Evolutionary Relationships Beyond Fold Boundaries

The comparative study of protein sequences and structures is traditionally used to better understand protein fold evolution. The insights we gain from our evolutionary analyses are applied in protein design projects. At the same time, we engineer proteins to test evolutionary assumptions; thus, we establish a feedback loop between both aspects of protein science. First, we compared Profile Hidden Markov Models, state of the art tools for homology detection, that represent all structures that adopt the (βα)8-barrel and the flavodoxin-like fold to discover an evolutionary relationship between these basic structural forms. Moreover, we located the region of the sequence space where both folds are most closely related. Having found this interface, we performed remote homologous searches and protein clustering to find sequences with intermediate features between the (βα)8-barrel and the flavodoxin-like fold. We determined the x-ray crystal structure of one of these sequences to learn possible scenarios of fold change during the evolution of these ancestral structures. The intermediate sequence, named NTM0182, displayed features towards both folds. Moreover, and by structurally superimposing the three structures, we found classical evidences of homology among the three folds: high sequence identity over long aligned fragments. Our approach then starts by using very sensitive novel tools for homology detection (probability scores), to find intermediary links that provide classical evidences of common ancestry (high sequence identity). Next, we extended the Profile Hidden Markov Model comparisons to include all folds classified as α/β in the Structural Classification of Proteins (SCOP). Our comparisons showed that high scoring pairwise alignments are correlated with high local structural similarities between different folds. This observation inspired us to look for interchangeable sub-domain size protein fragments, related by sequence and structure, to build chimeric proteins and mimic protein fold evolution. Our global comparisons revealed that both, the flavodoxin-like and the (βα)8-barrel folds were related to Periplasmic binding protein-like I proteins. We then employed sequence comparisons, structural superpositions, homology modeling and computational assessments to engineer a novel chimeric protein by fusing a flavodoxin-like fold protein into a PBP-like scaffold. The chimera turned out to be a well-folded protein with native-like properties. Thus, our research allowed us to gain evolutionary insights that are applied to protein engineering. In this respect, we establish a feedback loop between fold evolution and protein engineering. We finally contribute to an emergent vision where proteins from different folds are evolutionary related

Protein Structure and Enzyme Catalysis: Knowledge-Based Protein Loop Prediction and Ab Initio Equilibrium Constant Estimation

Prediction methods in the field of bioinformatics can be divided into ab initio and knowledge-based methods. The work in this thesis investigates the importance of anchor group positioning in knowledge-based protein loop prediction as well as the ab initio estimation of equilibrium constants using Density Functional Theory (DFT). The maximum possible prediction quality of knowledge-based loop prediction was examined for 595 insertions and 589 deletions with respect to gap length, fragment length, amino acid type, secondary structure and relative solvent accessibility while applying all possible anchor group positions for the fitting of loops between 3 and 12 residues in length. It was possible to predict 74.3 % of insertions and 83.7 % of deletions within an RMS deviation of < 1.5 Å between template and target structure using a knowledge-based fragment databank based on structures of the Protein Databank (PDB). The analysis showed that the importance of anchor group positioning increases with gap length and that medium fragments with lengths between 5-8 residues perform better than shorter or longer fragments. In addition, better predictions were obtained when anchor groups consisted of hydrophobic residues, were located within secondary structures such as helices and beta sheets, or had low relative solvent accessibilities. A test based on targeted anchor group selection using a combination of the above criteria showed an improvement in prediction quality compared to a random selection of anchor groups. Density Functional Theory (DFT) with a b3lyp/6-311g++ (d,p) basis set was used in combination with a preceding molecular mechanics conformational search to estimate the standard transformed Gibbs free energies of reaction (dGr°') for a set of 45 enzyme-catalyzed reactions at standard biochemical conditions (pH 7 and 298.15 K). For reactions from EC group 1 and EC groups 5 and 6, the calculated dGr°' values deviated from the experimental values by an average of 2.49 kcal/mol and 5.50 kcal/mol, respectively. This data was comparable to the values calculated using group contribution method by Mavrovouniotis (Mavrovouniotis, J.Biol.Chem 1991; 266:14440-45), where the mean error was 2.76 kcal/mol for reactions from EC group 1 and 4.76 kcal/mol for reactions from EC groups 5 and 6. The mean error for the entire set of reactions was 10.30 kcal/mol. These results are very promising, considering that purely structural information was used, and the method can be improved by further optimization

