Investigating the evolution of structural features in modern multidomain proteins helps to understand their immense diversity and functional versatility. The class of periplasmic binding proteins ...(PBPs) offers an opportunity to interrogate one of the main processes driving diversification: the duplication and fusion of protein sequences to generate new architectures. The symmetry of their two‐lobed topology, their mechanism of binding, and the organization of their operon structure led to the hypothesis that PBPs arose through a duplication and fusion event of a single common ancestor. To investigate this claim, we set out to reverse the evolutionary process and recreate the structural equivalent of a single‐lobed progenitor using ribose‐binding protein (RBP) as our model. We found that this modern PBP can be deconstructed into its lobes, producing two proteins that represent possible progenitor halves. The isolated halves of RBP are well folded and monomeric proteins, albeit with a lower thermostability, and do not retain the original binding function. However, the two entities readily form a heterodimer in vitro and in‐cell. The x‐ray structure of the heterodimer closely resembles the parental protein. Moreover, the binding function is fully regained upon formation of the heterodimer with a ligand affinity similar to that observed in the modern RBP. This highlights how a duplication event could have given rise to a stable and functional PBP‐like fold and provides insights into how more complex functional structures can evolve from simpler molecular components.
Protein stability can be fine‐tuned by modifying different structural features such as hydrogen‐bond networks, salt bridges, hydrophobic cores, or disulfide bridges. Among these, stabilization by ...salt bridges is a major challenge in protein design and engineering since their stabilizing effects show a high dependence on the structural environment in the protein, and therefore are difficult to predict and model. In this work, we explore the effects on structure and stability of an introduced salt bridge cluster in the context of three different de novo TIM barrels. The salt bridge variants exhibit similar thermostability in comparison with their parental designs but important differences in the conformational stability at 25°C can be observed such as a highly stabilizing effect for two of the proteins but a destabilizing effect to the third. Analysis of the formed geometries of the salt bridge cluster in the crystal structures show either highly ordered salt bridge clusters or only single salt bridges. Rosetta modeling of the salt bridge clusters results in a good prediction of the tendency on stability changes but not the geometries observed in the three‐dimensional structures. The results show that despite the similarities in protein fold, the salt bridge clusters differently influence the structural and stability properties of the de novo TIM barrel variants depending on the structural background where they are introduced.
PDB Code(s): 7OSU, 7OT7, 7OSV, 7OT8 and 7P12;
Over the past decades, the TIM‐barrel fold has served as a model system for the exploration of how changes in protein sequences affect their structural, stability, and functional characteristics, and ...moreover, how this information can be leveraged to design proteins from the ground up. After numerous attempts to design de novo proteins with this specific fold, sTIM11 was the first validated de novo design of an idealized four‐fold symmetric TIM barrel. Subsequent efforts to enhance the stability of this initial design resulted in the development of DeNovoTIMs, a family of de novo TIM barrels with various stabilizing mutations. In this study, we present an investigation into the biophysical and thermodynamic effects upon introducing a varying number of stabilizing mutations per quarter along the sequence of a four‐fold symmetric TIM barrel. We compared the base design DeNovoTIM0 without any stabilizing mutations with variants containing mutations in one, two, three, and all four quarters—designated TIM1q, TIM2q, TIM3q, and DeNovoTIM6, respectively. This analysis revealed a stepwise and nonlinear change in the thermodynamic properties that correlated with the number of mutated quarters, suggesting positive nonadditive effects. To shed light on the significance of the location of stabilized quarters, we engineered two variants of TIM2q which contain the same number of mutations but positioned in different quarter locations. Characterization of these TIM2q variants revealed that the mutations exhibit varying effects on the overall protein stability, contingent upon the specific region in which they are introduced. These findings emphasize that the amount and location of stabilized interfaces among the four quarters play a crucial role in shaping the conformational stability of these four‐fold symmetric TIM barrels. Analysis of de novo proteins, as described in this study, enhances our understanding of how sequence variations can finely modulate stability in both naturally occurring and computationally designed proteins.
El artículo analiza los cambios que se han producido en las estrategias narrativas utilizadas para la escritura y la producción de series audiovisuales en el marco del escenario transmedial actual. ...En tal sentido, primero hacemos referencia a los efectos producidos en el desarrollo de la estructura y los formatos de las series, incluyendo la aparición de un nuevo rol: el Showrunner. Luego analizamos el modo en que los cambios se vieron complementados por la inclusión de nuevas estrategias en el proceso de desarrollo y diseño narrativo de las series y marcan el escenario transmedial.
Substrate-binding proteins (SBPs) are used by organisms from the three domains of life for transport and signalling. SBPs are composed of two domains that collectively trap ligands with high affinity ...and selectivity. To explore the role of the domains and the integrity of the hinge region between them in the function and conformation of SBPs, here, we describe the ligand binding, conformational stability and folding kinetics of the Lysine Arginine Ornithine (LAO) binding protein from Salmonella thiphimurium and constructs corresponding to its two independent domains. LAO is a class II SBP formed by a continuous and a discontinuous domain. Contrary to the expected behaviour based on their connectivity, the discontinuous domain shows a stable native-like structure that binds l-arginine with moderate affinity, whereas the continuous domain is barely stable and shows no detectable ligand binding. Regarding folding kinetics, studies of the entire protein revealed the presence of at least two intermediates. While the unfolding and refolding of the continuous domain exhibited only a single intermediate and simpler and faster kinetics than LAO, the folding mechanism of the discontinuous domain was complex and involved multiple intermediates. These findings suggest that in the complete protein the continuous domain nucleates folding and that its presence funnels the folding of the discontinuous domain avoiding nonproductive interactions. The strong dependence of the function, stability and folding pathway of the lobes on their covalent association is most likely the result of the coevolution of both domains as a single unit.
