The photoactivated dynamics of dGpdC and dCpdG sequences in DNA: a comprehensive quantum mechanical study.

Study of alternating DNA GC sequences by different time-resolved spectroscopies has provided fundamental information on the interaction between UV light and DNA, a process of great biological importance. Multiple decay paths have been identified, but their interplay is still poorly understood. Here, we characterize the photophysics of GC-DNA by integrating different computational approaches, to study molecular models including up to 6 bases described at a full quantum mechanical level. Quantum dynamical simulations, exploiting a nonadiabatic linear vibronic coupling (LVC) model, coupled with molecular dynamics sampling of the initial structures of a (GC)5 DNA duplex, provide new insights into the photophysics in the sub-picosecond time-regime. They indicate a substantial population transfer, within 50 fs, from the spectroscopic states towards G → C charge transfer states involving two stacked bases (CTintra), thus explaining the ultrafast disappearance of fluorescence. This picture is consistent with that provided by quantum mechanical geometry optimizations, using time dependent-density functional theory and a polarizable continuum model, which we use to parametrize the LVC model and to map the main excited state deactivation pathways. For the first time, the infrared and excited state absorption signatures of the various states along these pathways are comprehensively mapped. The computational models suggest that the main deactivation pathways, which, according to experiment, lead to ground state recovery on the 10-50 ps time scale, involve CTintra followed by interstrand proton transfer from the neutral G to C-. Our calculations indicate that CTintra is populated to a larger extent and more rapidly in GC than in CG steps and suggest the likely involvement of monomer-like and interstrand charge transfer decay routes for isolated and less stacked CG steps. These findings underscore the importance of the DNA sequence and thermal fluctuations for the dynamics. They will also aid the interpretation of experimental results on other sequences.

A comprehensive analysis of SARS-CoV-2 missense mutations indicates that all possible amino acid replacements in the viral proteins occurred within the first two-and-a-half years of the pandemic.

The surveillance of COVID-19 pandemic has led to the determination of millions of genome sequences of the SARS-CoV-2 virus, with the accumulation of a wealth of information never collected before for an infectious disease. Exploring the information retrieved from the GISAID database reporting at that time >13 million genome sequences, we classified the 141,639 unique missense mutations detected in the first two-and-a-half years (up to October 2022) of the pandemic. Notably, our analysis indicates that 98.2 % of all possible conservative amino acid replacements occurred. Even non-conservative mutations were highly represented (73.9 %). For a significant number of residues (3 %), all possible replacements with the other nineteen amino acids have been observed. These observations strongly indicate that, in this time interval, the virus explored all possible alternatives in terms of missense mutations for all sites of its polypeptide chain and that those that are not observed severely affect SARS-CoV-2 integrity. The implications of the present findings go well beyond the structural biology of SARS-CoV-2 as the huge amount of information here collected and classified may be valuable for the elucidation of the sequence-structure-function relationships in proteins.

A Comprehensive Analysis of the Structural Recognition between KCTD Proteins and Cullin 3.

KCTD ((K)potassium Channel Tetramerization Domain-containing) proteins constitute an emerging class of proteins involved in fundamental physio-pathological processes. In these proteins, the BTB domain, which represents the defining element of the family, may have the dual role of promoting oligomerization and favoring functionally important partnerships with different interactors. Here, by exploiting the potential of recently developed methodologies for protein structure prediction, we report a comprehensive analysis of the interactions of all KCTD proteins with their most common partner Cullin 3 (Cul3). The data here presented demonstrate the impressive ability of this approach to discriminate between KCTDs that interact with Cul3 and those that do not. Indeed, reliable and stable models of the complexes were only obtained for the 15 members of the family that are known to interact with Cul3. The generation of three-dimensional models for all KCTD-Cul3 complexes provides interesting clues on the determinants of the structural basis of this partnership as clear structural differences emerged between KCTDs that bind or do not bind Cul3. Finally, the availability of accurate three-dimensional models for KCTD-Cul3 interactions may be valuable for the ad hoc design and development of compounds targeting specific KCTDs that are involved in several common diseases.

