bluuebunny/crossref_metadata_2025_split huggingface dataset:
Engineering antibody fragments to fold in the absence of disulfide bonds
Authors: Seo Min Jeong , Jeong Ki Jun , Leysath Clinton E. , Ellington Andrew D. , Iverson Brent L. , Georgiou George 
Published: February 2009

AbstractDisulfide bonds play a critical role in the stabilization of the immunoglobulin β‐sandwich sandwich. Under reducing conditions, such as those that prevail in the cytoplasm, disulfide bonds do not normally form and as a result most antibodies expressed in that compartment (intrabodies) accumulate in a misfolded and inactive state. We have developed a simple method for the quantitative isolation of antibody fragments that retain full activity under reducing conditions from large mutant libraries. In E. coli, inactivation of the cysteine oxidoreductase DsbA abolishes protein oxidation in the periplasm, which leads to the accumulation of scFvs and other disulfide‐containing proteins in a reduced form. Libraries of mutant scFvs were tethered onto the inner membrane of dsbA cells and mutants that could bind fluorescently labeled antigen in the reducing periplasm were screened by Anchored Periplasmic Expression (APEx; Harvey et al., Proc Natl Acad Sci USA 2004;101:9193–9198.). Using this approach, we isolated scFv antibody variants that are fully active when expressed in the cytoplasm or when the four Cys residues that normally form disulfides are substituted by Ser residues.
Discovery of amyloid‐beta aggregation inhibitors using an engineered assay for intracellular protein folding and solubility
Authors: Lee Li Ling , Ha HyungHo , Chang Young‐Tae , DeLisa Matthew P. 
Published: February 2009

AbstractGenetic and biochemical studies suggest that Alzheimer's disease (AD) is caused by a series of events initiated by the production and subsequent aggregation of the Alzheimer's amyloid β peptide (Aβ), the so‐called amyloid cascade hypothesis. Thus, a logical approach to treating AD is the development of small molecule inhibitors that either block the proteases that generate Aβ from its precursor (β‐ and γ‐secretases) or interrupt and/or reverse Aβ aggregation. To identify potent inhibitors of Aβ aggregation, we have developed a high‐throughput screen based on an earlier selection that effectively paired the folding quality control feature of the Escherichia coli Tat protein export system with aggregation of the 42‐residue AD pathogenesis effecter Aβ42. Specifically, a tripartite fusion between the Tat‐dependent export signal ssTorA, the Aβ42 peptide and the β‐lactamase (Bla) reporter enzyme was found to be export incompetent due to aggregation of the Aβ42 moiety. Here, we reasoned that small, cell‐permeable molecules that inhibited Aβ42 aggregation would render the ssTorA‐Aβ42‐Bla chimera competent for Tat export to the periplasm where Bla is active against β‐lactam antibiotics such as ampicillin. Using a fluorescence‐based version of our assay, we screened a library of triazine derivatives and isolated four nontoxic, cell‐permeable compounds that promoted efficient Tat‐dependent export of ssTorA‐Aβ42‐Bla. Each of these was subsequently shown to be a bona fide inhibitor of Aβ42 aggregation using a standard thioflavin T fibrillization assay, thereby highlighting the utility of our bacterial assay as a useful screen for antiaggregation factors under physiological conditions.
Residual structure within the disordered C‐terminal segment of p21Waf1/Cip1/Sdi1 and its implications for molecular recognition
Authors: Yoon Mi‐Kyung , Venkatachalam Veena , Huang Austin , Choi Byong‐Seok , Stultz Collin M. , Chou James J. 
Published: February 2009

AbstractProbably the most unusual class of proteins in nature is the intrinsically unstructured proteins (IUPs), because they are not structured yet play essential roles in protein‐protein signaling. Many IUPs can bind different proteins, and in many cases, adopt different bound conformations. The p21 protein is a small IUP (164 residues) that is ubiquitous in cellular signaling, for example, cell cycle control, apoptosis, transcription, differentiation, and so forth; it binds to approximately 25 targets. How does this small, unstructured protein recognize each of these targets with high affinity? Here, we characterize residual structural elements of the C‐terminal segment of p21 encompassing residues 145–164 using a combination of NMR measurements and molecular dynamics simulations. The N‐terminal half of the peptide has a significant helical propensity which is recognized by calmodulin while the C‐terminal half of the peptide prefers extended conformations that facilitate binding to the proliferating cell nuclear antigen (PCNA). Our results suggest that the final bound conformations of p21 (145–164) pre‐exist in the free peptide even without its binding partners. While the conformational flexibility of the p21 peptide is essential for adapting to diverse binding environments, the intrinsic structural preferences of the free peptide enable promiscuous yet high affinity binding to a diverse array of molecular targets.
An approach to characterizing single‐subunit mutations in multimeric prepores and pores of anthrax protective antigen
Authors: Janowiak Blythe E. , Finkelstein Alan , Collier R. John 
Published: February 2009

