Kinetic stability and temperature adaptation. Observations from a cold adapted subtilisin-like serine protease.

Life on earth is found everywhere where water is found, meaning that life has adapted to extremely varied environments. Thus, protein structures must adapt to a myriad of environmental stressors while maintaining their functional forms. In the case of enzymes, temperature is one of the main evolutionary pressures, affecting both the stability of the structure and the rate of catalysis. One of the solutions Nature has come up with to maintain activity and stability in harsh environments over biological relevant timescales, are kinetically stable proteins. This thesis will outline work carried out on the kinetically stable VPR, a cold active subtilisin-like serine protease and discuss our current understanding of protein kinetic stability, temperature adaptation and our current hypothesis of the molecular interactions contributing to the stability of VPR. The research model that we have used to study these attributes consists of the cold active VPR and its thermostable structural homolog AQUI. The results discussed in this thesis will be on the importance of calcium, the role of prolines in loops, the role of a conserved N-terminal tryptophan residue and lastly primary observations on differences in active site dynamics between VPR and AQUI. A model is proposed of a native structure that unfolds in a highly cooperative manner. This cooperativity can be disrupted, however, by modifying calcium binding of the protein or via mutations that affect how the N-terminus interacts with the rest of the protein. The N-terminus likely acts as a kinetic lock that infers stability to the rest of the structure through many different interactions. Some of these interactions may be strengthened via proline residues, that seemingly act as anchor points that tend to maintain correct orientation between these parts of the protein as thermal energy is increased in the system. Our results give a deeper insight into the nature of the kinetic stability, the importance of cooperativity during unfolding of kinetically stable proteases, synergy between distant parts of the protein through proline mutations and how different calcium binding sites have vastly differing roles. The results provide a solid ground for continuing work in designing enzyme variants with desired stabilities and activities and improve our understanding of kinetically stable systems.The Icelandic Research Fund [grant number 162977-051

Computational Design of Protein Structure and Prediction of Ligand Binding

Proteins perform a tremendous array of finely-tuned functions which are not only critical in living organisms, but can be used for industrial and medical purposes. The ability to rationally design these molecular machines could provide a wealth of opportunities, for example to improve human health and to expand the range and reduce cost of many industrial chemical processes. The modularity of a protein sequence combined with many degrees of structural freedom yield a problem that can frequently be best tackled using computational methods. These computational methods, which include the use of: bioinformatics analysis, molecular dynamics, empirical forcefields, statistical potentials, and machine learning approaches, amongst others, are collectively known as Computational Protein Design (CPD). Here CPD is examined from the perspective of four different goals: successful design of an intended structure, the prediction of folding and unfolding kinetics from structure (kinetic stability in particular), engineering of improved stability, and prediction of binding sites and energetics. A considerable proportion of protein folds, and the majority of the most common folds ("superfolds"), are internally symmetric, suggesting emergence from an ancient repetition event. CPD, an increasingly popular and successful method for generating de novo folded sequences and topologies, suffers from exponential scaling of complexity with protein size. Thus, the overwhelming majority of successful designs are of relatively small proteins (< 100 amino acids). Designing proteins comprised of repeated modular elements allows the design space to be partitioned into more manageable portions. Here, a bioinformatics analysis of a "superfold", the beta-trefoil, demonstrated that formation of a globular fold via repetition was not only an ancient event, but an ongoing means of generating diverse and functional sequences. Modular repetition also promotes rapid evolution for binding multivalent targets in the "evolutionary arms race" between host and pathogen. Finally, modular repetition was used to successfully design, on the first attempt, a well-folded and functional beta-trefoil, called ThreeFoil. Improving protein design requires understanding the outcomes of design and not simply the 3D structure. To this end, I undertook an extensive biophysical characterization of ThreeFoil, with the key finding that its unfolding is extraordinarily slow, with a half-life of almost a decade. This kinetic stability grants ThreeFoil near-immunity to common denaturants as well as high resistance to proteolysis. A large scale analysis of hundreds of proteins, and coarse-grained modelling of ThreeFoil and other beta-trefoils, indicates that high kinetic stability results from a folded structure rich in contacts between residues distant in sequence (long-range contacts). Furthermore, an analysis of unrelated proteins known to have similar protease resistance, demonstrates that the topological complexity resulting from these long-range contacts may be a general mechanism by which proteins remain folded in harsh environments. Despite the wonderful kinetic stability of ThreeFoil, it has only moderate thermodynamic stability. I sought to improve this in order to provide a stability buffer for future functional engineering and mutagenesis. Numerous computational tools which predict stability change upon point mutation were used, and 10 mutations made based on their recommendations. Despite claims of >80% accuracy for these predictions, only 2 of the 10 mutations were stabilizing. An in-depth analysis of more than 20 such tools shows that, to a large extent, while they are capable of recognizing highly destabilizing mutations, they are unable to distinguish between moderately destabilizing and stabilizing mutations. Designing protein structure tests our understanding of the determinants of protein folding, but useful function is often the final goal of protein engineering. I explored protein-ligand binding using molecular dynamics for several protein-ligand systems involving both flexible ligand binding to deep pockets and more rigid ligand binding to shallow grooves. I also used various levels of simulation complexity, from gas-phase, to implicit solvent, to fully explicit solvent, as well as simple equilibrium simulations to interrogate known interactions to more complex energetically biased simulations to explore diverse configurations and gain novel information

