Structural Characterization of an Analog of the Major Rate-Determining Disulfide Folding Intermediate of Bovine Pancreatic Ribonuclease A

Laity, John H.; Lester, Cathy; Shimotakahara, Sakurako; Zimmerman, Diane E.; Montelione, Gaetano T.; Scheraga, Harold A.

doi:10.1021/bi970878b

Cited by 78 publications

(151 citation statements)

References 103 publications

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“…This mutation eliminates a disulfide bridge with Cys92 that is conserved throughout the pancreatic RNase superfamily. Removal of the corresponding disulfide bond in RNase A decreases enzymatic activity by 11-fold (Ala mutations) or 20-fold (Ser mutations) and lowers T m by 21.8°C (Ala mutations), similar to the present findings for C39W-ANG (45). In addition, our modeling study predicts that the new Trp residue in C39W might cause perturbations that have an effect on catalytic activity beyond that due to simple loss of the stabilizing disulfide.…”

Section: Discussionsupporting

confidence: 86%

Characterization of Human Angiogenin Variants Implicated in Amyotrophic Lateral Sclerosis

et al. 2007

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Human angiogenin (ANG), the first member of the angiogenin family (from the pancreatic ribonuclease A superfamily) to be identified, is an angiogenic factor that induces neovascularization. It has received much attention due to its involvement in the growth of tumors and its elevated expression level in pancreatic and several other cancers. Recently the biological role of ANG has been shown to extend to the nervous system. Mutations in ANG have been linked with familial as well as sporadic forms of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder characterized by selective destruction of motor neurons. Furthermore, mouse angiogenin-1 has been shown to be expressed in the developing nervous system and during the neuronal differentiation of pluripotent stem cells. We have now characterized the seven variants of ANG reported in ALS patients with respect to the known biochemical properties of ANG and further studied the biological properties of three of these variants. Our results show that the ribonucleolytic activity of six of the seven ANG-ALS implicated variants is significantly reduced or lost and some variants also show altered thermal stability. We report a significant reduction in the cell proliferative and angiogenic activities of the three variants that we chose to investigate further. Our studies on the biochemical and structural features of these ANG variants now form the basis for further investigations to determine their role(s) in ALS.Amyotrophic lateral sclerosis (ALS) 1 is a fatal neurodegenerative disorder characterized by selective destruction of motor neurons (1). In the past decade, a small number of genes involved in the etiology of the disease have been identified (for a recent review see ref 2). The best studied of these is SOD1 (3), the gene for Cu/Zn superoxide dismutase. More than a hundred SOD1 mutations have now been linked with ALS, and motor neuron death from many of these mutations has been shown to result from a toxic gain of function rather than loss of dismutase activity. However, mutations in SOD1 account for only 1-2% of all cases of ALS and 20% of the familial cases. Some of the other proteins implicated in ALS are the vesicle-trafficking protein VAPB (4); ALSIN, a putative guanine nucleotide factor for GTPase (5); and senataxin (6). In addition, vascular endothelial growth factor (VEGF), an angiogenic factor that plays an important role in motor neuron survival, has been linked with ALS (7-10). However, the causes and molecular mechanisms underlying ALS are still largely unclear, and effective therapies do not appear to be imminent. Angiogenin (ANG), which encodes an angiogenic protein, was recently identified as a candidate susceptibility gene for ALS in the Irish and Scottish population by Greenway et al. (11). In a further study of over 2500 individuals from five independent populations from northern Europe and North America, Greenway et al. (12) found seven missense mutations (Figure 1) in 11 unrelated individuals with sporadic ALS and in f...

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Section: Discussionsupporting

confidence: 86%

Characterization of Human Angiogenin Variants Implicated in Amyotrophic Lateral Sclerosis

et al. 2007

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“…They appear soon after the start of the folding and have significant abundance up to 150 min. The restoration of the native state is confirmed by the Trp 5 and Tyr 27,28 (H3,H5) resonances. It can be seen that from 45 min onward there is some accumulation of the native state, which prevails from about 120 min onward.…”

Section: Oxidative Folding At the Level Of Individual Residuesmentioning

confidence: 84%

“…5C) of the three milestone species, all four aromatic residues (Tyr 21 , Tyr 27 , Tyr 28 , and Trp 5 ) contribute. In the photo-CIDNP spectrum of the N species Tyr 27 and Tyr 28 are clearly accessible, but Tyr 21 , which has a much weaker enhancement, seems less so. MFI shows accessibility for all three tyrosine residues, both in its major and minor conformations.…”

Section: Oxidative Folding At the Level Of Individual Residuesmentioning

confidence: 96%

“…5A. Here the NMR signals of four aromatic residues (Tyr 21 , Tyr 27 , Tyr 28 , and Trp 5 ) are shown for the equilibrium species and the time-resolved samples. There are several examples of how a peak with a native chemical shift develops through the folding reaction, for example Tyr 27 (H2,H6) and Trp 5 (H7), which are distinguishable already after 45 and 90 min of the folding reaction, respectively.…”