Unveiling CD59-Antibody Interactions to Design Paratope-Mimicking Peptides for Complement Modulation.

CD59 is an abundant immuno-regulatory human protein that protects cells from damage by inhibiting the complement system. CD59 inhibits the assembly of the Membrane Attack Complex (MAC), the bactericidal pore-forming toxin of the innate immune system. In addition, several pathogenic viruses, including HIV-1, escape complement-mediated virolysis by incorporating this complement inhibitor in their own viral envelope. This makes human pathogenic viruses, such as HIV-1, not neutralised by the complement in human fluids. CD59 is also overexpressed in several cancer cells to resist the complement attack. Consistent with its importance as a therapeutical target, CD59-targeting antibodies have been proven to be successful in hindering HIV-1 growth and counteracting the effect of complement inhibition by specific cancer cells. In this work, we make use of bioinformatics and computational tools to identify CD59 interactions with blocking antibodies and to describe molecular details of the paratope-epitope interface. Based on this information, we design and produce paratope-mimicking bicyclic peptides able to target CD59. Our results set the basis for the development of antibody-mimicking small molecules targeting CD59 with potential therapeutic interest as complement activators.

Computing the electronic circular dichroism spectrum of DNA quadruple helices of different topology: A critical test for a generalized excitonic model based on a fragment diabatization.

In this study, we exploit a recently developed fragment diabatization-based excitonic model, FrDEx, to simulate the electronic circular dichroism (ECD) spectra of three guanine-rich DNA sequences arranged in guanine quadruple helices with different topologies: thrombin binding aptamer (antiparallel), c-Myc promoter (parallel), and human telomeric sequence (3+1 hybrid). Starting from time-dependent density functional theory (TD-DFT) calculations with the M052X functional, we apply our protocol to parameterize the FrDEX Hamiltonian, which accounts for electron density overlap and includes both the coupling with charge transfer transitions and the effect of the surrounding bases on the local excitation of each chromophore. The TD-DFT/M052X spectral shapes are in good agreement with the experimental ones, the main source of discrepancy being related to the intrinsic error on the computed transition energies of guanine monomer. FrDEx spectra are fairly close to the reference TD-DFT ones, allowing a significant advance with respect to a more standard excitonic Hamiltonian. We also show that the ECD spectra are sensitive to the inclusion of the inner K + cation in the calculation.

Effect of the Thermal Fluctuations of the Photophysics of GC and CG DNA Steps: A Computational Dynamical Study.

Here we refine and assess two computational procedures aimed to include the effect of thermal fluctuations on the electronic spectra and the ultrafast excited state dynamics of multichromophore systems, focusing on DNA duplexes. Our approach is based on a fragment diabatization procedure that, from a given Quantum Mechanical (QM) reference method, can provide the parameters (energy and coupling) of the reference diabatic states on the basis of the isolated fragments, either for a purely electronic excitonic Hamiltonian (FrDEx) or a linear vibronic coupling Hamiltonian (FrD-LVC). After having defined the most cost-effective procedure for DNA duplexes on two smaller fragments, FrDEx is used to simulate the absorption and Electronic Circular Dichroism (ECD) spectra of (GC)5 sequences, including the coupling with the Charge Transfer (CT) states, on a number of structures extracted from classical Molecular Dynamics (MD) simulations. The computed spectra are close to the reference TD-DFT calculations and fully consistent with the experimental ones. We then couple MD simulations and FrD-LVC to simulate the interplay between local excitations and CT transitions, both intrastrand and interstrand, in GC and CG steps when included in a oligoGC or in oligoAT DNA sequence. We predict that for both sequences a substantial part of the photoexcited population on G and C decays, within 50-100 fs, to the corresponding intrastrand CT states. This transfer is more effective for GC steps that, on average, are more closely stacked than CG ones.

