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Browsing by Subject "Protein folding"
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Item Combating Parkinson's disease-associated toxicity by modulating proteostasis(National Academy of Sciences, 2017-01-31) Park, Yangshin; Hoang, Quyen Q.; Biochemistry and Molecular Biology, School of MedicineItem 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 Constructing Kinetically Controlled Denaturation Isotherms of Folded Proteins Using Denaturant-Pulse Chaperonin Binding(Springer Nature, 2018-10-20) O’Neil, Pierce T.; Machen, Alexandra J.; Thompson, Jackie A.; Wang, Wei; Hoang, Quyen Q.; Baldwin, Michael R.; Khar, Karen R.; Karanicolas, John; Fisher, Mark T.; Biochemistry and Molecular Biology, School of MedicineMethods to assess the kinetic stability of proteins, particularly those that are aggregation prone, are very useful in establishing ligand induced stabilizing effects. Because aggregation prone proteins are by nature difficult to work with, most solution based methods are compromised by this inherent instability. Here, we describe a label-free method that examines the denaturation of immobilized proteins where the dynamic unfolded protein populations are captured and detected by chaperonin binding.Item Evolution of insulin at the edge of foldability and its medical implications(National Academy of Sciences, 2020-11-24) Rege, Nischay K.; Liu, Ming; Yang, Yanwu; Dhayalan, Balamurugan; Wickramasinghe, Nalinda P.; Chen, Yen-Shan; Rahimi, Leili; Guo, Huan; Haataja, Leena; Sun, Jinhong; Ismail-Beigi, Faramarz; Phillips, Nelson B.; Arvan, Peter; Weiss, Michael A.; Biochemistry and Molecular Biology, School of MedicineProteins have evolved to be foldable, and yet determinants of foldability may be inapparent once the native state is reached. Insight has emerged from studies of diseases of protein misfolding, exemplified by monogenic diabetes mellitus due to mutations in proinsulin leading to endoplasmic reticulum stress and β-cell death. Cellular foldability of human proinsulin requires an invariant Phe within a conserved crevice at the receptor-binding surface (position B24). Any substitution, even related aromatic residue TyrB24, impairs insulin biosynthesis and secretion. As a seeming paradox, a monomeric TyrB24 insulin analog exhibits a native-like structure in solution with only a modest decrement in stability. Packing of TyrB24 is similar to that of PheB24, adjoining core cystine B19-A20 to seal the core; the analog also exhibits native self-assembly. Although affinity for the insulin receptor is decreased ∼20-fold, biological activities in cells and rats were within the range of natural variation. Together, our findings suggest that the invariance of PheB24 among vertebrate insulins and insulin-like growth factors reflects an essential role in enabling efficient protein folding, trafficking, and secretion, a function that is inapparent in native structures. In particular, we envision that the para-hydroxyl group of TyrB24 hinders pairing of cystine B19-A20 in an obligatory on-pathway folding intermediate. The absence of genetic variation at B24 and other conserved sites near this disulfide bridge-excluded due to β-cell dysfunction-suggests that insulin has evolved to the edge of foldability. Nonrobustness of a protein's fitness landscape underlies both a rare monogenic syndrome and "diabesity" as a pandemic disease of civilization.Item Intrinsically disordered proteins in molecular recognition and structural proteomics(2014-05) Oldfield, Christopher John; Janga, Sarath Chandra; Dunker, A. Keith; Shen, Li; Xia, Yuni; Uversky, Vladimir N.Intrinsically disordered proteins (IDPs) are abundant in nature, being more prevalent in the proteomes of eukaryotes than those of bacteria or archaea. As introduced in Chapter I, these proteins, or portions of these proteins, lack stable equilibrium structures and instead have dynamic conformations that vary over time and population. Despite the lack of preformed structure, IDPs carry out many and varied molecular functions and participate in vital biological pathways. In particular, IDPs play important roles in cellular signaling that is, in part, enabled by the ability of IDPs to mediate molecular recognition. In Chapter II, the role of intrinsic disorder in molecular recognition is examined through two example IDPs: p53 and 14-3-3. The p53 protein uses intrinsically disordered regions at its N- and C-termini to interact with a large number of partners, often using the same residues. The 14-3-3 protein is a structured domain that uses the same binding site to recognize multiple intrinsically disordered partners. Examination of the structural details of these interactions highlights the importance of intrinsic disorder and induced fit in molecular recognition. More generally, many intrinsically disordered regions that mediate interactions share similar features that are identifiable from protein sequence. Chapter IV reviews several models