The present article discusses some aspects concerning the inclusion of proton isomerism in simulations of the global protonation equilibrium of protein molecules. In the context of continuum electrostatic methods, the usual basis for these simulations, this isomerism can be treated as a coexistence of tautomeric forms in equilibrium in a rigid structure; furthermore, it can be formally extended to nontitrable sites with proton isomerism, such as alcohol groups and water molecules. We follow the previously adopted approach of transforming the real system of tautomeric sites into a thermodynamically equivalent one of nontautomeric pseudosites, establishing a proper relation between the two systems. The necessary energetic and entropic modifications of model compound pK a values are also discussed. Additionally, we discuss the new entropy term, named tautomeric entropy, that results from the explicit inclusion of tautomerism in the simulations and how it can be computed together with the occupational entropy. Simulations using tautomerism were done for hen egg white lysozyme (HEWL) using a simple set of tautomers at dihedral energy minima. A very good overall prediction of pK a values was obtained, presumably the best in the literature for HEWL, using a high value for the dielectric constant assigned to the protein region, p . The explicit inclusion of water molecules treated under the extended tautomer formalism further improved the prediction, in contrast with previous works using rigid water molecules. In all calculations performed, the region with p ≈ 20 is shown the to be the optimal one. Some aspects of the somewhat controversial issue of the "proper" p value are also discussed.

Protein structure and dynamics in nonaqueous solvents are here investigated using molecular dynamics simulation studies, by considering two model proteins (ubiquitin and cutinase) in hexane, under varying hydration conditions. Ionization of the protein groups is treated assuming "pH memory," i.e., using the ionization states characteristic of aqueous solution. Neutralization of charged groups by counterions is done by considering a counterion for each charged group that cannot be made neutral by establishing a salt bridge with another charged group; this treatment is more physically reasonable for the nonaqueous situation, contrasting with the usual procedures. Our studies show that hydration has a profound effect on protein stability and flexibility in nonaqueous solvents. The structure becomes more nativelike with increasing values of hydration, up to a certain point, when further increases render it unstable and unfolding starts to occur. There is an optimal amount of water, approximately 10% (w/w), where the protein structure and flexibility are closer to the ones found in aqueous solution. This behavior can explain the experimentally known bell-shaped dependence of enzyme catalysis on hydration, and the molecular reasons for it are examined here. Water and counterions play a fundamental and dynamic role on protein stabilization, but they also seem to be important for protein unfolding at high percentages of bound water.

Side-chain modeling has a widespread application in many current methods for protein tertiary structure determination, prediction, and design. Of the existing side-chain modeling methods, rotamer-based methods are the fastest and most efficient. Classically, a rotamer is conceived as a single, rigid conformation of an amino acid sidechain. Here, we present a flexible rotamer model in which a rotamer is a continuous ensemble of conformations that cluster around the classic rigid rotamer. We have developed a thermodynamically based method for calculating effective energies for the flexible rotamer. These energies have a one-to-one correspondence with the potential energies of the rigid rotamer. Therefore, the flexible rotamer model is completely general and may be used with any rotamer-based method in substitution of the rigid rotamer model. We have compared the performance of the flexible and rigid rotamer models with one side-chain modeling method in particular (the self-consistent mean field theory method) on a set of 20 high quality crystallographic protein structures. For the flexible rotamer model, we obtained average predictions of 85.8% for chi1, 76.5% for chi1+2 and 1.34 A for root-mean-square deviation (RMSD); the corresponding values for core residues were 93.0%, 87.7% and 0.70 A, respectively. These values represent improvements of 7.3% for chi1, 8.1% for chi1+2 and 0.23 A for RMSD over the predictions obtained with the rigid rotamer model under otherwise identical conditions; the corresponding improvements for core residues were 6.9%, 10.5% and 0.43 A, respectively. We found that the predictions obtained with the flexible rotamer model were also significantly better than those obtained for the same set of proteins with another state-of-the-art side-chain placement method in the literature, especially for core residues. The flexible rotamer model represents a considerable improvement over the classic rigid rotamer model. It can, therefore, be used with considerable advantage in all rotamer-based methods commonly applied to protein tertiary structure determination, prediction, and design and also in predictions of free energies in mutational studies.