Alphafold Predictions Provide Insights into the Structural Features of the Functional Oligomers of All Members of the KCTD Family.

Oligomerization endows proteins with some key properties such as extra-stabilization, long-range allosteric regulation(s), and partnerships not accessible to their monomeric counterparts. How oligomerization is achieved and preserved during evolution is a subject of remarkable scientific relevance. By exploiting the abilities of the machine-learning algorithms implemented in AlphaFold (AF) in predicting protein structures, herein, we report a comprehensive analysis of the structural states of functional oligomers of all members of the KCTD protein family. Interestingly, our approach led to the identification of reliable three-dimensional models for the pentameric states of KCNRG, KCTD6, KCTD4, KCTD7, KCTD9, and KCTD14 and possibly for KCTD11 and KCTD21 that are involved in key biological processes and that were previously uncharacterized from a structural point of view. Although for most of these proteins, the CTD domains lack any sequence similarity, they share some important structural features, such as a propeller-like structure with a central cavity delimited by five exposed and regular ß-strands. Moreover, the structure of the related proteins KCTD7 and KCTD14, although pentameric, appears to be characterized by a different organization of the CTD region, with the five chains forming a circle-like structure with a large cavity. Our predictions also suggest that other members of the family, such as KCTD10, KCTD13, and TNFAIP1, present a strong propensity to assume dimeric states. Although the structures of the functional oligomers reported herein represent models that require additional validations, they provide a consistent and global view of KCTD protein oligomerization.

Local Backbone Geometry Plays a Critical Role in Determining Conformational Preferences of Amino Acid Residues in Proteins.

The definition of the structural basis of the conformational preferences of the genetically encoded amino acid residues is an important yet unresolved issue of structural biology. In order to gain insights into this intricate topic, we here determined and compared the amino acid propensity scales for different (φ, ψ) regions of the Ramachandran plot and for different secondary structure elements. These propensities were calculated using the Chou-Fasman approach on a database of non-redundant protein chains retrieved from the Protein Data Bank. Similarities between propensity scales were evaluated by linear regression analyses. One of the most striking and unexpected findings is that distant regions of the Ramachandran plot may exhibit significantly similar propensity scales. On the other hand, contiguous regions of the Ramachandran plot may present anticorrelated propensities. In order to provide an interpretative background to these results, we evaluated the role that the local variability of protein backbone geometry plays in this context. Our analysis indicates that (dis)similarities of propensity scales between different regions of the Ramachandran plot are coupled with (dis)similarities in the local geometry. The concept that similarities of the propensity scales are dictated by the similarity of the NCαC angle and not necessarily by the similarity of the (φ, ψ) conformation may have far-reaching implications in the field.

Structure-Based Development of SARS-CoV-2 Spike Interactors.

Coronaviruses, including SARS-CoV-2 (the etiological agent of the current COVID-19 pandemic), rely on the surface spike glycoprotein to access the host cells, mainly through the interaction of their receptor-binding domain (RBD) with the human angiotensin-converting enzyme 2 (ACE2). Therefore, molecular entities able to interfere with the binding of the SARS-CoV-2 spike protein to ACE2 have great potential to inhibit viral entry. Starting from the available structural data on the interaction between SARS-CoV-2 spike protein and the host ACE2 receptor, we engineered a set of soluble and stable spike interactors, here denoted as S-plugs. Starting from the prototype S-plug, we adopted a computational approach by combining stability prediction, associated to single-point mutations, with molecular dynamics to enhance both S-plug thermostability and binding affinity to the spike protein. The best developed molecule, S-plug3, possesses a highly stable α-helical con-formation (with melting temperature Tm of 54 °C) and can interact with the spike RBD and S1 domains with similar low nanomolar affinities. Importantly, S-plug3 exposes the spike RBD to almost the same interface as the human ACE2 receptor, aimed at the recognition of all ACE2-accessing coronaviruses. Consistently, S-plug3 preserves a low nanomolar dissociation constant with the delta B.1.617.2 variant of SARS-CoV-2 spike protein (KD = 29.2 ± 0.6 nM). Taken together, we provide valid starting data for the development of therapeutical and diagnostic tools against coronaviruses accessing through ACE2.

