Protein engineering using circular permutation - structure, function, stability, and applications.

Protein engineering is important for creating novel variants from natural proteins, enabling a wide range of applications. Approaches such as rational design and directed evolution are routinely used to make new protein variants. Computational tools like de novo design can introduce new protein folds. Expanding the amino acid repertoire to include unnatural amino acids with non-canonical side chains in vitro by native chemical ligation and in vivo via codon expansion methods broadens sequence and structural possibilities. Circular permutation (CP) is an invaluable approach to redesigning a protein by rearranging the amino acid sequence, where the connectivity of the secondary structural elements is altered without changing the overall structure of the protein. Artificial CP proteins (CPs) are employed in various applications such as biocatalysis, sensing of small molecules by fluorescence, genome editing, ligand-binding protein switches, and optogenetic engineering. Many studies have shown that CP can lead to either reduced or enhanced stability or catalytic efficiency. The effects of CP on a protein's energy landscape cannot be predicted a priori. Thus, it is important to understand how CP can affect the thermodynamic and kinetic stability of a protein. In this review, we discuss the discovery and advancement of techniques to create protein CP, and existing reviews on CP. We delve into the plethora of biological applications for designed CP proteins. We subsequently discuss the experimental and computational reports on the effects of CP on the thermodynamic and kinetic stabilities of proteins of various topologies. An understanding of the various aspects of CP will allow the reader to design robust CP proteins for their specific purposes.

Circular permutation at azurin's active site slows down its folding.

Circular permutation (CP) is a technique by which the primary sequence of a protein is rearranged to create new termini. The connectivity of the protein is altered but the overall protein structure generally remains unperturbed. Understanding the effect of CP can help design robust proteins for numerous applications such as in genetic engineering, optoelectronics, and improving catalytic activity. Studies on different protein topologies showed that CP usually affects protein stability as well as unfolding rates. Though a significant number of proteins contain metals or other cofactors, reports of metalloprotein CPs are rare. Thus, we chose a bacterial metalloprotein, azurin, and its CP within the metal-binding site (cpF114). We studied the stabilities, folding, and unfolding rates of apo- and Zn2+-bound CP azurin using fluorescence and circular dichroism. The introduced CP had destabilizing effects on the protein. Also, the folding of the Zn2+-CP protein was much slower than that of the Zn2+-WT or apo-protein. We compared this study to our previously reported azurin-cpN42, where we had observed an equilibrium and kinetic intermediate. cpF114 exhibits an apparent two-state equilibrium unfolding but has an off-pathway kinetic intermediate. Our study hinted at CP as a method to modify the energy landscape of proteins to alter their folding pathways. WT azurin, being a faster folder, may have evolved to optimize the folding rate of metal-bound protein compared to its CPs, albeit all of them have the same structure and function. Our study underscores that protein sequence and protein termini positions are crucial for metalloproteins. TOC Figure. (Top) Zn2+-azurin WT structure (PDB code: 1E67) and 2-D topology diagram of Zn2+-cpF114 azurin. (Bottom) Cartoon diagram representing folding (red arrows) and unfolding (blue arrows) of apo- and Zn2+- WT and cpF114 azurins. The width of the arrows represents the rate of the corresponding processes.

Metal-binding and circular permutation-dependent thermodynamic and kinetic stability of azurin.

Native topology is known to determine the folding kinetics and the energy landscape of proteins. Furthermore, the circular permutation (CP) of proteins alters the order of the secondary structure connectivity while retaining the three-dimensional structure, making it an elegant and powerful approach to altering native topology. Previous studies elucidated the influence of CP in proteins with different folds such as Greek key ß-barrel, ß-sandwich, ß-α-ß, and all α-Greek key. CP mainly affects the protein stability and unfolding kinetics, while folding kinetics remains mostly unaltered. However, the effect of CP on metalloproteins is yet to be elaborately studied. The active site of metalloproteins poses an additional complexity in studying protein folding. Here, we investigate a CP variant (cpN42) of azurin-in both metal-free and metal-bound (holo) forms. As observed earlier in other proteins, apo-forms of wild-type (WT) and cpN42 fold with similar rates. In contrast, zinc-binding accelerates the folding of WT but decelerates the folding of cpN42. On zinc-binding, the spontaneous folding rate of WT increases by >250 times that of cpN42, which is unprecedented and the highest for any CP to date. On the other hand, zinc-binding reduces the spontaneous unfolding rate of cpN42 by ~100 times, making the WT and CP azurins unfold at similar rates. Our study demonstrates metal binding as a novel way to modulate the unfolding and folding rates of CPs compared to their WT counterparts. We hope our study increases the understanding of the effect of CP on the folding mechanism and energy landscape of metalloproteins.