Structural analysis of the protein shell of the propanediol utilisation metabolosome

PhDPropanediol metabolism occurs within a proteinaceous organelle in several bacterial species including Citrobacter freundii and Lactobacillus reuteri. The propanediol utilisation (Pdu) microcompartment shell is built from thousands of hexagonal shaped protein oligomers made from seven different types of protein subunits. In this Thesis, I investigate and analyse the structure and assembly of the bacterial microcompartment shell proteins. One of the shell proteins characterised in this work, PduT, has a tandem canonical bacterial microcompartment (BMC) repeat within the subunit and forms trimers with pseudo-hexagonal symmetry. This trimeric assembly forms a flat approximately hexagonally shaped disc with a central pore that is suitable for binding a 4Fe-4S cluster. The essentially cubic shaped 4Fe-4S cluster conforms to the threefold symmetry of the trimer with one free iron, the role of which could be to supply electrons to an associated microcompartment enzyme, PduS. The major shell protein PduB has a tandem permuted BMC repeat within the subunit and also forms trimers with pseudo-hexagonal symmetry. This shell protein closely resembles its homologous counterpart, EtuB; both possess three small pores formed within the subunits rather than a single pore at the centre of the pseudo-hexameric disc. The crystal structure of PduB provides insights into how substrates such as glycerol are able to use these pores as substrate channels. PduB appears to be able to pack within a sheet of PduA molecules, suggesting how the facet of the shell may be assembled. The higher order packing of shell proteins was investigated using PduA. Residues important for the packing of molecules into sheets were mutated and the effects on crystal morphology and on the shape of structures formed within the bacterial cell were assessed. PduA appears to assemble into straws in the bacterial cell and mutation of these residues has a profound influence on the structures produced.School of Biological and Chemical Sciences graduate teaching schem

Characterization of proteorhodopsin 2D crystals by electron microscopy and solid state nuclear magnetic resonance

