most commonly used techniques to characterize proteins in biological samples. With rapid technological developments allowing increasing throughput, thousands of proteins can now be monitored in a matter of hours. However, these advances brought a whole new set of analytical challenges. At the moment, it is no longer possible to rely on human experts to process the data. Instead, accurate computational tools are required.In line with these observations, my research work has involved development of computational methods to facilitate the analysis of mass spectrometry-based experiments. In particular, the projects included in this thesis revolve around the chromatography step of such experiments, where peptides are separated according to their hydrophobicity.The first part of the thesis describes an algorithm to predict retention time from peptide sequences. The method provides more accurate predictions compared to previous approaches, while being easily transferable to other chromatography setups. In addition, it gives equally good predictions for peptides carrying arbitrary posttranslational modifications as for unmodified peptides.The second part of the thesis includes two applications of retention time predictions in the context of mass spectrometry-based proteomics experiments. First, we show how theoretical calculations of masses and retention times can be used to infer proteins in shotgun proteomics experiments. Secondly, we illustrate the use of retention time predictions to calculate optimized gradient functions for reversed-phase liquid chromatography.
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| 7. |
- Sagit, Rauan, 1985-
(author)
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Variation in length of proteins by repeats and disorder regions
- 2013
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Doctoral thesis (other academic/artistic)abstract
- Protein-coding genes evolve together with their genome and acquire changes, some of which affect the length of their protein products. This explains why equivalent proteins from different species can exhibit length differences. Variation in length of proteins during evolution arguably presents a large number of possibilities for improvement and innovation of protein structure and function. In order to contribute to an increased understanding of this process, we have studied variation caused by tandem domain duplications and insertions or deletions of intrinsically disordered residues.The study of two proteins, Nebulin and Filamin, together with a broader study of long repeat proteins (>10 domain repeats), began by confirming that tandem domains evolve by internal duplications. Next, we show that vertebrate Nebulins evolved by duplications of a seven-domain unit, yet the most recent duplications utilized different gene parts as duplication units. However, Filamin exhibits a checkered duplication pattern, indicating that duplications were followed by similarity erosions that were hindered at particular domains due to the presence of equivalent binding motifs. For long repeat proteins, we found that human segmental duplications are over-represented in long repeat genes. Additionally, domains that have formed long repeats achieved this primarily by duplications of two or more domains at a time.The study of homologous protein pairs from the well-characterized eukaryotes nematode, fruit fly and several fungi, demonstrated a link between variation in length and variation in the number of intrinsically disordered residues. Next, insertions and deletions (indels) estimated from HMM-HMM pairwise alignments showed that disordered residues are clearly more frequent among indel than non-indel residues. Additionally, a study of raw length differences showed that more than half of the variation in fungi proteins is composed of disordered residues. Finally, a model of indels and their immediate surroundings suggested that disordered indels occur in already disordered regions rather than in ordered regions.
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| 8. |
- Björklund, Åsa, 1976-
(author)
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Creation of new proteins - domain rearrangements and tandem duplications
- 2010
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Doctoral thesis (other academic/artistic)abstract
- Proteins are modular entities with domains as their building blocks. The domains are recurrent protein fragments with a distinct structure, function and evolutionary history. During evolution, proteins with new functions have been invented through rearrangements as well as differentiation of domains. The focus of this thesis is to gain better understanding of the processes that govern domain rearrangements. In particular, the rearrangements that create long protein domain repeats have been investigated in detail.We estimate that about 65% of the eukaryotic and 40% of the prokaryotic proteins are of the multidomain type. Further, we find that the eukaryotic multidomain proteins are mainly created through insertion of a single domain at the N- or C-terminus. However, domain repeats differ from other domain rearrangements in the aspect that they are created from internal tandem duplications. We show that such duplications often involve several domains simultaneously, and that different repeated domain families show distinct evolutionary patterns. Finally, we have investigated how large repeat regions are created using a specific example; the Actin binding nebulin domain. The analysis reveals several tandem duplications of both single nebulin domains and super repeats of seven nebulins in a number of vertebrates. We see that the duplication breakpoints vary between the species and that multiple duplications of the same region are common.