Structural and Binding Properties of the Active Cell Wall Hydrolase RipA from M. tuberculosis, a Promising Biosensing Molecule for Early Warning Bacterial Detection.

BACKGROUND: Peptidoglycan is an essential component of the cell wall in all bacteria. In particular, the cell walls of Gram-positive bacteria are composed mostly of a thick layer of peptidoglycan. Its accessibility has important implications for their sensing in whole bacterial detection methodologies. Indeed, there is an urgent demand for rapid tests which can identify whole bacteria, e.g., directly at the point of care. OBJECTIVE: The aim of this work is to explore the suitability of RipA, a key cell division protein of M. tuberculosis, for whole cell biosensing of Gram-positive bacteria. METHODS: We here conducted Molecular Dynamics (MD) studies aimed at the understanding of the structural and dynamic features of active RipA and at the design of a suitable bioreceptor. Based on these studies, we engineered a RipA variant for covalent oriented immobilisation on golden surfaces and are able to bind peptidoglycan, albeit without degrading it. Surface Plasmon Resonance (SPR) was employed to check the ability of functionalized golden chips to recognize whole bacteria. RESULTS: MD analyses elucidated the structural details of the active form of RipA and suggested that this enzyme, once inactivated, presents a rigid and well-exposed peptidoglycan recognition cleft. We engineered RipA for proper oriented immobilisation on golden chips for SPR studies. Results show that once chemically coupled to a golden chip, the developed RipA-based bioreceptor is able to detect B. subtilis, used as a model in a concentration-dependent mode. CONCLUSION: Results highlight the potential of the engineered molecule to be integrated in the development of early warning biosensors for Gram-positive contamination in clinical diagnosis or food-borne infections.

Early steps of oxidative damage in DNA quadruplexes are position-dependent: Quantum mechanical and molecular dynamics analysis of human telomeric sequence containing ionized guanine.

Guanine radical cation (G•+) is a key intermediate in many oxidative processes occurring in nucleic acids. Here, by combining mixed Quantum Mechanical/Molecular Mechanics calculations and Molecular Dynamics (MD) simulations, we study how the structural behaviour of a tract GGG(TTAGGG)3 (hereafter Tel21) of the human telomeric sequence, folded in an antiparallel quadruple helix, changes when one of the G bases is ionized to G•+ (Tel21+). Once assessed that the electron-hole is localized on a single G, we perform MD simulations of twelve Tel21+ systems, differing in the position of G•+ in the sequence. When G•+ is located in the tetrad adjacent to the diagonal loop, we observe substantial structural rearrangements, which can decrease the electrostatic repulsion with the inner Na+ ions and increase the solvent exposed surface of G•+. Analysis of solvation patterns of G•+ provides new insights on the main reactions of G•+, i.e. the deprotonation at two different sites and hydration at the C8 atom, the first steps of the processes producing 8oxo-Guanine. We suggest the main structural determinants of the relative reactivity of each position and our conclusions, consistent with the available experimental trends, can help rationalizing the reactivity of other G-quadruplex topologies.

A global analysis of conservative and non-conservative mutations in SARS-CoV-2 detected in the first year of the COVID-19 world-wide diffusion.

