The Dimerization of an α/β-Knotted Protein Is Essential for Structure and Function

Summaryα/β-Knotted proteins are an extraordinary example of biological self-assembly; they contain a deep topological trefoil knot formed by the backbone polypeptide chain. Evidence suggests that all are dimeric and function as methyltransferases, and the deep knot forms part of the active site. We investigated the significance of the dimeric structure of the α/β-knot protein, YibK, from Haemophilus influenzae by the design and engineering of monomeric versions of the protein, followed by examination of their structural, functional, stability, and kinetic folding properties. Monomeric forms of YibK display similar characteristics to an intermediate species populated during the formation of the wild-type dimer. However, a notable loss in structure involving disruption to the active site, rendering it incapable of cofactor binding, is observed in monomeric YibK. Thus, dimerization is vital for preservation of the native structure and, therefore, activity of the protein

Knots in soft condensed matter

Understanding the mechanism by which a polypeptide chain thread itself spontaneously to attain a knotted conformation has been a major challenge in the field of protein folding. HP0242 is a homodimeric protein from Helicobacter pylori with intertwined helices to form a unique pseudo-knotted folding topology. A tandem HP0242 repeat has been constructed to become the first engineered trefoil-knotted protein. Its small size renders it a model system for computational analyses to examine its folding and knotting pathways. Here we report a multi-parametric study on the folding stability and kinetics of a library of HP0242 variants, including the trefoil-knotted tandem HP0242 repeat, using far-UV circular dichroism and fluorescence spectroscopy. Equilibrium chemical denaturation of HP0242 variants shows the presence of highly populated dimeric and structurally heterogeneous folding intermediates. Such equilibrium folding intermediates retain significant amount of helical structures except those at the N- and C-terminal regions in the native structure. Stopped-flow fluorescence measurements of HP0242 variants show that spontaneous refolding into knotted structures can be achieved within seconds, which is several orders of magnitude faster than previously observed for other knotted proteins. Nevertheless, the complex chevron plots indicate that HP0242 variants are prone to misfold into kinetic traps, leading to severely rolled-over refolding arms. The experimental observations are in general agreement with the previously reported molecular dynamics simulations. Based on our results, kinetic folding pathways are proposed to qualitatively describe the complex folding processes of HP0242 variants.The project is supported by a Career Development Award of the International Human Frontier Science Program, and funding from the Ministry of Science and Technology, National Tsing Hua University and Academia Sinica, Taiwan. Yu-Nan Liu was a recipient of a short-term EMBO fellowship to carry out preliminary experiments in Dr Sophie Jackson’s laboratory at the Department of Chemistry, University of Cambridge.This is the author accepted manuscript. The final version is available at http://dx.doi.org/10.1088/0953-8984/27/35/350301

Is an intermediate state populated on the folding pathway of ubiquitin?

AbstractIn the last couple of years, there has been increasing debate as to the presence and role of intermediate states on the folding pathways of several small proteins, including the 76-residue protein ubiquitin. Here, we present detailed kinetic studies to establish whether an intermediate state is ever populated during the folding of this protein. We show that the differences observed in previous studies are attributable to the transient aggregation of the protein during folding. Using a highly soluble construct of ubiquitin, which does not aggregate during folding, we establish the conditions in which an intermediate state is sufficiently stable to be observed by kinetic measurements

Glucagon-like peptide 1 aggregates into low-molecular-weight oligomers off-pathway to fibrillation

The physical stability of peptide-based drugs is of great interest to the pharmaceutical industry. Glucagon-like peptide 1 (GLP-1) is a 31-amino acid peptide hormone, the analogs of which are frequently used in the treatment of type 2 diabetes. We investigated the physical stability of GLP-1 and its C-terminal amide derivative, GLP-1-Am, both of which aggregate into amyloid fibrils. While off-pathway oligomers have been proposed to explain the unusual aggregation kinetics observed previously for GLP-1 under specific conditions, these oligomers have not been studied in any detail. Such states are important as they may represent potential sources of cytotoxicity and immunogenicity. Here, we identified and isolated stable, low-molecular-weight oligomers of GLP-1 and GLP-1-Am, using size-exclusion chromatography. Under the conditions studied, isolated oligomers were shown to be resistant to fibrillation or dissociation. These oligomers contain between two and five polypeptide chains and they have a highly disordered structure as indicated by a variety of spectroscopic techniques. They are highly stable with respect to time, temperature, or agitation despite their noncovalent character, which was established using liquid chromatography-mass spectrometry and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results provide evidence of stable, low-molecular-weight oligomers that are formed by an off-pathway mechanism which competes with amyloid fibril formation

The Knotted Protein UCH-L1 Exhibits Partially Unfolded Forms under Native Conditions that Share Common Structural Features with Its Kinetic Folding Intermediates.