Proteorhodopsin (PR) originally isolated from uncultivated &#947;-Proteobacterium as a result of biodiversity screens, is highly abundant ocean wide. PR, a Type I retinal binding protein with 26% sequence identity, is a bacterial homologue of Bacteriorhodopsin (BR). The members within this family share about 78% of sequence identity and display a 40 nm difference in the absorption spectra. This property of the PR family members provides an excellent model system for understanding the mechanism of spectral tuning. Functionally PR is a photoactive proton pump and is suggested to exhibit a pH dependent vectorality of proton transfer. This raises questions about its potential role as pH dependent regulator. The abundance of PR in huge numbers within the cell, its widespread distribution ocean wide at different depths hints towards the involvement of PR in utilization of solar energy, energy metabolism and carbon recycling in the Sea. Contrary to BR, which is known to be a natural 2D crystal, no such information is available for PR til date. Neither its functional mechanism nor its 3D structure has been resolved so far. This PhD project is an attempt to gain a deeper insight so as to understand structural and functional characterization of PR. The approach combines the potentials of 2D crystallography, Atomic Force Microscopy and Solid State NMR techniques for characterization of this protein. Wide range of crystalline conditions was obtained as a result of 2D crystallization screens. This hints towards dominant protein protein interactions. Considering the high number of PR molecules reported per cell, it is likely that driven by such interactions, the protein has a native dense packing in the environment. The projection map represented low resolution of these crystals but suggested a donut shape oligomeric arrangement of protein in a hexagonal lattice with unit cell size of 87Å*87Å. Preliminary FTIR measurements indicated that the crystalline environment does not obstruct the photocycle of PR and K as well as M intermediate states could be identified. Single molecule force spectroscopy and atomic force microscopy on these 2D crystals was used to probe further information about the oligomeric state and nature of unfolding. The data revealed that protein predominantly exists as hexamers in crystalline as well as densely reconstituted regions but a small percentage of pentamers is also observed. The unfolding mechanism was similar to the other relatively well-characterized members of rhodopsin family. A good correlation of the atomic force microscopy and the electron microscopy data was achieved. Solid State NMR of the isotopically labeled 2D crystalline preparations using uniformly and selectively labeling schemes, allowed to obtain high quality SSNMR spectra with typical 15N line width in the range of 0.6-1.2 ppm. The measured 15N chemical shift value of the Schiff base in the 2D crystalline form was observed to be similar to the Schiff base chemical shift values for the functionally active reconstituted samples. This provides an indirect evidence for the active functionality of the protein and hence the folding. The first 15N assignment has been achieved for the Tryptophan with the help of Rotational Echo Double Resonance experiments. The 2D Cross Polarization Lee Goldberg measurements reflect the dynamic state of the protein inspite of restricted mobility in the crystalline state. The behavior of lipids as measured by 31P from the lipid head group showed that the lipids are not tightly bound to the protein but behave more like the lipid bilayer. The 13C-13C homonulear correlation experiments with optimized mixing time based on build up curve analysis, suggest that it is possible to observe individual resonances as seen in case of glutamic acid. The signal to noise was good enough to record a decent spectrum in a feasible period. The selective unlabeling is an efficient method for reduction in the spectral overlap. However, more efficient labeling schemes are required for further characterization. The present spectral resolution is good for individual amino acid investigation but for uniformly labeled samples, further improvement is required.Proteorhodopsin (PR) wurde ursprünglich aus nicht kultivierten &#947;-Proteobakterium isoliert und ist in großen Mengen in den Ozeanen enthalten. PR ist wie sein homolog Bakteriorhodopsin (BR) ein TypI Retinal Bindeprotein und die Sequenzen sind zu 26% identisch. Innerhalb der PR Familie haben die Mitglieder eine Sequenzhomologie zu ungefähr 78% und zeigen einen Unterschied von 40 nm im absorptions spektrum. Diese Eigenschaft bietet ein gutes Modelsystem um zu verstehen durch welchen Mechanismus das Absorptionsspektrum moduliert wird. PR ist ein photoaktive Protonenpumpe und es wird angenommen, dass die Richtung des Protonentransfers vom pH-wert abhängt, was auf eine Rolle als ein pH abhängiger Regulator hindeutet. Da PR sowohl in der Zelle in hoher Zahl, als auch in den Ozeanen in unterschiedlichen Tiefen weit verbreitet ist, wird angenommen, dass PR bei der Verwertung von Sonnenlicht, im Energiestoffwechsel und beim Kohlenstoffumsatz beteiligt ist. Im Gegensatz zu BR, welches bekannterweise 2D Kristalle bildet, ist etwas vergleichbares für PR bis heute nicht bekannt. Weder der Mechanismus von PR noch seine 3D Struktur sind bisher gelöst. Die vorliegende Doktorarbeit versucht offene Punkte zum Mechanismus und zur Struktur von PR zu klären. Für die Charakterisierung werden 2D Kristallographie, "Atomic Force Microscopy" und Festkörper NMR verwendet. Für die Bildung von 2D Kristallen konnte eine große Auswahl an Kristallisationbedingungen ermittelt werden, was auf deutliche Protein Protein Wechselwirkungen hindeutet. Zieht man die hohe Zahl an PR Molekülen pro zelle in betracht, ist es wahrscheinlich, dass durch diese Interaktionen auch in der natürlichen Membran eine dichte Packung der Proteine auftritt. Elektronenmikroskopische Aufnahmen mit geringer Auflösung deuten auf eine ringförmige Anordnung der Proteine in einem hexagonalen Gitter mit einer Einheitszelle von 87Å * 87Å. Vorläufige FTIR Messungen deuten darauf hin, dass diese Anordnung den Photozyklus nicht behindert und sowohl K als auch M Zustand konnten identifiziert werden. Um weitere Informationen über den Oligomerisierungszustand der 2D Kristalle zu gewinnen wurden Einzelmolekül - und Rasterkraft Mikroskopie durchgeführt. Hierbei zeigte sich, dass das Protein in kristallinen und dicht rekonstituierten Regionen überwiegend als Hexamer vorliegt. Daneben kann zu einem geringen Anteil auch ein pentamerer Zustand beobachtet werden. Der Mechanismus der Proteinentfaltung war vergleichbar zu anderen, besser untersuchten Mitgliedern der Rhodopsinfamilie. Zwischen den Daten aus der "Atomic Force Microscopy" und der Elektronenmikroskopie zeigt sich eine gute Korrelation. Festkörper NMR an vollständig und selektiv markierten 2D Kristallen ergaben Spektren mit einer typischen 15N Linienbreite von 0,6 bis 1,2 ppm. Die 15N chemische Verschiebung der Schiffschen Base hat im Kristall den gleichen Wert wie funktional aktiv rekonstitutierte Proben, was indirekt die Funktionalität und die korrekte Faltung bestätigt. Die Zuordnung der 15N Signale für Tryptophan wurde durch "Rotational Echo Double Resonance" Experimente vorgenommen. 2D kreuzpolarisation Lee Goldburg Messungen zeigen den dynamischen Zustand des Proteins trotz der eingeschränkten Mobilität im kristallinen Zustand. Das Verhalten der Lipide wurde mit 31P messungen der Lipidkopfgruppe untersucht und zeigt, dass diese nicht fest gebunden sind, sondern sich mehr wie in einer Lipiddoppelschicht verhalten. Für 13C-13C homonukleare korrelations Experimente wurde die Mischzeit durch die Analyse von Aufbaukurven optimiert. Diese Versuche deuten darauf hin, dass es möglich ist einzelne Resonanzen aufzulösen, wie im Fall des Glutamat gezeigt mit einem gutem Signal zu Rauschen Verhältnis. Selektives "unlabeling" ist eine effizente Methode um die Ueberlappung der Signal zu reduzieren. Darüberhinaus sind für eine weitere Chrakterisisierung effizentere Markierungsschemata notwendig. Die bisherige spektrale Auflösung ist gut genug für die Untersuchung einzelner Aminosäuren, für vollständig markierte Proben sind weitere Verbesserungen notwendig