of IDP mediated protein-protein interactions that use completely different parameterizations. Each model has its relative strengths in identifying novel interaction regions, and all suggest that IDP mediated interactions are common in nature. In addition to the biologic importance of IDPs, they are also practically important in the structural study of proteins. The presence of intrinsic disordered regions can inhibit crystallization and solution NMR studies of otherwise well-structured proteins. This problem is compounded in the context of high throughput structure determination. In Chapter III, the effect of IDPs on structure determination by X-ray crystallography is examined. It is found that protein crystals are intolerant of intrinsic disorder by examining existing crystal structures from the PDB. A retrospective analysis of Protein Structure Initiative data indicates that prediction of intrinsic disorder may be useful in the prioritization and improvement of targets for structure determination.Item Peptide Model of the Mutant Proinsulin Syndrome. I. Design and Clinical Correlation(Frontiers Media, 2022-03-01) Dhayalan, Balamurugan; Glidden, Michael D.; Zaykov, Alexander N.; Chen, Yen-Shan; Yang, Yanwu; Phillips, Nelson B.; Ismail-Beigi, Faramarz; Jarosinski, Mark A.; DiMarchi, Richard D.; Weiss, Michael A.; Biochemistry and Molecular Biology, School of MedicineThe mutant proinsulin syndrome is a monogenic cause of diabetes mellitus due to toxic misfolding of insulin's biosynthetic precursor. Also designated mutant INS-gene induced diabetes of the young (MIDY), this syndrome defines molecular determinants of foldability in the endoplasmic reticulum (ER) of β-cells. Here, we describe a peptide model of a key proinsulin folding intermediate and variants containing representative clinical mutations; the latter perturb invariant core sites in native proinsulin (LeuB15→Pro, LeuA16→Pro, and PheB24→Ser). The studies exploited a 49-residue single-chain synthetic precursor (designated DesDi), previously shown to optimize in vitro efficiency of disulfide pairing. Parent and variant peptides contain a single disulfide bridge (cystine B19-A20) to provide a model of proinsulin's first oxidative folding intermediate. The peptides were characterized by circular dichroism and redox stability in relation to effects of the mutations on (a) in vitro foldability of the corresponding insulin analogs and (b) ER stress induced in cell culture on expression of the corresponding variant proinsulins. Striking correlations were observed between peptide biophysical properties, degree of ER stress and age of diabetes onset (neonatal or adolescent). Our findings suggest that age of onset reflects the extent to which nascent structure is destabilized in proinsulin's putative folding nucleus. We envisage that such peptide models will enable high-resolution structural studies of key folding determinants and in turn permit molecular dissection of phenotype-genotype relationships in this monogenic diabetes syndrome. Our companion study (next article in this issue) employs two-dimensional heteronuclear NMR spectroscopy to define site-specific perturbations in the variant peptides.Item Peptide Model of the Mutant Proinsulin Syndrome. II. Nascent Structure and Biological Implications(Frontiers Media, 2022-03-01) Yang, Yanwu; Glidden, Michael D.; Dhayalan, Balamurugan; Zaykov, Alexander N.; Chen, Yen-Shan; Wickramasinghe, Nalinda P.; DiMarchi, Richard D.; Weiss, Michael A.; Biochemistry and Molecular Biology, School of MedicineToxic misfolding of proinsulin variants in β-cells defines a monogenic diabetes syndrome, designated mutant INS-gene induced diabetes of the young (MIDY). In our first study (previous article in this issue), we described a one-disulfide peptide model of a proinsulin folding intermediate and its use to study such variants. The mutations (LeuB15→Pro, LeuA16→Pro, and PheB24→Ser) probe residues conserved among vertebrate insulins. In this companion study, we describe 1H and 1H-13C NMR studies of the peptides; key NMR resonance assignments were verified by synthetic 13C-labeling. Parent spectra retain nativelike features in the neighborhood of the single disulfide bridge (cystine B19-A20), including secondary NMR chemical shifts and nonlocal nuclear Overhauser effects. This partial fold engages wild-type side chains LeuB15, LeuA16 and PheB24 at the nexus of nativelike α-helices α1 and α3 (as defined in native proinsulin) and flanking β-strand (residues B24-B26). The variant peptides exhibit successive structural perturbations in order: parent (most organized) > SerB24 >> ProA16 > ProB15 (least organized). The same order pertains to (a) overall α-helix content as probed by circular dichroism, (b) synthetic yields of corresponding three-disulfide insulin analogs, and (c) ER stress induced in cell culture by corresponding mutant proinsulins. These findings suggest that this and related peptide models will provide a general platform for classification of