The thermal stability of trimeric lectin BC2L-CN was investigated and found to be considerably altered when mutating residue 83, originally a threonine, located at the fucose-binding loop. Mutants were analyzed using differential scanning calorimetry and isothermal microcalorimetry. Although most mutations decreased the affinity of the protein for oligosaccharide H type 1, six mutations increased the melting temperature (Tm) by >5 °C; one mutation, T83P, increased the Tm value by 18.2 °C(T83P, Tm = 96.3 °C). In molecular dynamic simulations, the investigated thermostable mutants, T83P, T83A, and T83S, had decreased fluctuations in the loop containing residue 83. In the T83S mutation, the side-chain hydroxyl group of serine formed a hydrogen bond with a nearby residue, suggesting that the restricted movement of the side-chain resulted in fewer fluctuations and enhanced thermal stability. Residue 83 is located at the interface and near the upstream end of the equivalent loop in a different protomer; therefore, fluctuations by this residue likely propagate throughout the loop. Our study of the dramatic change in thermal stability by a single amino acid mutation provides useful insights into the rational design of protein structures, especially the structures of oligomeric proteins.

Hypertrophic cardiomyopathy (HCM) is an autosomal dominant cardiomyopathy, which is one of the most common reasons for cardiac arrest in children or adolescents. It is characterized by ventricular hypertrophy (usually left ventricle), small ventricular cavity, and reduced ventricular diastolic compliance found by echocardiography in the absence of abnormal load (such as hypertension or aortic stenosis). HCM is usually caused by mutations in genes encoding sarcomere or sarcomere-related genes. Whole exome sequencing (WES) is performed to identify probable causative genes. Through WES, we identified LIM domain-binding protein 3 (LDB3) mutations (R547Q and P323S) respectively in an 11-year-old HCM girl and a 6-year-old HCM boy. Neural network analyses showed that the LDB3 (R547Q and P323S) mutation decreased its protein stability, with confidence scores of -0.9211 and -0.8967. The STRUM server also confirmed that the mutation decreased its protein stability. Thus, LDB3 mutation may be associated with heritable HCM. To our knowledge, this is the first time to report LDB3 heterozygous variants (R547Q and P323S) responsible for heritable HCM.

Protein mutations may lead to pathologies by causing protein misfunction or propensity to degradation. For this reason, several studies have been performed over the years to determine the capability of proteins to retain their native conformation under stress condition as well as factors to explain protein stabilization and the mechanisms behind unfolding. In this review, we explore the paradigmatic example of frataxin, an iron binding protein involved in Fe-S cluster biogenesis, and whose impairment causes a neurodegenerative disease called Friedreich\'s Ataxia (FRDA). We summarize what is known about most common point mutations identified so far in heterozygous FRDA patients, their effects on frataxin structure and function and the consequences of its binding with partners.

Understanding how proteins retain structural stability is not only of fundamental importance in biophysics but also critical to industrial production of antibodies and vaccines. Protein stability is known to depend mainly on two effects: internal hydrophobicity and H-bonding between the protein surface and solvent. A challenging task is to identify their individual contributions to a protein. Here, we investigate the structural stability of the apoptotic Bid protein in solutions containing various concentrations of guanidinium hydrochloride and urea using a combination of recently developed methods including the QTY (glutamine, threonine, and tyrosine) code and electron spin resonance-based peak-height analysis. We show that when the internal hydrophobicity of Bid is broken down using the QTY code, the surface H-bonding alone is sufficient to retain the structural stability intact. When the surface H-bonding is disrupted, Bid becomes sensitive to the temperature-dependent internal hydrophobicity such that it exhibits a reversible cold unfolding above water\'s freezing point. Using the combined approach, we show that the free-energy contributions of the two effects can be more reliably obtained. The surface H bonds are more important than the other effect in determining the structural stability of Bid protein.

Noncovalent interactions play a pivotal role in regulating protein conformation, stability and dynamics. Among the quantum mechanical (QM) overlap-based noncovalent interactions, n→π* is the best understood with studies ranging from small molecules to β-turns of model proteins such as GB1. However, these investigations do not explore the interplay between multiple overlap interactions in contributing to local structure and stability. In this work, we identify and characterize all noncovalent overlap interactions in the β-turn, an important secondary structural element that facilitates the folding of a polypeptide chain. Invoking a QM framework of natural bond orbitals, we demonstrate the role of several additional interactions such as n→σ* and π→π* that are energetically comparable to or larger than n→π*. We find that these interactions are sensitive to changes in the side chain of the residues in the β-turn of GB1, suggesting that the n→π* may not be the only component in dictating β-turn conformation and stability. Furthermore, a database search of n→σ* and π→π* in the PDB reveals that they are prevalent in most proteins and have significant interaction energies (∼1 kcal/mol). This indicates that all overlap interactions must be taken into account to obtain a comprehensive picture of their contributions to protein structure and energetics. Lastly, based on the extent of QM overlaps and interaction energies, we propose geometric criteria using which these additional interactions can be efficiently tracked in broad database searches.