The ability of SARS-CoV-2 to rapidly mutate represents a remarkable complicancy. Quantitative evaluations of the effects that these mutations have on the virus structure/function is of great relevance and the availability of a large number of SARS-CoV-2 sequences since the early phases of the pandemic represents a unique opportunity to follow the adaptation of the virus to humans. Here, we evaluated the SARS-CoV-2 amino acid mutations and their progression by analyzing publicly available viral genomes at three stages of the pandemic (2020 March 15th and October 7th, 2021 February 7th). Mutations were classified in conservative and non-conservative based on the probability to be accepted during the evolution according to the Point Accepted Mutation substitution matrices and on the similarity of the encoding codons. We found that the most frequent substitutions are T > I, L > F, and A > V and we observe accumulation of hydrophobic residues. These findings are consistent among the three stages analyzed. We also found that non-conservative mutations are less frequent than conservative ones. This finding may be ascribed to a progressive adaptation of the virus to the host. In conclusion, the present study provides indications of the early evolution of the virus and tools for the global and genome-specific evaluation of the possible impact of mutations on the structure/function of SARS-CoV-2 variants.

AlphaFold-Predicted Structures of KCTD Proteins Unravel Previously Undetected Relationships among the Members of the Family.

One of the most striking features of KCTD proteins is their involvement in apparently unrelated yet fundamental physio-pathological processes. Unfortunately, comprehensive structure-function relationships for this protein family have been hampered by the scarcity of the structural data available. This scenario is rapidly changing due to the release of the protein three-dimensional models predicted by AlphaFold (AF). Here, we exploited the structural information contained in the AF database to gain insights into the relationships among the members of the KCTD family with the aim of facilitating the definition of the structural and molecular basis of key roles that these proteins play in many biological processes. The most important finding that emerged from this investigation is the discovery that, in addition to the BTB domain, the vast majority of these proteins also share a structurally similar domain in the C-terminal region despite the absence of general sequence similarities detectable in this region. Using this domain as reference, we generated a novel and comprehensive structure-based pseudo-phylogenetic tree that unraveled previously undetected similarities among the protein family. In particular, we generated a new clustering of the KCTD proteins that will represent a solid ground for interpreting their many functions.

Electronic Circular Dichroism Spectra of DNA Quadruple Helices Studied by Molecular Dynamics Simulations and Excitonic Calculations including Charge Transfer States.

We here investigate the Electronic Circular Dichroism (ECD) Spectra of two representative Guanine-rich sequences folded in a Quadruple helix (GQ), by using a recently developed fragment diabatisation based excitonic model (FrDEx). FrDEx can include charge transfer (CT) excited states and consider the effect of the surrounding monomers on the local excitations (LEs). When applied to different structures generated by molecular dynamics simulations on a fragment of the human telomeric sequence (Tel21/22), FrDEx provides spectra fully consistent with the experimental one and in good agreement with that provided by quantum mechanical (QM) method used for its parametrization, i.e., TD-M05-2X. We show that the ECD spectrum is moderately sensitive to the conformation adopted by the bases of the loops and more significantly to the thermal fluctuations of the Guanine tetrads. In particular, we show how changes in the overlap of the tetrads modulate the intensity of the ECD signal. We illustrate how this correlates with changes in the character of the excitonic states at the bottom of the La and Lb bands, with larger LE and CT involvement of bases that are more closely stacked. As an additional test, we utilised FrDEx to compute the ECD spectrum of the monomeric and dimeric forms of a GQ forming sequence T30695 (5'TGGGTGGGTGGGTGGG3'), i.e., a system containing up to 24 Guanine bases, and demonstrated the satisfactory reproduction of the experimental and QM reference results. This study provides new insights on the effects modulating the ECD spectra of GQs and, more generally, further validates FrDEx as an effective tool to predict and assign the spectra of closely stacked multichromophore systems.

Structural features of HtpGMtb and HtpG-ESAT6Mtb vaccine antigens against tuberculosis: Molecular determinants of antigenic synergy and cytotoxicity modulation.