AbstractHeptameric pores formed by the protective antigen (PA) moiety of anthrax toxin translocate the intracellular effector moieties of the toxin across the endosomal membrane to the cytosol of mammalian cells. We devised a protocol to characterize the effects of individual mutations in a single subunit of heptameric PA prepores (pore precursors) or pores. We prepared monomeric PA containing a test mutation plus an innocuous Cys‐replacement mutation at a second residue (Lys563, located on the external surface of the prepore). The introduced Cys was biotinylated, and the protein was allowed to cooligomerize with a 20‐fold excess of wild‐type PA. Finally, biotinylated prepores were freed from wild‐type prepores by avidin affinity chromatography. For the proof of principle, we examined single‐subunit mutations of Asp425 and Phe427, two residues where Ala replacements have been shown to cause strong inhibitory effects. The single‐subunit D425A mutation inhibited pore formation by >104 and abrogated activity of PA almost completely in our standard cytotoxicity assay. The single‐subunit F427A mutation caused ∼100‐fold inhibition in the cytotoxicity assay, and this effect was shown to result from a combination of strong inhibition of translocation and smaller effects on pore formation and ligand affinity. Our results show definitively that replacing a single residue in one subunit of the heptameric PA prepore can inhibit the transport activity of the oligomer almost completely—and by different mechanisms, depending on the specific residue mutated.
Nonenzymatic biotinylation of histone H2A
Authors: Healy Shannon , Heightman Tom D. , Hohmann Laura , Schriemer David , Gravel Roy A. 
Published: February 2009

AbstractHolocarboxylase synthetase (HCS, eukaryotic enzyme) and BirA (prokaryotic) are biotin protein ligases that catalyze the ATP‐dependent attachment of biotin to apocarboxylases via the reactive intermediate, bio‐5′‐AMP. In this study, we examined the in vitro mechanism of biotin attachment to histone H2A in the presence of HCS and BirA. The experiment derives from our observations that HCS is found in the nucleus of cells in addition to the cytoplasm, and it has the ability to attach biotin to histones in vitro (Narang et al., Hum Mol Genet 2004; 13:15–23). Using recombinant HCS or BirA, the rate of biotin attachment was considerably slower with histone H2A than with the biotin binding domain of an apocarboxylase. However, on incubation of recombinant H2A with chemically synthesized bio‐5′‐AMP, H2A was observed to be rapidly labeled with biotin in the absence of enzyme. Nonenzymatic biotinylation of a truncated apocarboxylase (BCCP87) has been previously reported (Streaker and Beckett, Protein Sci 2006; 15:1928–1935), though at a much slower rate than we observe for H2A. The specific attachment sites of nonenzymatically biotinylated recombinant H2A at different time points were identified using mass spectrometry, and were found to consist of a similar pattern of biotin attachment as seen in the presence of HCS, with preference for lysines in the highly basic N‐terminal region of the histone. None of the lysine sites within H2A resembles the biotin attachment consensus sequence seen in carboxylases, suggesting a novel mechanism for histone biotinylation.
StoneHinge: Hinge prediction by network analysis of individual protein structures
Authors: Keating Kevin S. , Flores Samuel C. , Gerstein Mark B. , Kuhn Leslie A. 
Published: February 2009