Understanding SARS-CoV-2 evolution and host immunity is critical to control COVID-19 pandemics. At the core is an arms-race between SARS-CoV-2 antibody and angiotensin-converting enzyme 2 (ACE2) recognition, a function of the viral protein spike. Mutations in spike impacting antibody and/or ACE2 binding are appearing worldwide, imposing the need to monitor SARS-CoV2 evolution and dynamics in the population. Determining signatures in SARS-CoV-2 that render the virus resistant to neutralizing antibodies is critical. We engineered 25 spike-pseudotyped lentiviruses containing individual and combined mutations in the spike protein, including all defining mutations in the variants of concern, to identify the effect of single and synergic amino acid substitutions in promoting immune escape. We confirmed that E484K evades antibody neutralization elicited by infection or vaccination, a capacity augmented when complemented by K417N and N501Y mutations. In silico analysis provided an explanation for E484K immune evasion. E484 frequently engages in interactions with antibodies but not with ACE2. Importantly, we identified a novel amino acid of concern, S494, which shares a similar pattern. Using the already circulating mutation S494P, we found that it reduces antibody neutralization of convalescent and post-immunization sera, particularly when combined with E484K and with mutations able to increase binding to ACE2, such as N501Y. Our analysis of synergic mutations provides a signature for hotspots for immune evasion and for targets of therapies, vaccines and diagnostics.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has triggered a global pandemic with devastating consequences for health-care and social-economic systems. Thus, the understanding of fundamental aspects of SARS-CoV-2 is of extreme importance.In this work, we have focused our attention on the viral ribonuclease (RNase) nsp14, since this protein was considered one of the most interferon antagonists from SARS-CoV-2, and affects viral replication. This RNase is a multifunctional protein that harbors two distinct activities, an N-terminal 3’-to-5’ exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), both with critical roles in coronaviruses life cycle. Namely, SARS-CoV-2 nsp14 ExoN knockout mutants are non-viable, indicating nsp14 as a prominent target for the development of antiviral drugs.Nsp14 ExoN activity is stimulated through the interaction with the nsp10 protein, which has a pleiotropic function during viral replication. In this study, we have performed the first biochemical characterization of the complex nsp14-nsp10 from SARS-CoV-2. Here we confirm the 3’-5’ exoribonuclease and MTase activities of nsp14 in this new Coronavirus, and the critical role of nsp10 in upregulating the nsp14 ExoN activity in vitro. Furthermore, we demonstrate that SARS-CoV-2 nsp14 N7-MTase activity is functionally independent of the ExoN activity. The nsp14 MTase activity also seems to be independent of the presence of nsp10 cofactor, contrarily to nsp14 ExoN.Until now, there is no available structure for the SARS-CoV-2 nsp14-nsp10 complex. As such, we have modelled the SARS-CoV-2 nsp14-nsp10 complex based on the 3D structure of the complex from SARS-CoV (PDB ID 5C8S). We also have managed to map key nsp10 residues involved in its interaction with nsp14, all of which are also shown to be essential for stimulation of the nsp14 ExoN activity. This reinforces the idea that a stable interaction between nsp10 and nsp14 is strictly required for the nsp14-mediated ExoN activity of SARS-CoV-2, as observed for SARS-CoV.We have studied the role of conserved DEDD catalytic residues of SARS-CoV-2 nsp14 ExoN. Our results show that motif I of ExoN domain is essential for the nsp14 function contrasting to the functionality of these conserved catalytic residues in SARS-CoV, and in the Middle East respiratory syndrome coronavírus (MERS). The differences here revealed can have important implications regarding the specific pathogenesis of SARS-CoV-2.The nsp10-nsp14 interface is a recognized attractive target for antivirals against SARS-CoV-2 and other coronaviruses. This work has unravelled a basis for discovering inhibitors targeting the specific amino acids here reported, in order to disrupt the assembly of this complex and interfere with coronaviruses replication.

Objective This study ascertained effects of cactus pear in association with different roughage in the diet of F1 Holstein/Zebu cows on intake, nutrient digestibility, nitrogen balance, ingestive behavior and performance. Methods Eight cows with 72±11 days of lactation were used. The experimental design was simultaneous in two 4×4 Latin squares. Four experimental diets were used: Diet 1, sorghum silage as exclusive roughage; Diet 2, sorghum silage associated with cactus pear in a proportion of 50% of the roughage (dry matter basis); Diet 3, elephant grass ( Pennisetum purpureum cv. Roxo) as exclusive roughage; Diet 4, elephant grass associated with cactus pear in a proportion of 50% of the roughage. The roughage:concentrate ratio was 75:25. Results Dry matter intake (p = 0.01) was higher with sorghum silage. There were differences in dry matter intake (p = 0.01), crude protein (p<0.01), ether extract (p = 0.01), non-fibrous carbohydrates (p<0.01) and total digestible nutrients (p = 0.01) among the diets. Cactus pear in the diet reduced water intake by 44.52% (p<0.01). The nitrogen balance was 59.71% and 27.49% lower in animals treated with exclusive sorghum silage and sorghum silage associated with cactus pear in relation to diets with elephant grass and elephant grass associated with cactus pear, respectively (p<0.01). The diets did not influence the milk production (p = 0.70), 3.5% fat corrected milk production (p = 0.72) or feed efficiency (p = 0.61). Conclusion The association of cactus pear with sorghum or elephant grass silage does not alter milk production, reduces the intake of dry matter and water and improves the digestibility of nutrients.