Insights into the stability of a therapeutic antibody Fab fragment by molecular dynamics and its stabilization by computational design

Successful development of protein therapeutics depends critically on achieving stability under a range of conditions, while retaining their specific mode of action. Gaining a deeper understanding of the drivers of instability across different stress conditions, will potentially enable the engineering of protein scaffolds that are inherently manufacturable and stable. Here, we compared the structural robustness of a humanized antibody fragment (Fab) A33 using atomistic molecular dynamics simulations under two different stresses of low pH and high temperature. RMSD calculations, structural alignments and contact analysis revealed that low pH unfolding was initiated through loss of contacts at the constant domain interface (CL-CH1), prior to CL domain unfolding. By contrast, thermal unfolding began with loss of contacts in both the CL-CH1 and variable domain interface (VL-VH), followed by domain unfolding of CL and also of VH, thus revealing divergent unfolding pathways. FoldX and Rosetta both agreed that mutations at the CL-CH1 interface have the greatest potential for increasing the stability of Fab A33. Additionally, packing density calculations found these residues to be under-packed relative to other inter-domain residues. Two salt bridges were identified that possibly drive the conformational change at low pH, while at high temperature, salt bridges were lost and reformed quickly, and not always with the same partner, thus contributing to an overall destabilization. Sequence entropy analysis of existing Fab sequences revealed considerable scope for further engineering, where certain natural mutations agreed with FoldX and Rosetta predictions. Lastly, the unfolding events at the two stress conditions exposed different predicted aggregation-prone regions (APR), which would potentially lead to different aggregation mechanisms. Overall, our results identified the early stages of unfolding and stability-limiting regions of Fab A33, which provide interesting targets for future protein engineering work aimed at stabilizing to both thermal and pH-stresses simultaneously

Metal ions and protein folding: conformational and functional interplay

Dissertation presented to obtain a PhD degree in Biochemistry at Instituto de Tecnologia Química e Biológica, Universidade Nova de LisboaMetal ions are cofactors in about 30% of all proteins, where they fulfill catalytical and structural roles. Due to their unique chemistry and coordination properties they effectively expand the intrinsic polypeptide properties (by participating in catalysis or electron transfer reactions), stabilize protein conformations (like in zinc fingers) and mediate signal transduction (by promoting functionally relevant protein conformational changes). However, metal ions can also exert have deleterious effects in living systems by incorporating in non-native binding sites, promoting aberrant protein aggregation or mediating redox cycling with generation of reactive oxygen and nitrogen species. For this reason, the characterization of the roles of metal ions as modulators of protein conformation and stability provides fundamental knowledge on protein folding properties and is instrumental in establishing the molecular basis of disease. In this thesis we have analyzed protein folding processes using model protein systems incorporating covalently bound metal cofactors – iron-sulfur (FeS) proteins – or where metal ion binding is reversible and associated conformational readjustments – the S100 proteins.(...