Role of Glycogen Synthase Kinase-3 in the Etiology of Type 2 Diabetes Mellitus: A Review.

The risk of type 2 diabetes mellitus (T2DM) is increasing abundantly due to lifestyle-related obesity and associated cardiovascular problems. Presently, Glycogen synthase kinase-3 (GSK-3) has gained considerable attention from biomedical scientists to treat diabetes. Phosphorylation of GSK-3 permits a number of cellular activities like regulation of cell signaling, cellular metabolism, cell proliferation and cellular transport. Inhibiting GSK-3 activity by pharmacological intervention has become an important strategy for the management of T2DM. This review focuses on the schematic representation of fundamental GSK-3 enzymology and encompasses the GSK-3 inhibitors as a future therapeutic lead target for the management of T2DM that may significantly regulate insulin sensitivity to insulin receptor, glycogen synthesis and glucose metabolism. The various signaling mechanisms of inhibiting the GSK-3 by describing insulin signaling through Insulin Receptor Substrate (IRS-1), Phosphatidylinositol-3 Kinase (PI3K) and Protein Kinase B (PKB/ AKT) pathways that may hopefully facilitate the pharmacologist to design for antidiabetic drug evaluation model in near future have also been highlighted.

Azurin-Derived Peptides: Comparison of Nickel- and Copper-Binding Properties.

Metalloproteins are an important class of proteins involved in metal uptake, transport, and electron-transfer reactions. Mimicking the active sites of these proteins through miniaturization is an active area of research with applications in biotechnology and medicine. Azurin is a 128-residue copper-binding cupredoxin protein involved in electron-transfer reactions. Previous studies have reported on the copper-binding-induced spectroscopic and structural properties of peptide loops (11 and 13 residues) from azurin. These azurin peptides exhibited novel stoichiometries. However, the underlying mechanism of fluorescence quenching upon copper binding remains to be understood, whether it is due to electron transfer, energy transfer, or both. Here, we report nickel-binding-associated spectroscopic and structural properties of the azurin peptides. They develop a ß-turn upon nickel binding as seen in circular dichroism and exhibit electronic transitions centered at 270 and 450 nm. Unlike copper, which exhibited 1:1 and 1:2 peptide:metal stoichiometries, nickel exhibited only a 1:1 stoichiometry. Tryptophan-containing peptides showed fluorescence quenching upon nickel binding, which is due to electron transfer. These results further suggest that the quenching in copper-bound peptides is also due to electron transfer, which could not be ascertained in previous studies. Overall, azurin peptides provide a platform for studying metal-induced structural and spectroscopic properties using transition-metal ions.

Copper-induced spectroscopic and structural changes in short peptides derived from azurin.

The active sites of metalloproteins may be mimicked by designing peptides that bind to their respective metal ions. Studying the binding of protein ligands to metal ions along with the associated structural changes is important in understanding metal uptake, transport and electron transfer functions of proteins. Copper-binding metalloprotein azurin is a 128-residue electron transfer protein with a redox-active copper cofactor. Here, we report the copper-binding associated spectroscopic and structural properties of peptide loops (11 and 13 residues) from the copper-binding site of azurin. These peptides develop a ß-turn upon copper-binding with a 1:1 Cu2+:peptide stoichiometry as seen in circular dichroism and exhibit electronic transitions centered at 340 nm and 540 nm. Further addition of copper develops a helical feature along with a shift in the absorption maxima to ~360 nm and ~580 nm at 2:1 Cu2+:peptide stoichiometry, indicating stoichiometric dependence of copper-binding geometry. Mass spectrometry indicates the copper-binding to cysteine, histidine and methionine in the peptide with 1:1 stoichiometry, and interestingly, dimerization through a disulfide linkage at 2:1 stoichiometry, as observed previously for denatured azurin. Fluorescence quenching studies on peptides with tryptophan further confirm the copper-binding induced changes in the two peptides are bi-phasic.

The evolution of trauma surgery at a high-volume Canadian centre: implications for public health, prevention, clinical care, education and recruitment.