AbstractHinge motions are important for molecular recognition, and knowledge of their location can guide the sampling of protein conformations for docking. Predicting domains and intervening hinges is also important for identifying structurally self‐determinate units and anticipating the influence of mutations on protein flexibility and stability. Here we present StoneHinge, a novel approach for predicting hinges between domains using input from two complementary analyses of noncovalent bond networks: StoneHingeP, which identifies domain‐hinge‐domain signatures in ProFlex constraint counting results, and StoneHingeD, which does the same for DomDecomp Gaussian network analyses. Predictions for the two methods are compared to hinges defined in the literature and by visual inspection of interpolated motions between conformations in a series of proteins. For StoneHingeP, all the predicted hinges agree with hinge sites reported in the literature or observed visually, although some predictions include extra residues. Furthermore, no hinges are predicted in six hinge‐free proteins. On the other hand, StoneHingeD tends to overpredict the number of hinges, while accurately pinpointing hinge locations. By determining the consensus of their results, StoneHinge improves the specificity, predicting 11 of 13 hinges found both visually and in the literature for nine different open protein structures, and making no false‐positive predictions. By comparison, a popular hinge detection method that requires knowledge of both the open and closed conformations finds 10 of the 13 known hinges, while predicting four additional, false hinges.
Selection and structural analysis of de novo proteins from an α3β3 genetic library
Authors: Jumawid Mariejoy Therese , Takahashi Tsuyoshi , Yamazaki Toshimasa , Ashigai Hiroshi , Mihara Hisakazu 
Published: February 2009

AbstractThe construction of novel functional proteins has been a key area of protein engineering. However, there are few reports of functional proteins constructed from artificial scaffolds. Here, we have constructed a genetic library encoding α3β3 de novo proteins to generate novel scaffolds in smaller size using a binary combination of simplified hydrophobic and hydrophilic amino acid sets. To screen for folded de novo proteins, we used a GFP‐based screening system and successfully obtained the proteins from the colonies emitting the very bright fluorescence as a similar intensity of GFP. Proteins isolated from the very bright colonies (vTAJ) and bright colonies (wTAJ) were analyzed by circular dichroism (CD), 8‐anilino‐1‐naphthalenesulfonate (ANS) binding assay, and analytical size‐exclusion chromatography (SEC). CD studies revealed that vTAJ and wTAJ proteins had both α‐helix and β‐sheet structures with thermal stabilities. Moreover, the selected proteins demonstrated a variety of association states existing as monomer, dimer, and oligomer formation. The SEC and ANS binding assays revealed that vTAJ proteins tend to be a characteristic of the folded protein, but not in a molten‐globule state. A vTAJ protein, vTAJ13, which has a packed globular structure and exists as a monomer, was further analyzed by nuclear magnetic resonance. NOE connectivities between backbone signals of vTAJ13 suggested that the protein contains three α‐helices and three β‐strands as intended by its design. Thus, it would appear that artificially generated α3β3 de novo proteins isolated from very bright colonies using the GFP fusion system exhibit excellent properties similar to folded proteins and would be available as artificial scaffolds to generate functional proteins with catalytic and ligand binding properties.
Dynamics of a truncated prion protein, PrP(113–231), from 15N NMR relaxation: Order parameters calculated and slow conformational fluctuations localized to a distinct region
Authors: O'Sullivan Denis B. D. , Jones Christopher E. , Abdelraheim Salama R. , Brazier Marcus W. , Toms Harold , Brown David R. , Viles John H. 
Published: February 2009

AbstractPrion diseases are associated with the misfolding of the prion protein (PrPC) from a largely α‐helical isoform to a β‐sheet rich oligomer (PrPSc). Flexibility of the polypeptide could contribute to the ability of PrPC to undergo the conformational rearrangement during PrPC–PrPSc interactions, which then leads to the misfolded isoform. We have therefore examined the molecular motions of mouse PrPC, residues 113–231, in solution, using 15N NMR relaxation measurements. A truncated fragment has been used to eliminate the effect of the 90‐residue unstructured tail of PrPC so the dynamics of the structured domain can be studied in isolation. 15N longitudinal (T1) and transverse relaxation (T2) times as well as the proton‐nitrogen nuclear Overhauser effects have been used to calculate the spectral density at three frequencies, 0, ωN, and 0.87ωH. Spectral densities at each residue indicate various time‐scale motions of the main‐chain. Even within the structured domain of PrPC, a diverse range of motions are observed. We find that removal of the tail increases T2 relaxation times significantly indicating that the tail is responsible for shortening of T2 times in full‐length PrPC. The truncated fragment of PrP has facilitated the determination of meaningful order parameters (S2) from the relaxation data and shows for the first time that all three helices in PrPC have similar rigidity. Slow conformational fluctuations of mouse PrPC are localized to a distinct region that involves residues 171 and 172. Interestingly, residues 170–175 have been identified as a segment within PrP that will form a steric zipper, believed to be the fundamental amyloid unit. The flexibility within these residues could facilitate the PrPC–PrPSc recognition process during fibril elongation.
Methionine oxidation in human IgG2 Fc decreases binding affinities to protein A and FcRn
Authors: Pan Hai , Chen Kenneth , Chu Liping , Kinderman Francis , Apostol Izydor , Huang Gang 
Published: February 2009

