- Browse by Subject
Browsing by Subject "Protein engineering"
Now showing 1 - 5 of 5
Results Per Page
Sort Options
Item Computational protein design: assessment and applications(2015) Li, Zhixiu; Zhou, YaoqiComputational protein design aims at designing amino acid sequences that can fold into a target structure and perform a desired function. Many computational design methods have been developed and their applications have been successful during past two decades. However, the success rate of protein design remains too low to be of a useful tool by biochemists whom are not an expert of computational biology. In this dissertation, we first developed novel computational assessment techniques to assess several state-of-the-art computational techniques. We found that significant progresses were made in several important measures by two new scoring functions from RosettaDesign and from OSCAR-design, respectively. We also developed the first machine-learning technique called SPIN that predicts a sequence profile compatible to a given structure with a novel nonlocal energy-based feature. The accuracy of predicted sequences is comparable to RosettaDesign in term of sequence identity to wild type sequences. In the last two application chapters, we have designed self-inhibitory peptides of Escherichia coli methionine aminopeptidase (EcMetAP) and de novo designed barstar. Several peptides were confirmed inhibition of EcMetAP at the micromole-range 50% inhibitory concentration. Meanwhile, the assessment of designed barstar sequences indicates the improvement of OSCAR-design over RosettaDesign.Item New Horizons: Next-Generation Insulin Analogues: Structural Principles and Clinical Goals(The Endocrine Society, 2022) Jarosinski, Mark A.; Chen, Yen-Shan; Varas, Nicolás; Dhayalan, Balamurugan; Chatterjee, Deepak; Weiss, Michael A.; Biochemistry and Molecular Biology, School of MedicineDesign of “first-generation” insulin analogues over the past 3 decades has provided pharmaceutical formulations with tailored pharmacokinetic (PK) and pharmacodynamic (PD) properties. Application of a molecular tool kit—integrating protein sequence, chemical modification, and formulation—has thus led to improved prandial and basal formulations for the treatment of diabetes mellitus. Although PK/PD changes were modest in relation to prior formulations of human and animal insulins, significant clinical advantages in efficacy (mean glycemia) and safety (rates of hypoglycemia) were obtained. Continuing innovation is providing further improvements to achieve ultrarapid and ultrabasal analogue formulations in an effort to reduce glycemic variability and optimize time in range. Beyond such PK/PD metrics, next-generation insulin analogues seek to exploit therapeutic mechanisms: glucose-responsive (“smart”) analogues, pathway-specific (“biased”) analogues, and organ-targeted analogues. Smart insulin analogues and delivery systems promise to mitigate hypoglycemic risk, a critical barrier to glycemic control, whereas biased and organ-targeted insulin analogues may better recapitulate physiologic hormonal regulation. In each therapeutic class considerations of cost and stability will affect use and global distribution. This review highlights structural principles underlying next-generation design efforts, their respective biological rationale, and potential clinical applications.Item Solution structure of an ultra-stable single-chain insulin analog connects protein dynamics to a novel mechanism of receptor binding(American Society for Biochemistry and Molecular Biology, 2018-01-05) Glidden, Michael D.; Yang, Yanwu; Smith, Nicholas A.; Phillips, Nelson B.; Carr, Kelley; Wickramasinghe, Nalinda P.; Ismail-Beigi, Faramarz; Lawrence, Michael C.; Smith, Brian J.; Weiss, Michael A.; Biochemistry and Molecular Biology, School of MedicineDomain-minimized insulin receptors (IRs) have enabled crystallographic analysis of insulin-bound "micro-receptors." In such structures, the C-terminal segment of the insulin B chain inserts between conserved IR domains, unmasking an invariant receptor-binding surface that spans both insulin A and B chains. This "open" conformation not only rationalizes the inactivity of single-chain insulin (SCI) analogs (in which the A and B chains are directly linked), but also suggests that connecting (C) domains of sufficient length will bind the IR. Here, we report the high-resolution solution structure and dynamics of such an active SCI. The hormone's closed-to-open transition is foreshadowed by segmental flexibility in the native state as probed by heteronuclear NMR spectroscopy and multiple conformer simulations of crystallographic protomers as described in the companion article. We propose a model of the SCI's IR-bound state based on molecular-dynamics simulations of a micro-receptor complex. In this model, a loop defined by the SCI's B and C domains encircles the C-terminal segment of the IR