Section: Oxidative Folding At the Level Of Individual Residuesmentioning

confidence: 99%

“…These intermediates (MFI, I-5, and I-6) appear to be the first folding intermediates observed to have a vicinal disulfide bond. MFI has been studied in detail for its tertiary structure and the solvent accessibility of aromatic residues Tyr 21 , Tyr 27 , Tyr 28 , and Trp 5 (33). Because of its "bead-like" disulfide structure (1-8, 17-18, 23-31) and the high contact order that it confers, MFI has been assigned a significant role in the folding process (33).…”

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confidence: 99%

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Oxidative Folding of Amaranthus α-Amylase Inhibitor

Cemazar

Zahariev

Pongor

et al. 2004

Journal of Biological Chemistry

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Oxidative folding is the fusion of native disulfide bond formation with conformational folding. This complex process is guided by two types of interactions: first, covalent interactions between cysteine residues, which transform into native disulfide bridges, and second, non-covalent interactions giving rise to secondary and tertiary protein structure. The aim of this work is to understand both types of interactions in the oxidative folding of Amaranthus ␣-amylase inhibitor (AAI) by providing information both at the level of individual disulfide species and at the level of amino acid residue conformation. The cystine-knot disulfides of AAI protein are stabilized in an interdependent manner, and the oxidative folding is characterized by a high heterogeneity of one-, two-, and three-disulfide intermediates. The formation of the most abundant species, the main folding intermediate, is favored over other species even in the absence of non-covalent sequential preferences. Time-resolved NMR and photochemically induced dynamic nuclear polarization spectroscopies were used to follow the oxidative folding at the level of amino acid residue conformation. Because this is the first time that a complete oxidative folding process has been monitored with these two techniques, their results were compared with those obtained at the level of an individual disulfide species. The techniques proved to be valuable for the study of conformational developments and aromatic accessibility changes along oxidative folding pathways. A detailed picture of the oxidative folding of AAI provides a model study that combines different biochemical and biophysical techniques for a fuller understanding of a complex process.Cracking the code for folding a string of amino acid residues into a biologically active three-dimensional structure is still a major challenge of both chemical and biological interest. The information that determines the folding of a protein is contained solely in its amino acid residues (1). In proteins that contain cysteine residues the formation of disulfide bonds is an additional degree of freedom in the folding process. In these proteins a fusion between the recovery of the native tertiary structure (conformational folding) and the regeneration of native disulfide bonds is coupled in the oxidative folding process (2).Disulfide formation is important for in vivo folding of a considerable number of eukaryotic proteins and is a key posttranslational modification for the stabilization of folded structures. The study of oxidative folding in vitro can provide a detailed structural description in terms of the disulfide intermediate species present along the pathway of the complex folding process. A thorough account of an oxidative folding reaction should combine a study of the formation of covalently stable intermediate species and their disulfide bond connectivity and an analysis of the conformational assembly of these species. An established method for elucidation of the exact role that disulfides play in the process of achieving...

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Structure and dynamic properties of the single disulfide-deficient α-amylase inhibitor [C45A/C73A]tendamistat: An NMR study

et al. 1998

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Covalent linkages such as disulfide bonds are important for the stabilization of proteins. In the present NMR study we compare the structure and the dynamics of the single disulfide-deficient variant C45A/C73A of the alpha-amylase inhibitor tendamistat and the wild-type protein, which contains two disulfide bonds (C11-C27 and C45-C73). Complete proton assignment was achieved by standard homonuclear 2D techniques for the variant. Chemical shift differences, intra-strand NOE effects and protected amide proton were used to compare the connectivity of the secondary structure elements of variant and wild-type. Dynamic properties of the wild-type protein were studied by 13C(alpha) heteronuclear NOE experiments with carbon in natural abundance. 15N isotope labeling was necessary to obtain the relaxation parameters of the variant, because of sample degradation. The 15N resonance assignment was achieved by a 15N 3D-NOESY-HMQC. Removal of the C45-C73 bond by the C45A/C73A mutation has no influence upon the beta-barrel structure of tendamistat beside very local changes at the mutation site. The relaxation data revealed only subtle differences between variant and wild-type on a subnanosecond time scale. Only the N-terminus and G62 in the connecting loop between the anti-parallel beta-sheets showed an increased mobility. The results are discussed in respect to thermodynamic stability and the secretion efficiency of tendamistat.

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Structural Characterization of an Analog of the Major Rate-Determining Disulfide Folding Intermediate of Bovine Pancreatic Ribonuclease A

Cited by 78 publications

References 103 publications

Characterization of Human Angiogenin Variants Implicated in Amyotrophic Lateral Sclerosis

Characterization of Human Angiogenin Variants Implicated in Amyotrophic Lateral Sclerosis

Oxidative Folding of Amaranthus α-Amylase Inhibitor

Structure and dynamic properties of the single disulfide-deficient α-amylase inhibitor [C45A/C73A]tendamistat: An NMR study

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