AbstractSusceptibility of methionine residues to oxidation is a significant issue of protein therapeutics. Methionine oxidation may limit the product's clinical efficacy or stability. We have studied kinetics of methionine oxidation in the Fc portion of the human IgG2 and its impact on the interaction with FcRn and Protein A. Our results confirm previously published observations for IgG1 that two analogous solvent‐exposed methionine residues in IgG2, Met 252 and Met 428, oxidize more readily than the other methionine residue, Met 358, which is buried inside the Fc. Met 397, which is not present in IgG1 but in IgG2, oxidizes at similar rate as Met 358. Oxidation of two labile methionines, Met 252 and Met 428, weakens the binding of the intact antibody with Protein A and FcRn, two natural protein binding partners. Both of these binding partners share the same binding site on the Fc. Additionally, our results shows that Protein A may serve as a convenient and inexpensive surrogate for FcRn binding measurements.
Structural plasticity of the phage P22 tail needle gp26 probed with xenon gas
Authors: Olia Adam S. , Casjens Sherwood , Cingolani Gino 
Published: February 2009

AbstractThe tail needle, gp26, is a highly stable homo‐trimeric fiber found in the tail apparatus of bacteriophage P22. In the mature virion, gp26 is responsible for plugging the DNA exit channel, and likely plays an important role in penetrating the host cell envelope. In this article, we have determined the 1.98 Å resolution crystal structure of gp26 bound to xenon gas. The structure led us to identify a calcium and a chloride ion intimately bound at the interior of α‐helical core, as well as seven small cavities occupied by xenon atoms. The two ions engage in buried polar interactions with gp26 side chains that provide specificity and register to gp26 helical core, thus enhancing its stability. Conversely, the distribution of xenon accessible cavities correlates well with the flexibility of the fiber observed in solution and in the crystal structure. We suggest that small internal cavities in gp26 between the helical core and the C‐terminal tip allow for flexible swinging of the latter, without affecting the overall stability of the protein. The C‐terminal tip may be important in scanning the bacterial surface in search of a cell‐envelope penetration site, or for recognition of a yet unidentified receptor on the surface of the host.
Conformational stability of porcine serum transferrin
Authors: Shen Zhi Min , Yang Jen Tsi , Feng You‐Min , Wu Chuen‐Shang C. 
Published: February 2009

AbstractThe conformation of porcine serum ferric transferrin (Tf) and its stability against denaturation were studied by circular dichroism. Tf was estimated to have 19–24% α‐helix and 50–55% β‐sheet based on the methods of Chang et al. (Chang, C.T., Wu, C.‐S.C., & Yang, J.T., 1978, Anal. Biochem. 91, 13–31) and Provencher and Glöckner (Provencher, S.W. & Glöckner, J., 1981, Biochemistry 20, 33–37). Removal of the bound ferric ions (apo‐Tf) did not alter the overall conformation, but there were subtle changes in local conformation based on its near‐UV CD spectrum. The Tfs were stable between pH 3.5 and 11. Denaturation by guanidine hydrochloride (Gu‐HCl) showed two transitions at 1.6 and 3.4 M denaturant. The process of denaturation by acid and base was reversible, whereas that by Gu‐HCl was partially reversible. The irreversible thermal unfolding of Tfs began at temperatures above 60 °C and was not complete even at 80 °C. The bound irons (based on absorbance at 460 nm) were completely released at pH <4 or in Gu‐HCl solution above 1.7 M, when the protein began to unfold, but they remained intact in neutral solution even at 85 °C. The NH2‐ and COOH‐terminal halves of the Tf molecule obtained by limited trypsin digestion had CD spectra similar to the spectrum of native Tf, and the COOH‐terminal fragment had more stable secondary structure than the NH2‐terminal fragment.
Kinetic studies of the refolding of yeast phosphoglycerate kinase: Comparison with the isolated engineered domains
Authors: Missiakas Dominique , Betton Jean‐Michel , Chaffotte Alain , Minard Philippe , Yon Jeannine M. 
Published: February 2009