MIDY mutations based on molecular mechanisms by which nascent disulfide pairing is impaired. We propose that the syndrome's variable phenotypic spectrum-onsets ranging from the neonatal period to later in childhood or adolescence-reflects structural features of respective folding intermediates.Item Per Aspera ad Chaos: Vladimir Uversky’s Odyssey through the Strange World of Intrinsically Disordered Proteins(MDPI, 2023-06-19) Kulkarni, Prakash; Brocca, Stefania; Dunker, A. Keith; Longhi, Sonia; Biochemistry and Molecular Biology, School of MedicineItem “Register-shift” insulin analogs uncover constraints of proteotoxicity in protein evolution(Elsevier, 2020-03-06) Rege, Nischay K.; Liu, Ming; Dhayalan, Balamurugan; Chen, Yen-Shan; Smith, Nicholas A.; Rahimi, Leili; Sun, Jinhong; Guo, Huan; Yang, Yanwu; Haataja, Leena; Phillips, Nelson F. B.; Whittaker, Jonathan; Smith, Brian J.; Arvan, Peter; Ismail-Beigi, Faramarz; Weiss, Michael A.; Biochemistry and Molecular Biology, School of MedicineGlobular protein sequences encode not only functional structures (the native state) but also protein foldability, i.e. a conformational search that is both efficient and robustly minimizes misfolding. Studies of mutations associated with toxic misfolding have yielded insights into molecular determinants of protein foldability. Of particular interest are residues that are conserved yet dispensable in the native state. Here, we exploited the mutant proinsulin syndrome (a major cause of permanent neonatal-onset diabetes mellitus) to investigate whether toxic misfolding poses an evolutionary constraint. Our experiments focused on an invariant aromatic motif (PheB24-PheB25-TyrB26) with complementary roles in native self-assembly and receptor binding. A novel class of mutations provided evidence that insulin can bind to the insulin receptor (IR) in two different modes, distinguished by a "register shift" in this motif, as visualized by molecular dynamics (MD) simulations. Register-shift variants are active but defective in cellular foldability and exquisitely susceptible to fibrillation in vitro Indeed, expression of the corresponding proinsulin variant induced endoplasmic reticulum stress, a general feature of the mutant proinsulin syndrome. Although not present among vertebrate insulin and insulin-like sequences, a prototypical variant ([GlyB24]insulin) was as potent as WT insulin in a rat model of diabetes. Although in MD simulations the shifted register of receptor engagement is compatible with the structure and allosteric reorganization of the IR-signaling complex, our results suggest that this binding mode is associated with toxic misfolding and so is disallowed in evolution. The implicit threat of proteotoxicity limits sequence variation among vertebrate insulins and insulin-like growth factors.Item Role of ATF4 in directing gene expression in the basal state and during the unfolded protein response in liver(2016-08) Fusakio, Michael Edward; Wek, Ronald C.Disturbances in membrane composition and protein folding in the endoplasmic reticulum (ER) trigger the unfolded protein response (UPR). Three UPR sensory proteins, PERK (PEK/EIF2AK3), IRE1, and ATF6 are each activated by ER stress. PERK phosphorylation of the alpha subunit of eIF2 represses global protein synthesis, lowering influx of nascent polypeptides into the stressed ER, coincident with the preferential translation of ATF4 (CREB2). Results from cultured cells demonstrate that ATF4 induces transcriptional expression of genes directed by the PERK arm of the UPR, including genes involved in amino acid metabolism, resistance to oxidative stress, and the proapoptotic transcription factor CHOP (GADD153/DDIT3). In this study, we characterized two ATF4 knockout mouse models and show in liver exposed to ER stress that ATF4 is not required for CHOP expression, but rather ATF6 is a primary inducer. RNA-sequence analysis indicated that ATF4 was responsible for a small portion of the PERK-dependent genes in the UPR. This smaller than expected subset of gene expression lends itself to the relevance of UPR crosstalk, with ATF6, XBP1, and CHOP being capable of upregulating UPR genes in the absence of ATF4. RNA-sequence analysis also revealed a requirement for expression of ATF4 for expression of a comparable number of genes basally, including those involved in oxidative stress response and cholesterol metabolism. Consistent with this pattern of gene expression, loss of ATF4 in our mouse model resulted in enhanced oxidative damage and increased free cholesterol in liver under stress accompanied by lowered cholesterol in sera. Taken together, this study highlights both an expansion of the role of ATF4 in transcriptional regulation of genes involved in metabolism in the basal state and a more specialized role during ER stress. These findings are important for understanding the variances of the UPR signaling between cell culture and in vivo and for a greater understanding of all the roles ATF4 plays within the cell.