The human ubiquitin C-terminal hydrolase, UCH-L1, is an abundant neuronal deubiquitinase that is associated with Parkinson's disease. It contains a complex Gordian knot topology formed by the polypeptide chain alone. Using a combination of fluorescence-based kinetic measurements, we show that UCH-L1 has two distinct kinetic folding intermediates that are transiently populated on parallel pathways between the denatured and native states. NMR hydrogen-deuterium exchange (HDX) experiments indicate the presence of partially unfolded forms (PUFs) of UCH-L1 under native conditions. HDX measurements as a function of urea concentration were used to establish the structure of the PUFs and pulse-labelled HDX NMR was used to show that the PUFs and the folding intermediates are likely the same species. In both cases, a similar stable core encompassing most of the central β-sheet is highly structured and α-helix 3, which is partially formed, packs against it. In contrast to the stable β-sheet core, the peripheral α-helices display significant local fluctuations leading to rapid exchange. The results also suggest that the main difference between the two kinetic intermediates is structure and packing of α-helices 3 and 7 and the degree of structure in β-strand 5. Together, the fluorescence and NMR results establish that UCH-L1 neither folds through a continuum of pathways nor by a single discrete pathway. Its folding is complex, the β-sheet core forms early and is present in both intermediate states, and the rate-limiting step which is likely to involve the threading of the chain to form the 52-knot occurs late on the folding pathway.This work was supported by the National Science Council (99-2911-I-007-034 and 104-2113-M-001-016), National Tsing Hua University and Academia Sinica, Taiwan. S.-T.D.H. was supported by a Career Development Award (CDA- 00025/2010-C) from the International Human Frontier Science Program. The NMR spectra were obtained at the NMR facility of the Department of Chemistry, University of Cambridge and at the Core Facility for Protein Structural Analysis, supported by the National Core Facility Program for Biotechnology, Taiwan.This is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.jmb.2016.04.00

Context-dependent nature of destabilizing mutations on the stability of fkbp12

ABSTRACT: The context-dependent nature in which mutations affect protein stability was investigated using the FK506-binding protein, FKBP12. Thirty-four mutations were made at sites throughout the protein, including residues located in the hydrophobic core, the -sheet, and the solvent-exposed face of the R-helix. Urea-induced denaturation experiments were used to measure the change in stability of the mutants relative to that of the wild type (∆∆G U-F ). The results clearly show that the extent of destabilization, or stabilization, is highly context-dependent. Correlations were sought in order to link ∆∆G U-F to various structural parameters. The strongest correlation found was between ∆∆G U-F and N, the number of methyl-(ene) groups within a 6 Å radius of the group(s) deleted. For mutations of buried hydrophobic residues, a correlation coefficient of 0.73 (n ) 16,where n is the number of points) was obtained. This increased to 0.81 (n ) 24) on inclusion of mutations of partially buried hydrophobic residues. These data could be superimposed on data obtained for other proteins for which similarly detailed studies have been performed. Thus, the contribution to stability from hydrophobic side chains, independent of the extent to which a side chain is buried, can be estimated quantitatively using N. This correlation appears to be a general feature of all globular proteins. The effect on stability of mutating polar and charged residues in the R-helix and -sheet was also found to be highly context-dependent. Previous experimental and statistical studies have shown that specific side chains can stabilize the N-caps of R-helices in proteins. Substitutions of Ile56 to Thr and Asp at the N-cap of the R-helix of FKBP12, however, were found to be highly destabilizing. Thus, the intrinsic propensities of an amino acid for a particular element of secondary structure can easily be outweighed by tertiary packing factors. This study highlights the importance of packing density in determining the contribution of a residue to protein stability. This is the most important factor that should be taken into consideration in protein design. To design novel proteins, or rationally alter existing ones, a quantitative understanding of the factors that affect the stability of the native state is required. For proteins without disulfide bonds, noncovalent interactionsssuch as hydrophobic interactions, hydrogen bonds, and electrostatic interactionssdetermine protein stability (1). Protein engineering studies have provided an abundance of information on the relationship between protein structure and stability. Studies on hydrophobic groups (2-13) have shown that the packing of nonpolar groups and burial of hydrophobic surface area are the dominant forces in the stabilization of proteins. Studies on both fully and partially buried hydrophobic residues in barnase, CI2, and staphylococcal nuclease have shown correlations between the change in protein stability upon mutation (∆∆G U-F ) and both the packing density [number of methyl(ene) groups within a certain radius of the nonpolar groups removed

The Amyloid Fibril-Forming β-Sheet Regions of Amyloid β and α-Synuclein Preferentially Interact with the Molecular Chaperone 14-3-3ζ.