AbstractUnfolding and refolding kinetics of yeast phosphoglycerate kinase were studied by following the time‐dependent changes of two signals: the ellipticity at 218 nm and 222 nm, and the fluorescence emission at 330 nm (following excitation at 295 nm). The protein is composed of two similar‐sized structural domains. Each domain has been produced by recombinant DNA techniques. It has been previously demonstrated that the engineered isolated domains are able to fold into a quasinative structure (Minard, P., et al., 1989b, Protein Eng. 3, 55–60; Missiakas, D., Betton, J.M., Minard, P., & Yon, J.M., 1990, Biochemistry 29, 8683–8689). The behavior of the isolated domains was studied using the same two conformational probes as for the whole enzyme. We found that the refolding kinetics of each domain are multiphasic. In the whole protein, domain folding and pairing appeared to be simultaneous events. However, it was found that some refolding steps occurring during the refolding of the isolated C‐domain are masked during the refolding of yeast phosphoglycerate kinase. The N‐domain was also found to refold faster when it was isolated than when integrated.
Comparison of the X‐ray structure of native rubredoxin from pyrococcus furiosus with the NMR structure of the zinc‐substituted protein
Authors: Blake Paul R. , Day Michael W. , Hsu Barbara T. , Joshua‐Tor Leemor , Park Jae‐Bum , Hare Dennis R. , Adams Michael W. W. , Rees Douglas C. , Summers Michael F. 
Published: February 2009

AbstractThe three‐dimensional X‐ray structures of the oxidized and reduced forms of rubredoxin from Pyrococcus furiosus, determined at ‐161°C, and the NMR structure of the zinc‐substituted protein, determined in solution at 45°C, are compared. The NMR and X‐ray structures, which were determined independently, are very similar and lead to similar conclusions regarding the interactions that confer hyperthermostability.
The curious case of protein splicing: Mechanistic insights suggested by protein semisynthesis
Authors: Wallace CARMICHAEL J.A. 
Published: February 2009

AbstractThe gradual accumulation of examples of protein splicing, in which a nested intervening sequence is spliced out of the interior of a polyprotein precursor, suggests that this curious phenomenon might prove to have universal phylogenetic distribution and biological significance. The known examples are reviewed, with the aim of establishing underlying patterns, and a generalized mechanism of autocatalytic protein splicing is proposed. The testable consequences of such a proposal and the possible evolutionary origins of the phenomenon are discussed.
Covalent binding properties of the human complement protein C4 and hydrolysis rate of the internal thioester upon activation
Authors: Sepp Armin , Dodds Alister W. , Anderson Michael J. , Duncan Campbell R. , Willis Antony C. , Alex Law S.K. 
Published: February 2009

AbstractThe complement proteins C3 and C4 have an internal thioester. Upon activation on the surface of a target cell, the thioester becomes exposed and reactive to surface‐bound amino and hydroxyl groups, thus allowing covalent deposition of C3 and C4 on these targets. The two human C4 isotypes, C4A and C4B, which differ by only four amino acids, have different binding specificities. C4A binds more efficiently than C4B to amino groups, and C4B is more effective than C4A in binding to hydroxyl groups. By site‐directed mutagenesis, the four residues in a cDNA clone of C4B were modified. The variants were expressed and their binding properties studied. Variants with a histidine residue at position 1106 showed C4B‐like binding properties, and those with aspartic acid, alanine, or asparagine at the same position were C4A‐like. These results suggest that the histidine is important in catalyzing the reaction of the thioester with water and other hydroxyl group‐containing compounds. When substituted with other amino acids, this reaction is not catalyzed and the thioester becomes apparently more reactive with amino groups. This interpretation also predicts that the stability of the thioester in C4A and C4B, upon activation, will be different. We measured the time course of activation and binding of glycine to C4A and C4B. The lag in the binding curve behind the activation curve for C4A is significantly greater than that for C4B. The hydrolysis rates (k0) of the thioester in the activated proteins were estimated to be 0.068 s−1 (t1/2 of 10.3 s) for C4A and 1.08 s−1 (t1/2 of 0.64 s) for C4B. These results indicate that the difference in hydrolysis rate of the thioester accounts, at least in part, for the difference in the binding properties of C4A and C4B.