Stability and aggregation-prone conformations of an antibody fragment antigen-binding (Fab)

Antibody-based products have become the main drug class of approved biopharmaceuticals, with over 60 drugs on the market and many more in clinical development. However, many never reach the market because protein aggregates form during manufacturing and storage, which lower the efficacy of the product and may cause immune responses in patients. To date, very little is known about the structural conformers that initiate aggregation. Stability of the humanized fragment antigen-binding (Fab) A33 was first studied using molecular dynamic (MD) simulations under two stresses, low pH and high temperature. Results revealed different unfolding pathways, with CL domain partially unfolding at low pH, and CL and VH at high temperature. These conformational changes exposed different predicted aggregation-prone regions (APR), to suggest different aggregation mechanisms. Further salt bridge analysis provided insights into the ionizable residues likely to get protonated first. Mutational study with FoldX and Rosetta predicted that the constant domain interface can be stabilized further, backed by packing density calculations. To experimentally characterize the aggregation-prone conformers, solution structures of Fab A33 under different conditions of pH and salt concentration, were solved using small angle X-ray scattering (SAXS). SAXS revealed an expanded conformation at pH 5.5 and below, with an Rg increase of 2.2% to 4.1%, that correlated with accelerated aggregation. Scattering data were fitted using 45,000 structures obtained from the atomistic MD simulations under the same conditions, to locate the conformational change at low pH to the CL domain. The approach was then validated using intra-molecular single-molecule FRET with a dual-labelled Fab as an orthogonal detection method. The conformational changes were found to expose a predicted APR, which forms a mechanistic basis for subsequent aggregation. Overall, these findings provide a means by which aggregation-prone conformers can be determined experimentally, and thus potentially used to guide protein engineering, or ligand binding strategies, with the aim of stabilizing the protein against aggregation

Molecular and dynamic mechanisms of prokaryotic and eukaryotic flavoenzymes: insights into their implication in human metabolism and health