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| 9. |
- Nilsson, Daniel
(author)
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Insane in the membrane: : Insertion of marginally hyrdophobic transmembrane helices and global analysis of membrane protein topology
- 2014
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Licentiate thesis (other academic/artistic)abstract
- Proteins are responsible for carrying out most of the tasks in a living cell; transcription, translation, replication, movement, catalysis and communication to mention a few. A subset of proteins is the integral membrane proteins, IMPs, which play an important role in governing communication across membranes, whether it is the plasma membrane or an organelle membrane. As IMPs are often responsible for uptake of substances such as hormones and pharmaceuticals they are also very often prime targets in medical research. In order for an IMP to function correctly it must fold and insert into the membrane properly as well as display the correct motifs to its surroundings, on either side of the membrane. IMPs cannot spontaneously insert into the membrane, the insertion is assisted by the Sec-translocon machinery. The Sec-translocon creates a pore in the membrane enabling nascent polypeptides to translocate across the membrane. This thesis will cover both the insertion of IMP segments as well as an evaluation of different approaches on how to investigate the topology of integral membrane proteins.The first question to be addressed is whether there are any specific sequence motifs within the sequence context that can improve the co-translational insertion of a marginally hydrophobic transmembrane helix, mTMH, into the membrane. A mTMH is a protein segment that would not insert by itself into the membrane. It has however been shown that these mTMHs can insert effectively into the membrane using their neighboring helices and loops, referred to as its sequence context, to compensate for the unfavorable insertion of only the mTMH. We show for a number of mTMHs that disrupting the sequence context motifs, usually lowering the ΔG-values for insertion by introducing more hydrophobic residues through substitutions in the sequence context, does not by itself improve the insertion of a given mTMH. It can however be concluded that the positive inside rule is of great importance to improve recognition and co-translational insertion of these mTMHs as it provides an oriental preference of the subsequent helix. This oriental preference will enable the mTMH to insert. This means that the positive inside rule it stronger when followed by a transmembrane helix, at least for the insertion of mTMHs.The second question addressed is that of how to design a method to analyze the topology of membrane proteins in a high-throughput proteomic fashion. In order to extract information on membrane protein topology a protease can be used to degrade the exposed parts of the integral membrane protein, known as shaving. These peptides can then subsequently be degraded and analyzed using MS and bioinformatics. To compare different proteases, we first apply our shaving experiment on two over-expressed proteins and analyze the detected peptides using MS. Secondly; we run the same experiment on non-over-expressed Escherichia coli membrane proteins with known structure. Finally, the results from the above experiments were used to test the accuracy of a number of topology predictors. We can conclude that the use of the protease Thermolysin does show promising results when compared to for instance trypsin. Even though the two proteases show somewhat similar output on the proteins used in this study, Thermolysin does produce fewer peptides originating from the transmembrane region. This is most likely due to the milder, more native like reaction conditions combined with the shorter incubation time used for Thermolysin as compared to trypsin. These properties are believed to greatly improve the output and accuracy when applied on large scale global analysis.
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| 10. |
- Skwark, Marcin J., 1982-
(author)
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Ensemble methods for protein structure prediction
- 2013
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Doctoral thesis (other academic/artistic)abstract
- Proteins play an essential role in virtually all of life's processes. Their function is tightly coupled to the three-dimensional structure they adopt.Solving protein structures experimentally is a complicated, time- and resource-consuming endeavor. With the rapid growth of the amount of protein sequences known, it is very likely that only a small fraction of known proteins will ever have their structures solved experimentally. Recently, computational methods for protein structure prediction have become increasingly accurate and offer a promise for bridging this gap.In this work, we show the ways the rapidly growing amounts of available biological data can be used to improve the accuracy of protein structure prediction. We discuss the use of multiple sources of structural information to improve the quality of predicted models. The methods for assigning the estimated quality scores for predicted models are discussed as well. In particular we present a novel, successful approach to the clustering-based quality assessment, which runs nearly 50 times faster than other methods of comparable accuracy, allowing to tackle much larger problems.Additionally, this thesis discusses the impact the recent breakthroughs in sequencing and the consequent rapid growth of sequence data have on the prediction of residue-residue contacts. We propose a novel methodology, which allows for predicting such contacts with astonishing, previously unheard-of accuracy. These contacts in turn can be used to guide protein modeling, allowing for discovering protein structures that have been unattainable by conventional prediction methods.Finally, a considerable part of this dissertation discusses the community efforts in protein structure prediction, as embodied by CASP (Critical Assessment of protein Structure Prediction) experiments.
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