The present study investigates the impact of charge variants on bevacizumab\'s structure, stability, and biological activity. Five basic and one acidic charge variants were separated using semi-preparative cation exchange chromatography using linear pH gradient elution with purity > 85%. Based on the commercial biosimilar product\'s composition, two basic variants, one acidic and the main bevacizumab product, were chosen for further investigation. Intact mass analysis and tryptic peptide mapping established the basic variants\' identity as those originating from an incomplete clipping of either one or both C-terminal lysine residues in the heavy chain of bevacizumab. Based on peptide mapping data, the acidic variant formation was attributed to deamidation of asparagine residue (N84), oxidation of M258, and preservation of C-terminal lysine residue, located on the heavy chain of bevacizumab. None of the observed charge heterogeneities in bevacizumab were due to differences in glycosylation among the variants. The basic (lysine) variants exhibited similar structural, functional, and stability profiles as the bevacizumab main product. But it was also noted that both the variants did not improve bevacizumab\'s therapeutic utility when pooled in different proportions with the main product. The acidic variant was found to have an equivalent secondary structure with subtle differences in the tertiary structure. The conformational difference also translated into a ~ 62% decrease in biological activity. Based on these data, it can be concluded that different charge variants behave differently with respect to their structure and bioactivity. Hence, biopharmaceutical manufacturers need to incorporate this understanding into their process and product development guidelines to maintain consistency in product quality.

Insulin-like growth factor 1 (IGF-1) is indicated for growth failure in pediatric patients with primary IGF-1 deficiency and for patients with neutralizing antibodies to growth hormone. IGF-1 was cloned, expressed and purified in-house. Preliminary stability studies prior to the transdermal delivery experiments showed that although stable in contact with stratum corneum, the solution concentration of IGF-1 decreased to 23.63 ± 2.48 and 21.58 ± 2.62% of the initial value upon exposure for 8 h to porcine dermis of 250 and 750 µm thickness. This led to an investigation into how it might be possible to improve the stability of IGF-1 in the presence of porcine/human skin. The stability of IGF-1 in the presence of dermis improved upon heating the skin samples at 60 °C for 2 min suggesting that IGF-1 was subject to enzymatic degradation. Although addition of the protease inhibitor, phenylmethanesulfonyl fluoride (PMSF) alone, did not improve stability, the use of a protease inhibitor cocktail completely blocked proteolytic degradation of IGF-1; the solution concentration after an 8 h exposure to porcine skin was equivalent to the initial level (103.87 ± 9.15%). The results obtained with porcine skin were confirmed with human skin (IGF-1 recovery was 99.31 ± 9.98%). These findings suggest that the inclusion of protease inhibitor cocktails may be useful in limiting the degradation of therapeutic proteins during iontophoresis and transdermal delivery in general - this could be of particular interest for local delivery of peptide/protein therapeutics for dermatological applications.

Microtubules, polymers of the heterodimeric protein αβ-tubulin, are indispensable for many cellular activities such as maintenance of cell shape, division, migration, and ordered vesicle transport. In vitro assays to study microtubule functions and their regulation by associated proteins require the availability of assembly-competent purified tubulin. However, tubulin is a thermolabile protein that rapidly converts into a nonpolymerizing state. For this reason, it is usually stored at -80 °C or liquid nitrogen to preserve its conformation and polymerization properties. In this chapter, we describe a method for freeze-drying of assembly-competent tubulin in the presence of nonreducing sugar trehalose, and methods enabling the evaluation of tubulin functions in rehydrated samples.

We present a novel Java-based program denominated PeptiDesCalculator for computing peptide descriptors. These descriptors include: redefinitions of known protein parameters to suite the peptide domain, generalization schemes for the global descriptions of peptide characteristics, as well as empirical descriptors based on experimental evidence on peptide stability and interaction propensity. The PeptiDesCalculator software provides a user-friendly Graphical User Interface (GUI) and is parallelized to maximize the use of computational resources available in current work stations. The PeptiDesCalculator indices are employed in modeling 8 peptide bioactivity endpoints demonstrating satisfactory behavior. Moreover, we compare the performance of a support vector machine (SVM) classifier built using 15 PeptiDesCalculator indices with that of a recently reported deep neural network (DNN) antimicrobial activity classifier, demonstrating comparable test set performance notwithstanding the remarkably lower degree of freedom for the former. This software will facilitate the development of in silico models for the prediction of peptide properties.

Enzymes are key proteins performing the basic functional activities in cells. In humans, enzymes can be also responsible for diseases, and the molecular mechanisms underlying the genotype to phenotype relationship are under investigation for diagnosis and medical care. Here, we focus on highlighting enzymes that are active in different metabolic pathways and become relevant hubs in protein interaction networks. We perform a statistics to derive our present knowledge on human metabolic pathways (the Kyoto Encyclopaedia of Genes and Genomes (KEGG)), and we found that activity aldehyde dehydrogenase (NAD(+)), described by Enzyme Commission number EC 1.2.1.3, and activity acetyl-CoA C-acetyltransferase (EC 2.3.1.9) are the ones most frequently involved. By associating functional activities (EC numbers) to enzyme proteins, we found the proteins most frequently involved in metabolic pathways. With our analysis, we found that these proteins are endowed with the highest numbers of interaction partners when compared to all the enzymes in the pathways and with the highest numbers of predicted interaction sites. As specific enzyme protein test cases, we focus on Alpha-Aminoadipic Semialdehyde Dehydrogenase (ALDH7A1, EC 2.3.1.9) and Acetyl-CoA acetyltransferase, cytosolic and mitochondrial (gene products of ACAT2 and ACAT1, respectively; EC 2.3.1.9). With computational approaches we show that it is possible, by starting from the enzyme structure, to highlight clues of their multiple roles in different pathways and of putative mechanisms promoting the association of genes to disease.