Vaccine development against tuberculosis is an urgent need as the only available vaccine, M. bovis Bacillus Calmette Guerin (BCG), is unable to provide significant protection in adults. Among newly identified antigens, Rv2299c is an excellent candidate for the rational design of an effective multi-antigenic TB vaccine. Also, when fused to the T cell antigen ESAT6, it becomes highly effective in boosting BCG immunization and it adopts low cytotoxicity compared to ESAT6. We here characterize these proteins by coupling various biophysical techniques to cytofluorimetry and computational studies. Altogether, our data provide an experimental evidence of the role of Rv2299c as a dimeric and highly thermostable molecular chaperone, here denoted as HtpGMtb. Molecular dynamics simulations show that ATP rigidly anchors the ATP-binding loop in a conformation incompatible with the structure of the free enzyme. We also show that HtpGMtb dimeric state is an important molecular feature for the improved antigenic and cytotoxic properties of HtpG-ESAT6Mtb. Indeed, structural features of HtpG-ESAT6Mtb show that not only does this molecule combine the antigenic properties of HtpGMtb and ESAT6, but HtpGMtb locks ESAT6 in a dimeric state, thus improving its cytotoxicity properties. The data presented here provide solid basis for the rational design of upgraded antigens.

Studying the excited electronic states of guanine rich DNA quadruplexes by quantum mechanical methods: main achievements and perspectives.

The main insights into the photoactivated dynamics of guanine quadruplexes (G4s) recently provided by quantum mechanical computations are concisely reviewed here. The experimental steady state absorption and circular dichroism spectra of different topologies can be reproduced and assigned. After light absorption from excited states delocalized over multiple bases, the most important decay pathways involve localization of the excitation over a single base or on two stacked guanines, excimers with different degrees of charge transfer character. Two main photochemical reactions are discussed. One involves the photodimerization of two stacked guanine bases on the 'neutral' excimer path. The other, ionization of guanine, which triggers deprotonation of the resulting cation to form (G-H2)˙ and (G-H1)˙ radicals. Both the static and dynamical properties of G4 excited states are ruled by their topology and modulated by the inner coordinated metal ions.

The non-swapped monomeric structure of the arginine-binding protein from Thermotoga maritima.

Domain swapping is a widespread oligomerization process that is observed in a large variety of protein families. In the large superfamily of substrate-binding proteins, non-monomeric members have rarely been reported. The arginine-binding protein from Thermotoga maritima (TmArgBP), a protein endowed with a number of unusual properties, presents a domain-swapped structure in its dimeric native state in which the two polypeptide chains mutually exchange their C-terminal helices. It has previously been shown that mutations in the region connecting the last two helices of the TmArgBP structure lead to the formation of a variety of oligomeric states (monomers, dimers, trimers and larger aggregates). With the aim of defining the structural determinants of domain swapping in TmArgBP, the monomeric form of the P235GK mutant has been structurally characterized. Analysis of this arginine-bound structure indicates that it consists of a closed monomer with its C-terminal helix folded against the rest of the protein, as typically observed for substrate-binding proteins. Notably, the two terminal helices are joined by a single nonhelical residue (Gly235). Collectively, the present findings indicate that extending the hinge region and conferring it with more conformational freedom makes the formation of a closed TmArgBP monomer possible. On the other hand, the short connection between the helices may explain the tendency of the protein to also adopt alternative oligomeric states (dimers, trimers and larger aggregates). The data reported here highlight the importance of evolutionary control to avoid the uncontrolled formation of heterogeneous and potentially harmful oligomeric species through domain swapping.

A Functional Analysis of the Unclassified Pro2767Ser BRCA2 Variant Reveals Its Potential Pathogenicity that Acts by Hampering DNA Binding and Homology-Mediated DNA Repair.