Las flavoenzimas y flavoproteínas son biomoléculas versátiles y diversas que están implicadas en el metabolismo energético y otros procesos celulares como la transducción de señales, la síntesis de nucleótidos, el plegamiento de proteínas o la defensa frente al estrés oxidativo. Estas proteínas tienen como cofactores los derivados de riboflavina (RF, vitamina B2), mononucleótido de flavina (FMN) y/o dinucleótido de flavina y adenina (FAD), que les confieren propiedades únicas y versátiles. Todos los organismos contienen flavoproteínas y flavoenzimas clave, y muchas de ellas se han convertido en interesantes dianas terapéuticas o herramientas biotecnológicas. En esta tesis, se ha indagado en los mecanismos moleculares de flavoenzimas y flavoproteínas con funciones metabólicas clave en procariotas y eucariotas, como las enzimas humanas RF quinasa (Publicación I), FAD sintetasa (FADS) (Publicación II) o NAD(P)H:quinona oxidorreductasa 1 (Publicación III), o las FADS procariotas (Publicaciones IV y V). La caracterización detallada de estas enzimas contribuye a la mejor comprensión de sus patologías asociadas, y sienta las bases de nuevas estrategias terapéuticas y del diseño de compuestos dirigidos a estas dianas. Por ejemplo, aquí presentamos una primera aproximación a la búsqueda de inhibidores de las FADS procariotas que puede contribuir a su explotación farmacológica como potencial agentes antimicrobianos (Publicaciones IV y V).Esta Tesis Doctoral, presentada en la modalidad de compendio de publicaciones, incluye las siguientes publicaciones:− Publicación I. Anoz-Carbonell E, Ribero M, Polo V, Velázquez-Campoy A, Medina M. 2020. Human riboflavin kinase: species-specific traits in thebiosynthesis of the FMN cofactor. The FASEB Journal, 34:10871–10886.JCR Impact Factor 2019: 4.966. Rank: Q1 (57/297) Biochemistry and Molecular Biology; D1 (9/93) Biology; Q2 (58/195) Cell Biology.− Publicación II. Leone P, Galluccio M, Quarta S, Anoz-Carbonell E, Medina M, Indiveri C, Barile M. 2019. Mutation of aspartate 238 in FADsynthase isoform 6 increases the specific activity by weakening the FAD binding. International Journal of Molecular Sciences, 20(24):6203.JCR Impact Factor 2019: 4.556. Rank: Q1 (74/297) Biochemistry and Molecular Biology; Q2 (48/177) Chemistry (multidisciplinary). − Publicación III. Anoz-Carbonell E, Timson DJ, Pey AL, Medina M. 2020. The catalytic cycle of the antioxidant and cancer-associated human NQO1enzyme: hydride transfer, conformational dynamics and functional cooperativity. Antioxidants, 9(9):E772. JCR Impact Factor 2019: 5.014. Rank: Q1 (56/297) Biochemistry and Molecular Biology; Q1 (7/61) Medicinal Chemistry; D1 (10/139) Food Science & Technology.− Publicación IV. Sebastián M, Anoz-Carbonell E, Gracia B, Cossio P, Aínsa JA, Lans I, Medina M. 2018. Discovery of antimicrobial compounds targeting bacterial type FAD synthetases. Journal of Enzyme Inhibition and Medicinal Chemistry, 33:1, 241-254. JCR Impact Factor 2018: 4.027. Rank: Q1 (10/61) Medicinal Chemistry; Q2 (84/299) Biochemistry and Molecular Biology.− Publicación V. Lans I, Anoz-Carbonell E, Palacio-Rodríguez K, Aínsa JA, Medina M, Cossio P. 2020. In silico discovery and biological validation ofligands FAD synthase, a promising new antimicrobial target. PLOS Computational Biology, 16(8):e1007898. JCR Impact Factor 2019: 4.700. Rank: Q1 (9/77) Biochemical Research Methods; Q1 (6/59) Mathematical & Computational Biology.Flavoenzymes and flavoproteins are versatile and diverse biomolecules that are implicated in the energetic metabolism and other cellular processes such as signalling, nucleotide synthesis, protein folding or defense against oxidative stress. These proteins have as cofactors the riboflavin (RF, vitamin B2) derivatives flavin mononucleotide (FMN) and/or flavin adenine dinucleotide (FAD), which confer them their unique and versatile properties. All organisms contain key flavoproteins and flavoenzymes, and many of them are becoming interesting as therapeutic targets or biotechnological tools. In the present thesis, we have delved into the molecular mechanisms of flavoenzymes with key metabolic functions in prokaryotes and eukaryotes, such as the human RF kinase (Publication I) and FAD synthase (FADS) (Publication II), human NAD(P)H:quinone oxidoreductase 1 (Publication III), and prokaryotic FADS (Publications IV and V). The detailed characterization of these enzymes contributes to the better understanding of their associated pathologies, and provides a framework to novel therapeutic strategies and to the design of compounds targeting them. For instance, here we show a first approximation for identification of inhibitors of the prokaryotic FADS that might contribute to exploit them as pharmacological antimicrobial drugs (Publications IV and V). This Doctoral Thesis, presented in the form of a compendium of publications, comprises the following publications: − Anoz-Carbonell E, Ribero M, Polo V, Velázquez-Campoy A, Medina M. 2020. Human riboflavin kinase: species-specific traits in the biosynthesis of the FMN cofactor. The FASEB Journal, 34:10871–10886. − Leone P, Galluccio M, Quarta S, Anoz-Carbonell E, Medina M, Indiveri C, Barile M. 2019. Mutation of aspartate 238 in FAD synthase isoform 6 increases the specific activity by weakening the FAD binding. International Journal of Molecular Sciences, 20(24):6203. − Anoz-Carbonell E, Timson DJ, Pey AL, Medina M. 2020. The catalytic cycle of the antioxidant and cancer-associated human NQO1 enzyme: hydride transfer, conformational dynamics and functional cooperativity. Antioxidants, 9(9):E772. − Sebastián M, Anoz-Carbonell E, Gracia B, Cossio P, Aínsa JA, Lans I, Medina M. 2018. Discovery of antimicrobial compounds targeting bacterial type FAD synthetases. Journal of Enzyme Inhibition and Medicinal Chemistry, 33:1, 241-254. − Lans I, Anoz-Carbonell E, Palacio-Rodríguez K, Aínsa JA, Medina M, Cossio P. 2020. In silico discovery and biological validation of ligands FAD synthase, a promising new antimicrobial target. PLOS Computational Biology, 16(8):e1007898.<br /