14-3-3 proteins are abundant, intramolecular proteins that play a pivotal role in cellular signal transduction by interacting with phosphorylated ligands. In addition, they are molecular chaperones that prevent protein unfolding and aggregation under cellular stress conditions in a similar manner to the unrelated small heat-shock proteins. In vivo, amyloid β (Aβ) and α-synuclein (α-syn) form amyloid fibrils in Alzheimer's and Parkinson's diseases, respectively, a process that is intimately linked to the diseases' progression. The 14-3-3ζ isoform potently inhibited in vitro fibril formation of the 40-amino acid form of Aβ (Aβ40) but had little effect on α-syn aggregation. Solution-phase NMR spectroscopy of 15N-labeled Aβ40 and A53T α-syn determined that unlabeled 14-3-3ζ interacted preferentially with hydrophobic regions of Aβ40 (L11-H21 and G29-V40) and α-syn (V3-K10 and V40-K60). In both proteins, these regions adopt β-strands within the core of the amyloid fibrils prepared in vitro as well as those isolated from the inclusions of diseased individuals. The interaction with 14-3-3ζ is transient and occurs at the early stages of the fibrillar aggregation pathway to maintain the native, monomeric, and unfolded structure of Aβ40 and α-syn. The N-terminal regions of α-syn interacting with 14-3-3ζ correspond with those that interact with other molecular chaperones as monitored by in-cell NMR spectroscopy

Donacija Ivana Generalića iz 1980. godine Muzeju grada Koprivnice : uz 100. godišnjicu rođenja

For influenza A and B viruses to be infectious, they require eight viral RNA (vRNA) genome segments to be packaged into virions. For efficient packaging, influenza A viruses utilize cis-acting vRNA sequences, containing both non-coding and protein coding regions of each segment. Whether influenza B viruses have similar packaging signals is unknown. Here we show that coding regions at the 3' and 5' ends of the influenza B virus vRNA segment 4 are required for genome packaging, with the first 30 nt at each end essential for this process. Synonymous mutation of these regions led to virus attenuation, an increase in defective particle production and a reduction in packaging of multiple vRNAs. Overall, our data suggest that the influenza B virus vRNA gene segments likely interact with each other during the packaging process, which is driven by cis-acting packaging signals that extend into protein coding regions of the vRNA.PostprintPeer reviewe

Inter-model comparison of global hydroxyl radical (OH) distributions and their impact on atmospheric methane over the 2000–2016 period

The modeling study presented here aims to estimate how uncertainties in global hydroxyl radical (OH) distributions, variability, and trends may contribute to resolving discrepancies between simulated and observed methane (CH4) changes since 2000. A multi-model ensemble of 14 OH fields was analyzed and aggregated into 64 scenarios to force the offline atmospheric chemistry transport model LMDz (Laboratoire de Meteorologie Dynamique) with a standard CH4 emission scenario over the period 2000–2016. The multi-model simulated global volume-weighted tropospheric mean OH concentration ([OH]) averaged over 2000–2010 ranges between 8:7*10^5 and 12:8*10^5 molec cm-3. The inter-model differences in tropospheric OH burden and vertical distributions are mainly determined by the differences in the nitrogen oxide (NO) distributions, while the spatial discrepancies between OH fields are mostly due to differences in natural emissions and volatile organic compound (VOC) chemistry. From 2000 to 2010, most simulated OH fields show an increase of 0.1–0:3*10^5 molec cm-3 in the tropospheric mean [OH], with year-to-year variations much smaller than during the historical period 1960–2000. Once ingested into the LMDz model, these OH changes translated into a 5 to 15 ppbv reduction in the CH4 mixing ratio in 2010, which represents 7%–20% of the model-simulated CH4 increase due to surface emissions. Between 2010 and 2016, the ensemble of simulations showed that OH changes could lead to a CH4 mixing ratio uncertainty of > 30 ppbv. Over the full 2000–2016 time period, using a common stateof- the-art but nonoptimized emission scenario, the impact of [OH] changes tested here can explain up to 54% of the gap between model simulations and observations. This result emphasizes the importance of better representing OH abundance and variations in CH4 forward simulations and emission optimizations performed by atmospheric inversions