α-subunit. This binding mode predicts a conformational transition between an ultra-stable closed state (in the free hormone) and an active open state (on receptor binding). Optimization of this switch within an ultra-stable SCI promises to circumvent insulin's complex global cold chain. The analog's biphasic activity, which serendipitously resembles current premixed formulations of soluble insulin and microcrystalline suspension, may be of particular utility in the developing world.Item Structural principles of insulin formulation and analog design: A century of innovation(Elsevier, 2021-10) Jarosinski, Mark A.; Dhayalan, Balamurugan; Chen, Yen-Shan; Chatterjee, Deepak; Varas, Nicolás; Weiss, Michael A.; Biochemistry and Molecular Biology, School of MedicineBackground: The discovery of insulin in 1921 and its near-immediate clinical use initiated a century of innovation. Advances extended across a broad front, from the stabilization of animal insulin formulations to the frontiers of synthetic peptide chemistry, and in turn, from the advent of recombinant DNA manufacturing to structure-based protein analog design. In each case, a creative interplay was observed between pharmaceutical applications and then-emerging principles of protein science; indeed, translational objectives contributed to a growing molecular understanding of protein structure, aggregation and misfolding. Scope of review: Pioneering crystallographic analyses-beginning with Hodgkin's solving of the 2-Zn insulin hexamer-elucidated general features of protein self-assembly, including zinc coordination and the allosteric transmission of conformational change. Crystallization of insulin was exploited both as a step in manufacturing and as a means of obtaining protracted action. Forty years ago, the confluence of recombinant human insulin with techniques for site-directed mutagenesis initiated the present era of insulin analogs. Variant or modified insulins were developed that exhibit improved prandial or basal pharmacokinetic (PK) properties. Encouraged by clinical trials demonstrating the long-term importance of glycemic control, regimens based on such analogs sought to resemble daily patterns of endogenous β-cell secretion more closely, ideally with reduced risk of hypoglycemia. Major conclusions: Next-generation insulin analog design seeks to explore new frontiers, including glucose-responsive insulins, organ-selective analogs and biased agonists tailored to address yet-unmet clinical needs. In the coming decade, we envision ever more powerful scientific synergies at the interface of structural biology, molecular physiology and therapeutics.Item An ultra-stable single-chain insulin analog resists thermal inactivation and exhibits biological signaling duration equivalent to the native protein(American Society for Biochemistry and Molecular Biology, 2018-01-05) Glidden, Michael D.; Aldabbagh, Khadijah; Phillips, Nelson B.; Carr, Kelley; Chen, Yen-Shan; Whittaker, Jonathan; Phillips, Manijeh; Wickramasinghe, Nalinda P.; Rege, Nischay; Swain, Mamuni; Peng, Yi; Yang, Yanwu; Lawrence, Michael C.; Yee, Vivien C.; Ismail-Beigi, Faramarz; Weiss, Michael A.; Biochemistry and Molecular Biology, School of MedicineThermal degradation of insulin complicates its delivery and use. Previous efforts to engineer ultra-stable analogs were confounded by prolonged cellular signaling in vivo, of unclear safety and complicating mealtime therapy. We therefore sought an ultra-stable analog whose potency and duration of action on intravenous bolus injection in diabetic rats are indistinguishable from wild-type (WT) insulin. Here, we describe the structure, function, and stability of such an analog, a 57-residue single-chain insulin (SCI) with multiple acidic substitutions. Cell-based studies revealed native-like signaling properties with negligible mitogenic activity. Its crystal structure, determined as a novel zinc-free hexamer at 2.8 Å, revealed a native insulin fold with incomplete or absent electron density in the C domain; complementary NMR studies are described in the accompanying article. The stability of the analog (ΔGU 5.0(±0.1) kcal/mol at 25 °C) was greater than that of WT insulin (3.3(±0.1) kcal/mol). On gentle agitation, the SCI retained full activity for >140 days at 45 °C and >48 h at 75 °C. These findings indicate that marked resistance to thermal inactivation in vitro is compatible with native duration of activity in vivo Further, whereas WT insulin forms large and heterogeneous aggregates above the standard 0.6 mm pharmaceutical strength, perturbing the pharmacokinetic properties of concentrated formulations, dynamic light scattering, and size-exclusion chromatography revealed only limited SCI self-assembly and aggregation in the concentration range 1-7 mm Such a combination of favorable biophysical and biological properties suggests that SCIs could provide a global therapeutic platform without a cold chain.