The study of binding thermodynamics is essential to understand how affinity and selectivity are acquired in molecular complexes. Periplasmic binding proteins (PBPs) are macromolecules of ...biotechnological interest that bind a broad number of ligands and have been used to design biosensors. The lysine‐arginine‐ornithine binding protein (LAO) is a PBP of 238 residues that binds the basic amino acids l‐arginine and l‐histidine with nm and μm affinity, respectively. It has been shown that the affinity difference for arginine and histidine binding is caused by enthalpy, this correlates with the higher number of protein–ligand contacts formed with arginine. In order to elucidate the structural bases that determine binding affinity and selectivity in LAO, the contribution of protein–ligand contacts to binding energetics was assessed. To this end, an alanine scanning of the LAO‐binding site residues was performed and arginine and histidine binding were characterized by isothermal titration calorimetry and X‐ray crystallography. Although unexpected enthalpy and entropy changes were observed in some mutants, thermodynamic data correlated with structural information, especially, the binding heat capacity change. We found that selectivity is conferred by several residues rather than exclusive arginine–protein interactions. Furthermore, crystallographic structures revealed that protein–ligand contributions to binding thermodynamics are highly influenced by the solvent. Finally, we found a similar backbone conformation in all the closed structures obtained, but different structures in the open state, suggesting that the binding site residues of LAO play an important role in stabilizing not only the holo conformation, but also the apo state.
Database
Structural data are available in the Protein Data Bank database under the accession numbers 6MLE, 6MLN, 6MLG, 6MKX, 6MLI, 6MLA, 6MKU, 6MKW, 6ML0, 6MLD, 6MLV, 6MLO, 6MLP, 6ML9, 6MLJ.
Experimental data on binding affinity and selectivity are required to design new ligand‐binding capabilities. The periplasmic binding protein LAO binds l‐arginine with nanomolar affinity and l‐histidine in the micromolar range. Here, we used alanine scanning to define residues that are critical to affinity and selectivity. Thermodynamic and structural data suggest that these properties result from the interplay between protein–ligand and water‐mediated interactions, without significant changes in the backbone conformation of the closed state.
Recent evidence suggests that obstructive sleep apnea (OSA) may be a risk factor for developing mild cognitive impairment and Alzheimer's disease. However, how sleep apnea affects longitudinal risk ...for Alzheimer's disease is less well understood.
To test the hypothesis that there is an association between severity of OSA and longitudinal increase in amyloid burden in cognitively normal elderly.
Data were derived from a 2-year prospective longitudinal study that sampled community-dwelling healthy cognitively normal elderly. Subjects were healthy volunteers between the ages of 55 and 90, were nondepressed, and had a consensus clinical diagnosis of cognitively normal. Cerebrospinal fluid amyloid β was measured using ELISA. Subjects received Pittsburgh compound B positron emission tomography scans following standardized procedures. Monitoring of OSA was completed using a home sleep recording device.
We found that severity of OSA indices (AHIall F
= 4.26; P < 0.05 and AHI4% F
= 4.36; P < 0.05) were associated with annual rate of change of cerebrospinal fluid amyloid β
using linear regression after adjusting for age, sex, body mass index, and apolipoprotein E4 status. AHIall and AHI4% were not associated with increases in AD
-mask (Alzheimer's disease vulnerable regions of interest Pittsburg compound B positron emission tomography mask) most likely because of the small sample size, although there was a trend for AHIall (F
= 2.96, P = 0.09; and F
= 2.32, not significant, respectively).
In a sample of cognitively normal elderly, OSA was associated with markers of increased amyloid burden over the 2-year follow-up. Sleep fragmentation and/or intermittent hypoxia from OSA are likely candidate mechanisms. If confirmed, clinical interventions for OSA may be useful in preventing amyloid build-up in cognitively normal elderly.
Radiated emission tests are generally performed in either free space, reflection-free environments, such as an open area test site or semi- or full-anechoic chambers, or in reverberation chambers. ...This paper describes measurements in semireflecting environments such as an office, a workshop of a large industrial apparatus or installation. The objective is to develop test methods and associated correction factors and uncertainties for measurements which are performed on-site, near large industrial apparatus which cannot be moved to an EMC laboratory. The measurement technique is based on reverberation chambers and tools such as insertion loss, quality factor, as well as a goodness of fit test were used to perform the test site analysis. The advantages and drawbacks of on-site measurements of large apparatus are discussed. A simplified, but not limited to perfect reverberation chambers, method of finding the measurement uncertainties was used to calculate the errors associated with imperfect reverberation and field uniformity, as well as the influence of noise and equipment nonlinearities. The results are given in form of a guideline, concluding that such measurements are possible.
Client knowledge remains a key strategic point in hospitality management. However, the role that can be played by large amounts of available information in the Customer Relationship Management (CRM) ...systems, when addressed by using emerging Big Data techniques for efficient client profiling, is still in its early stages. In this work, we addressed the client profile of the data in a CRM system of an international hotel chain, by using Big Data technology and Bootstrap resampling techniques for Proportion Tests. Strong consistency was found on the most representative feature of repeaters being traveling without children. Profiles were more similar for British and German clients, and their main differences with Spanish clients were in the stay duration and in age. For a vacation chain, these results suggest further analysis on the target orientation towards new market segments. Big Data technologies can be extremely useful for analyzing indoor data available in CRM information systems from hospitality industry.
•Big Data technology is useful yielding specific guest profiles from CRM hotel information.•Proportion differences using Bootstrap resampling yield detailed characterization of the client profile.•Repeaters included features such as traveling without children.•Repeaters were similar between German and English but different from Spanish.•German and English included the elderly staying for longer periods whereas Spanish were short-term and younger ones.
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