Hydrogen bonding and the stability of the polypeptide backbone

The tertiary structures of globular proteins are crucial in determining reactivity and specificity as biological catalysts and signalling systems. The rules determining the final fold of a protein are still unknown, but some progress has been made in defining tertiary structure in terms of the secondary structure, the conformation of the polypeptide chain. Perhaps surprisingly, not all of the conformational properties of this backbone are known, and several new approaches to studying these are described. Most studies of peptide structure have focused on hydrogen bonding, and this is used as a starting point for this study. Different descriptions of the hydrogen bond, from geometric rules to ab initio calculations, are considered, and an approach based on analysing contributions of individual polar groups to the potential energy using semi empirical Lennard-Jones calculations is chosen on grounds of accuracy, flexibility, and ease of calculation. Using this approach, it is shown that electrostatic interactions between main chain atoms stabilise the right handed twist found in Beta-strands and similar interactions between main-chain atoms not hydrogen bonded to each other influence the geometries of hydrogen bonds in Alpha-helices and Beta-sheets. A role for water and tertiary hydrogen bonds in determining backbone conformation is suggested. The same technique makes it possible to investigate interatomic repulsions as well as attractions. A detailed analysis of the attractions and repulsions in an idealised polypeptide explains many of the features of helical structures in proteins, and suggests a hitherto unexpected directional helix forming pathway, which is supported by a range of kinetic and structural data. Software for automated searching of a hydrogen bond database is developed, and used to identify hydrogen bonded rings formed by amide side chains and main chain peptides. Integrating the database with novel visualisation techniques allows a previously unidentified property of beta sheets, the hydrophobic ridge, to be detected. A range of different computational approaches was used surging this research, from molecular modelling to database searching. Several pieces of software were developed, and these are described together with some observations about the types of software and working environments which were found to be useful in structural biochemistry, and what types of software technology could be developed to make this task easier