We studied the performance of Fusarium solani pisi cutinase, immobilized on a zeolite, in supercritical fluids. The catalytic activity of the enzyme was strongly dependent on water activity, was unaffected by pressure up to 300 bar, and was higher in supercritical ethylene than in supercritical carbon dioxide. The enzyme was very selective toward one of the isomers of 1-phenylethanol, with an enantiomeric excess of virtually 100%, regardless of water activity, pressure, solvent, and temperature. We used the X-ray crystal structure of the enzyme and did a computer modeling of the structures of the transition states formed by the two enantiomers. The differences between these structures helped elucidate the preference for the (R)-enantiomer.

RESUMOObjetivou-se avaliar a inclusão de ureia e glicerina bruta como aditivos na ensilagem da cana-de-açúcar, na composição químico-bromatológica, pH, N-amoniacal (N-NH 3 ) e digestibilidade in vitro (DIV). Os tratamentos foram quatro doses de ureia, 0, 10, 20 e 30 g de ureia por kg de cana-de-açúcar na ensilagem, e cinco doses de glicerina bruta, 0, 10, 20, 30 e 40g de glicerina bruta por kg de cana-de-açúcar na ensilagem. As silagens foram armazenadas por 180 dias. O tratamento com ureia afetou a maioria das variáveis relacionadas ao valor nutritivo, aumentando os teores de matéria seca (MS) e proteína (PB) (2,58; 7,76; 18,70 e 19,31%), reduzindo os teores de fibra em detergente neutro (FDN) e melhorando a DIV da MS (42,61; 48,53; 50,69 e 51,18%) e FDN (38,81; 39,23; 41,06 e 43,46%), e as características fermentativas da silagem, apresentando valores de pH de 3,49; 3,86; 4,18 e 3,93 e de N-NH 3 de 1,72; 3,80; 7,88 e 9,00, para as dose de 0, 10, 20 e 30 g, respectivamente. A glicerina bruta aumentou os teores de MS e extrato etéreo (1,45; 3,03; 3,62; 3,41 e 4,38%), melhorou a DIV da MS com valores de 49,61; 52,24; 53,28; 55,60 e 56,09% e reduziu perdas por gases durante o processo de fermentação, apresentando médias de 6,69; 5,97; 5,89; 5,51 e 5,48% da MS para as doses 0, 10, 20, 30 e 40g, respectivamente. Assim, a ureia e a glicerina bruta podem ser utilizadas como aditivos na ensilagem da cana-de-açúcar.Palavras-chave: conservação de forragem, glicerol, silagem sugar cane, in chemical composition, pH, ) and in vitro digestibility (IVD) (2.58, 7.76, 18.70 and 19.31%) and reduced levels of neutral detergent fiber (NDF) and improved IVDDM (42.61, 48.53, 50.69 and 51.18%) and NDF (38.81, 39.23, 41.06 and 43.46%) and fermentation characteristics of silage, with pH values of 3.49, 3.86, 4.18 and 3.93 and NH3 1.72, 3.80, 7.88 and 9.00 for the dose of 0, 10, 20 and 30 g, respectively. The crude glycerin increased in DM and ether extract (1.45, 3.03, 3.62, 3.41 and 4.38%), improved IVDDM with values of 49.61, 52.24, 53 28; 55.60 and 56.09% and reduced gas losses during the fermentation process with mean of 6.69, 5.97, 5.89, 5.51 and 5.48% of DM for the doses 0, 10 , 20, 30 and 40g, respectively. Urea and crude glycerin can be used as an additive in ensiling of sugar cane. ABSTRACT The aim of this study was to evaluate the inclusion of urea and crude glycerin as an additive in ensiling of