BACKGROUND: Trauma centres continue to evolve with respect to clinical care and their impact on public health. Despite improvements in patient outcomes, operative volumes, and therefore maintenance of surgical skills, has become a challenging issue. We sought to determine whether injury demographics and treatments at a high-volume centre changed over time. METHODS: We used the Alberta Trauma Registry to analyze all severely injured (injury severity score [ISS] ≥ 12) patient admissions over a 16-year period (1995-2011). RESULTS: Of the 12,879 severely injured patients requiring admission, there was a 1.5- fold increase in the annual admission rate despite population normalization (p = 0.001). Over the 16-year interval, patients were older with a subsequent lower mortality (p = 0.001) and length of hospital stay (p = 0.007). In patients with the most severe ISS (≥ 48), there was no change in mortality (27%, p = 0.26). In 2011, falls were the most common mechanism compared with motor vehicle crashes (41% v. 23%; p < 0.001); this was a complete reversal compared with 1995 (25% v. 41%). Motorized recreational vehicle and motorcycle injuries also increased (p < 0.001). The mean number of operations performed by trauma surgeons decreased (laparotomies: 67 [17%] in 1995 v. 47 [5%] in 2011, p < 0.001). Thoracotomies and tracheostomies remained unchanged (p = 0.19). CONCLUSION: Clinical care has improved despite an increasing overall volume of severely injured patient admissions. The number of operative interventions performed by trauma surgeons continues to decrease concurrent to a change in injury mechanisms. Despite these improvements, maintenance of technical skills among trauma surgeons has become an important issue.

CONTEXTE: Les centres de traumatologie continuent d'évoluer au plan des soins cliniques et de leur impact sur la santé publique. Malgré certaines améliorations, les résultats pour les patients, le volume opératoire et par conséquent, le maintien des habiletés chirurgicales sont devenus un enjeu délicat. Nous avons voulu déterminer si les caractéristiques démographiques et les traitements en traumatologie ont évolué avec le temps dans un centre qui traite un volume élevé de cas. MÉTHODES: Nous avons eu recours au Registre albertain des traumatismes pour analyser toutes les admissions de grands blessés (indice de gravité des blessures [IGB] ≥ 12) au cours d'une période de 16 ans (1995­2011). RÉSULTANTS: Chez les 12 879 grands blessés ayant dû être hospitalisés, nous avons noté une augmentation selon un facteur de 1,5 du taux annuel d'admissions, malgré une normalisation de la population (p = 0,001). Au cours de cet intervalle de 16 ans, les patients ont graduellement été plus âgés, et la mortalité (p = 0,001) et la durée des séjours hospitaliers (p = 0,007) ont subséquemment diminué. Chez les patients présentant les IGB les plus élevés (≥ 48), on n'a noté aucun changement de la mortalité (27 %, p = 0,26). En 2011, les chutes ont été la cause la plus fréquente des traumatismes, par rapport aux accidents de la route (41 % c. 23 %, p < 0,001), ce qui s'est révélé être un renversement complet par rapport à 1995 (25 % c. 41 %). Le nombre de blessures subies avec des véhicules motorisés récréatifs et des motocyclettes a aussi augmenté (p < 0,001). Le nombre moyen d'interventions effectuées par les chirurgiens en traumatologie a diminué (laparotomies : 67 [17 %] en 1995 c. 47 [5 %] en 2011, p < 0,001). Le nombre de thoracotomies et de trachéotomies est resté inchangé (p = 0,19). CONCLUSION: Les soins cliniques se sont améliorés malgré l'augmentation du volume global d'hospitalisations de patients grièvement blessés. Le nombre d'interventions chirurgicales effectuées par les chirurgiens en traumatologie continue de diminuer parallèlement à une évolution des causes de traumatismes. Malgré ces améliorations, le maintien des habiletés techniques des chirurgiens en traumatologie est devenu un enjeu important.

A booming economy means a bursting trauma system: association between hospital admission for major injury and indicators of economic activity in a large Canadian health region.

BACKGROUND: Injury epidemiology fluctuates with economic activity in many countries. These relationships remain unclear in Canada. METHODS: The annual risk of admission for major injury (Injury Severity Score ≥12) to a high-volume, level-1 Canadian trauma center was compared with indicators of economic activity over a 16-year period using linear regression. RESULTS: An increased risk of injured patient admissions was associated with rising mean gross domestic product (GDP [millions of chained 2002 dollars]) (.36 person increase per 100,000 population/$1,000 increase in GDP; P = .001) and annual gasoline prices (.47 person increase per 100,000 population/cent increase in gasoline price; P = .001). Recreation-related vehicle injuries were also associated with economic affluence. The risk of trauma patient mortality with increasing mean annual GDP (P = .72) and gasoline prices (P = .32) remained unchanged. CONCLUSION: Hospital admissions for major injury, but not trauma patient mortality, were associated with economic activity in a large Canadian health care region.