Structure and dynamics of membrane peptides from solid-state NMR

Solid-state NMR is among the most important analytical techniques to provide atomic-level structural and dynamic information of chemical and biological systems. Due to the insoluble and non-crystalline nature of most membrane peptides and proteins, SSNMR is particularly powerful to investigate their conformations, dynamics, domain assembly, oliogmerization, and the characteristic structural properties in lipid bilayers including insertion orientation and depth, residue-lipid interaction, and membrane per-turbation. Our research is to collect structural and dynamic information and correlate it with biological functions to elucidate the structure-bioactivity relation. In my PhD pro-jects, we have successfully applied various SSNMR techniques to study many interesting membrane peptides including the cell-penetrating peptide (CPP), antimicrobial peptides (AMP), antimicrobial oligomer (AMO), gating helix of K+ channel (KvAP) and trans-membrane 1H channel of influenza M2 protein (M2TM). We also developed a novel paramagnetic-ion-membrane bound paramagnetic relaxation enhancement (PRE) method to provide quantitative long-range distance constrain (~20 y) in membrane-active bio-systems and applied the method to obtain high-resolution residue-specific insertion depth of two membrane peptides, penetratin and M2TM. One main category of my research topics is the cationic membrane peptide. On the one hand, phospholipid membranes have highly hydrophobic interiors that cannot accommodate charged species, while on the other hand, cationic peptides need to insert or translocate across the membrane to conduct biological functions. So, we are motivated to uncover the structural basis of the membrane insertion and translocation. With this motivation, we have studied two kinds of cationic bio-macromolecules, including CPP and AMP. We have experimentally proved that all these Arg-rich peptides generally have strong guanidinium-phosphate interaction with the phospholipids. This charge-charge interaction causes headgroup reorientation and allows the peptide to insert. For CPPs, the guanidinium-phosphate ion pair helps to stabilize the unstructured peptide in the membrane-water interface. The observed peptide-water interaction further minimizes the peptide polarity and makes it more membrane-soluble. We find that two representative CPPs, penetratin and TAT, have highly dynamic and plastic conformations, proposed to facilitate the movement within the membrane. In the penetratin study, the one-side Mn2+-bound PRE method has been developed and applied to study the pep-tide-concentration dependent insertion depth and symmetry in the outer and inner leaflets of the POPC/POPG bilayer. Another important kind of cationic membrane peptides is AMP. Taking PG-1 and its charge reduced mutant IB484 as model AMPs, we have stud-ied the antimicrobial mechanism, and for the first time, provided high-resolution struc-tural information to elucidate the bacterial Gram-selectivity. We find that the interaction manifests the manner of peptide insertion in terms of orientation and depth, which in turn determined the antimicrobial ability in gram positive and negative bacterial membranes. The antimicrobial mechanism of a guanidinium-rich AMO, PMX30016, has also been investigated. The finding of drug-concentration dependant lipid 31P CSA change and the fast uniaxial motion in the interfacial membrane region suggest a subtle and combined antimcicrobial mechanism of membrane potential perturbation and in-plane disruption. Another category of my research topics is the transmembrane ion-conductive channel study, including the gating mechanism of a K+ channel (KvAP) and the blocking mechanism of the M2TM 1H channel by the metal ion inhibitor (Cu2+). We have deter-mined the topology of an isolated gating helix (S4) of KvAP and compared the orientation with that of an intact K+ channel, Kv1.2-Kv2.1 paddle chimera. The identical tilted and rotational angles of the S4 helix in the isolated form and intact protein, and the observed interaction suggest the channel gating might be manifested by the pep-tide-lipid interaction rather than the interaction among different helical domains. Finally, we applied PRE techniques to study the Cu2+-inhibited M2TM channel and obtained high resolution Cu2+ binding structure and long-range distance constraints for the binding structure refinement

Synthetic biology approaches to direct recombinant protein filament assembly

The design of self-assembling modular nanoscale devices from biological materials has long been a goal of synthetic biology. Filamentous protein assemblies have been investigated as modular scaffolds that can be functionalised through genetic or chemical fusion of the protein subunits to other molecules. The bioengineer’s toolbox is replete with diverse natural protein filaments, with each being suited to applications for different functions and at different scales. However, the length distribution inherent within filamentous protein assemblies has precluded their application for precise nanoscale designs. Efforts to control the length of protein filaments have included the application of capping proteins, which prevent further elongation when incorporated into an assembling filament. To extend and perfect this strategy, immobilising capping proteins within a defined cavity, where they would serve as nucleation sites for filament assembly, would enable precise control over the length of the resulting filament. To create such a confined space, the predictable nature of Watson-Crick base pairing could be harnessed to design and produce nanoscale moulds using DNA origami. Within this thesis, we addressed several challenges in protein and DNA engineering that must be overcome to achieve this vision. In the first results chapter, an existing capping protein was converted into a split-protein system with the vision of directing protein filament nucleation. In the second results chapter, novel capping proteins for two thermostable protein filaments were designed, produced, and tested. In the third results chapter, a nanoscale mould was constructed using DNA origami. Together, the results presented in this thesis suggest that guiding protein filament assembly with nucleation proteins is a plausible means of controlling filament length

Statistical Mechanics of Conformational Transitions in Biopolymers.