BRCA1 and BRCA2 are the genes most frequently associated with hereditary breast and ovarian cancer (HBOC). They are crucial for the maintenance of genome stability, particularly in the homologous recombination-mediated repair pathway of DNA double-strand breaks (HR-DSBR). Widespread BRCA1/2 next-generation sequencing (NGS) screening has revealed numerous variants of uncertain significance. Assessing the clinical significance of these variants is challenging, particularly regarding the clinical management of patients. Here, we report the functional characterization of the unclassified BRCA2 c.8299C > T variant, identified in a young breast cancer patient during BRCA1/2 NGS screening. This variant causes the change of Proline 2767 to Serine in the DNA binding domain (DBD) of the BRCA2 protein, necessary for the loading of RAD51 on ssDNA during the HR-DSBR. Our in silico analysis and 3D-structure modeling predicted that the p.Pro2767Ser substitution is likely to alter the BRCA2 DBD structure and function. Therefore, to evaluate the functional impact of the p.Pro2767Ser variant, we used a minigene encoding a truncated protein that contains the BRCA2 DBD and the nearby nuclear localization sequence. We found that the ectopically expressed truncated protein carrying the normal DBD, which retains the DNA binding function and lacks the central RAD51 binding domain, interferes with endogenous wild-type BRCA2 mediator functions in the HR-DSBR. We also demonstrated that the BRCA2 Pro2767Ser DBD is unable to compete with endogenous BRCA2 DNA binding, thereby suggesting that the p.Pro2767Ser substitution in the full-length protein causes the functional loss of BRCA2. Consequently, our data suggest that the p.Pro2767Ser variant should be considered pathogenic, thus supporting a revision of the ClinVar interpretation. Moreover, our experimental strategy could be a valid method with which to preliminarily evaluate the pathogenicity of the unclassified BRCA2 germline variants in the DBD and their risk of predisposing to HBOC.

Structure, stability and aggregation propensity of a Ribonuclease A-Onconase chimera.

Structural roles of loop regions are frequently overlooked in proteins. Nevertheless, they may be key players in the definition of protein topology and in the self-assembly processes occurring through domain swapping. We here investigate the effects on structure and stability of replacing the loop connecting the last two ß-strands of RNase A with the corresponding region of the more thermostable Onconase. The crystal structure of this chimeric variant (RNaseA-ONC) shows that its terminal loop size better adheres to the topological rules for the design of stabilized proteins, proposed by Baker and coworkers [43]. Indeed, RNaseA-ONC displays a thermal stability close to that of RNase A, despite the lack of Pro at position 114, which, due to its propensity to favor a cis peptide bond, has been identified as an important stabilizing factor of the native protein. Accordingly, RNaseA-ONC is significantly more stable than RNase A variants lacking Pro114; RNaseA-ONC also displays a higher propensity to form oligomers in native conditions when compared to either RNase A or Onconase. This finding demonstrates that modifications of terminal loops should to be carefully controlled in terms of size and sequence to avoid unwanted and/or potentially harmful aggregation processes.

Loop size optimization induces a strong thermal stabilization of the thioredoxin fold.

The definition of the structural basis of protein thermostability represents a major topic in structural biology and protein chemistry. We have recently observed that proteins isolated from thermophilic organisms show a better adherence to the fundamental rules of protein topology previously unveiled by Baker and coworkers (Koga et al. Nature. 2012; 491: 222-227). Here, we explored the possibility that ad hoc modifications of a natural protein following these rules could represent an efficient tool to stabilize its structure. Hence, we here designed and characterized novel variants of Escherichia coli thioredoxin (EcTrx) using a repertoire of biophysical/structural techniques. Trx chimeric variants were prepared by replacing the loop of EcTrx with the corresponding ones present in the Trxs isolated from Sulfolobus solfataricus and Sulfolobus tokodaii that show a better adherence to the topological rules. Interestingly, although the loop sequences of these proteins did not display any significant similarity, their insertion in EcTrx induced a remarkable stabilization of the protein (≥10 °C). The crystallographic structure of one of these variants corroborates the hypothesis that the optimization of the loop size is the driving force of the observed stabilization. The remarkable stabilization of the two novel chimeric Trxs, generated by applying the topological rules, represents the proof of concept that these rules may be used to stabilize natural proteins through the ad hoc optimization of the loop size. Based on the present results, we propose a novel protocol of protein stabilization that can be potentially applied to other proteins.