Protein folding, metal ions and conformational states: the case of a di-cluster ferredoxin

Dissertation presented to obtain the PhD degree in Biochemistry at the Instituto de Tecnologia Química e Biológica, Universidade Nova de LisboaMetal ions are present in over thirty percent of known proteins. Apart from a well established function in catalysis and electron transfer, metals and metal centres are also important structural elements which may as well play a key role in modulating protein folding and stability. In this respect, cofactors can act not only as local structural stabilizing elements in the native state, contributing to the maintenance of a given specific structural fold, but may also function as potential nucleation points during the protein folding process...Fundação para a Ciência e Tecnologia is acknowledged for financial support, by awarding a PhD Grant SFRH/BD/18653/2004. This work has been funded by the projects POCTI/QUI/37521; POCTI/QUI/45758 and PTDC/QUI/70101 all to Cláudio M. Gomes

Minimal model for the secondary structures and conformational conversions in proteins

Better understanding of protein folding process can provide physical insights on the function of proteins and makes it possible to benefit from genetic information accumulated so far. Protein folding process normally takes place in less than seconds but even seconds are beyond reach of current computational power for simulations on a system of all-atom detail. Hence, to model and explore protein folding process it is crucial to construct a proper model that can adequately describe the physical process and mechanism for the relevant time scale. We discuss the reduced off-lattice model that can express &alpha;-helix and &beta;-hairpin conformations defined solely by a given sequence in order to investigate a protein folding mechanism of conformations such as a &beta;-hairpin and also to investigate conformational conversions in proteins. The first two chapters introduce and review essential concepts in protein folding modelling physical interaction in proteins, various simple models, and also review computational methods, in particular, the Metropolis Monte Carlo method, its dynamic interpretation and thermodynamic Monte Carlo algorithms. Chapter 3 describes the minimalist model that represents both &alpha;-helix and &beta;-sheet conformations using simple potentials. The native conformation can be specified by the sequence without particular conformational biases to a reference state. In Chapter 4, the model is used to investigate the folding mechanism of &beta;-hairpins exhaustively using the dynamic Monte Carlo and a thermodynamic Monte Carlo method an effcient combination of the multicanonical Monte Carlo and the weighted histogram analysis method. We show that the major folding pathways and folding rate depend on the location of a hydrophobic. The conformational conversions between &alpha;-helix and &beta;-sheet conformations are examined in Chapter 5 and 6. First, the conformational conversion due to mutation in a non-hydrophobic system and then the conformational conversion due to mutation with a hydrophobic pair at a different position at various temperatures are examined

Single molecule studies of protein unfolding in highly saline solutions

Life on earth has been found thriving in a number of extreme environments, including those of high salinity, high and low temperatures and pH, and high pressure. Organisms which live in the presence of large quantities of salt are known as halophilic (meaning salt-loving), such as in the Dead Sea. Proteins are fundamental components of all living organisms. They are large, complex molecules that carry out many processes within a cell. Halophilic proteins are of great interest due to their ability to remain soluble, flexible and functional under highly saline conditions. Intriguingly, these proteins are unstable in a low saline environment, suggesting a delicate balance between the intermolecular interactions of the protein, salt and solvent. How have halophilic proteins adapted to survive in highly saline environments? To probe the effect of salt on the mechanical stability of a protein, a combination of molecular biology and single molecule force spectroscopy (SMFS) was used. Protein engineering was utilised to create chimeric polyprotein constructs including a obligate halophilic and a mesophilic protein. SMFS experiments have been carried out using these polyprotein constructs in 0.5 M and 2 M KCl. The studies suggest that an increase in the hydrophobic interactions of a mesophilic protein cause an increase in its mechanical stability. The results also indicate that an obligate halophilic protein does not have an increased mechanical stability in the increased salt concentration. Further studies in combination with molecular dynamics simulations have the potential to gain atomistic information on the mechanical unfolding behaviour of a halophilic protein