Recently extended theoretical methods and existent experimental methods have been employed for the determination of conformational-dependent properties of polymers of biological interest like synthetic polyamino acids, natural polypeptides and proteins. Calculation of theoretical properties, like for example average helical content and average helical length, as derived from the configuration partition function has been achieved by making use of the Rotational Isomeric State approximation through modifications and innovations of a nearest-neighbor interaction version of the Zimm-Bragg model of helix-coil transitions as presented in a matrix format suitable for use in an electronic computer. The scope of the theory allows for the numerical evaluation of properties devoid of experimental counterparts, like for example helix initiation and helix propagation probabilities. Application was made as well of various theoretical formalisms (developed elsewhere) to the analysis of the thermal denaturation profiles of a two chain coiled-coil crosslinked (alpha)-tropomyosin dimer. In addition, the conformations of a synthetic copolymer of L-lysine and L-glutamic acid, and of the natural pituitary opioid peptide dynorphine-(1-13) have been determined in the presence of various detergents and lipids. This has been achieved by means of ultraviolet circular dichroism spectroscopic methods

Structural Changes and Chain Mobility During Processing of Bloodmeal-Based Thermoplastics

The purpose of this study was to use concepts from classical polymer physics to develop a fundamental understanding of the interdependent relationship between structure, properties and processing in NovateinTM Thermoplastic Protein (NTP). NTP is produced from bloodmeal, a by-product of the meat industry which is 95% protein, by extruding with sodium sulphite, urea, sodium dodecyl sulphate, water and tri-ethylene glycol. Dynamic mechanical thermal analysis (DMA) and differential scanning calorimetry (DSC) were used to investigate thermal transitions and chain relaxation, accompanied by synchrotron based Fourier transform infrared spectroscopy (FT-IR) to investigate chain architecture and structural changes. Extrusion, injection moulding and mechanical testing were used to investigate macroscopic properties, such as processability and mechanical properties. Material pocket DMA enabled detection of glass transition temperatures not only for moulded NTP test pieces, but also for each processing step: bloodmeal, NTP prior to extrusion, extruded NTP, and conditioned NTP. The pockets increased resolution for injection moulded and conditioned samples, revealing multiple transitions which indicated the presence of more than one phase. Spatially resolved FT-IR experiments were used to characterise the relative content and distribution of protein secondary structures in bloodmeal and NTP after each processing step. Increased chain mobility was observed due to the additives used, and also drastic structural rearrangement consistent with consolidation into a thermoplastic material after extrusion. Viscoelastic phenomena such as creep and stress relaxation further confirmed processed NTP was a consolidated thermoplastic, exhibiting time dependent behaviour characteristic of thermoplastic polymers. The thermal properties of NTP suggested an underlying semi-crystalline structure, with protein secondary structures such as α-helices and β-sheets constraining motion in the amorphous phase, contributing to a broad glass transition region. These structures do not melt at typical temperatures encountered during extrusion processing, but are dispersed more evenly throughout an amorphous matrix. Other than plasticisation, strong hydrogen bonding interactions stabilising secondary structures were the dominant influence on mechanical properties of the processed NTP. When TEG was included as a plasticiser, the degree of crystallinity decreased, and the fraction of randomly coiled protein chains was greater at all stages of processing compared to NTP without TEG. TEG competes with protein side groups for hydrogen bonding sites on the protein backbone, reducing secondary structure formation. β-sheets increased when NTP was heated in the absence of shear, displacing TEG and causing it to migrate out of the particles. The short residence time and presence of shear and mixing during extrusion and injection moulding prevented migration during processing, but an overall increase in β-sheet content was still observed after these processing steps. The relationship between structure, properties and processing of NTP is therefore characterised by its semi crystalline nature, in which additives overcome protein-protein interactions in the amorphous phase enabling β-sheet dispersion throughout the material during processing. Future attempts at modifying properties should be informed by this understanding