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

Tying up the Loose Ends : A Mathematically Knotted Protein

Knots have attracted scientists in mathematics, physics, biology, and engineering. Long flexible thin strings easily knot and tangle as experienced in our daily life. Similarly, long polymer chains inevitably tend to get trapped into knots. Little is known about their formation or function in proteins despite >1,000 knotted proteins identified in nature. However, these protein knots are not mathematical knots with their backbone polypeptide chains because of their open termini, and the presence of a "knot" depends on the algorithm used to create path closure. Furthermore, it is generally not possible to control the topology of the unfolded states of proteins, therefore making it challenging to characterize functional and physicochemical properties of knotting in any polymer. Covalently linking the amino and carboxyl termini of the deeply trefoil-knotted YibK from Pseudomonas aeruginosa allowed us to create the truly backbone knotted protein by enzymatic peptide ligation. Moreover, we produced and investigated backbone cyclized YibK without any knotted structure. Thus, we could directly probe the effect of the backbone knot and the decrease in conformational entropy on protein folding. The backbone cyclization did not perturb the native structure and its cofactor binding affinity, but it substantially increased the thermal stability and reduced the aggregation propensity. The enhanced stability of a backbone knotted YibK could be mainly originated from an increased ruggedness of its free energy landscape and the destabilization of the denatured state by backbone cyclization with little contribution from a knot structure. Despite the heterogeneity in the side-chain compositions, the chemically unfolded cyclized YibK exhibited several macroscopic physico-chemical attributes that agree with theoretical predictions derived from polymer physics.Peer reviewe

Predicted CP probabilities (CPred scores) for <i>Npu</i> DnaE (NpuInt), <i>Ssp</i> DnaE and <i>Ssp</i> DnaB inteins (<i>Npu: Nostoc punctiforme</i> and <i>Ssp: Synechchotcystis sp.</i>) plotted <i>versus</i> residue number.

<p>The coordinates of NpuInt (PDB code: 2KEQ) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043820#pone.0043820-Oeemig1" target="_blank">[23]</a>, <i>Ssp</i> DnaE (PDB code: 1ZD7) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043820#pone.0043820-Sun1" target="_blank">[25]</a> and <i>Ssp</i> DnaB (PDB code: 1MI8) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043820#pone.0043820-Ding1" target="_blank">[24]</a> were submitted to CPred to estimate the CP probability as a function of residue number. The locations of reported functional split sites (efficiency >50%) are indicated by closed black triangles, and asterisks indicate the naturally occurring split sites. The reported non-functional split sites in <i>Ssp</i> DnaE and <i>Ssp</i> DnaB are marked by closed red triangles. Two newly predicted <i>Npu</i> DnaE intein CP sites at residues 12 and 36 are indicated by open black triangles.</p

Correspondence of reported split sites and CPred scores.

a<p>The PDB coordinates used for prediction were as follows: GFP, 1GFL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043820#pone.0043820-Yang1" target="_blank">[45]</a>; β-lactamase, 1BTL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043820#pone.0043820-Jelsch1" target="_blank">[46]</a>; DHFR, 1HFR <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043820#pone.0043820-Cody1" target="_blank">[47]</a>; ubiquitin, 1UBQ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043820#pone.0043820-VijayKumar1" target="_blank">[48]</a>; firefly luciferase, 2D1S <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043820#pone.0043820-Nakatsu1" target="_blank">[49]</a>; RNase, 1FS3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043820#pone.0043820-Chatani1" target="_blank">[50]</a>.</p

Thermodynamics and stability of protein unfolding of single-chain and split NpuInts.

<p>Thermodynamics and stability of protein unfolding of single-chain and split NpuInts.</p

Superposition of the ¹H-¹⁵N HSQC of NpuInt SP and the corresponding CP variant showing the overall correspondence between two spectra.

<p>(A) NpuInt CP36 (blue) and NpuInt SP36 (red). (B) NpuInt CP102 (blue) and NpuInt SP102 (red).</p

<i>In vitro</i> protein <i>trans</i>-splicing (PTS) assay.

<p>Time course of the protein ligation of GB1 and GB1 by (A) the naturally occurring split intein SP102 and (B) the engineered split intein SP36. (C) PTS kinetic analysis of the ligated product of GB1 duplication from SDS-PAGE after reaction using SP102 (blue line), SP36 (red line) and SP12 (green line). The schematic plot of the PTS reactions is depicted at the side.</p

Thermal stability of intein variants monitored by CD ellipticity at 224 nm.

<p>(A) Far-UV CD spectra of NpuInt variants at 25°C. (B) The two-state thermal denaturation profiles of NpuInt variants. (C) Temperature dependence of the denaturation thermodynamics using van’t Hoff analysis.</p

Secondary structure of the CP variants (A) CP36 and (B) CP102, evaluated according to the parameter Δδ_Cα−Δδ_Cβ.

<p>The chemical shift values for ¹³C_α and ¹³C_β of CP36 and CP102 were obtained, and Δδ_Cα and Δδ_Cβ were calculated from the differences between the experimental values and random coil values. The value of Δδ_Cα−Δδ_Cβ for each residue represents the average of three consecutive residues, centered at the particular residue. The Δδ_Cα−Δδ_Cβ value derived from native NpuInt C1G (indicated by closed circles) is overlaid onto the CP results for comparison. The corresponding secondary structure of C1G is depicted at the top. The difference (ΔΔδ) in (Δδ_Cα−Δδ_Cβ) between each CP variant and C1G was calculated and is indicated at the bottom of the figure.</p

Circular Permutation Prediction Reveals a Viable Backbone Disconnection for Split Proteins: An Approach in Identifying a New Functional Split Intein

<div><p>Split-protein systems have emerged as a powerful tool for detecting biomolecular interactions and reporting biological reactions. However, reliable methods for identifying viable split sites are still unavailable. In this study, we demonstrated the feasibility that valid circular permutation (CP) sites in proteins have the potential to act as split sites and that CP prediction can be used to search for internal permissive sites for creating new split proteins. Using a protein ligase, intein, as a model, CP predictor facilitated the creation of circular permutants in which backbone opening imposes the least detrimental effects on intein folding. We screened a series of predicted intein CPs and identified stable and native-fold CPs. When the valid CP sites were introduced as split sites, there was a reduction in folding enthalpy caused by the new backbone opening; however, the coincident loss in entropy was sufficient to be compensated, yielding a favorable free energy for self-association. Since split intein is exploited in protein semi-synthesis, we tested the related protein <em>trans</em>-splicing (PTS) activities of the corresponding split inteins. Notably, a novel functional split intein composed of the N-terminal 36 residues combined with the remaining C-terminal fragment was identified. Its PTS activity was shown to be better than current reported two-piece intein with a short N-terminal segment. Thus, the incorporation of <em>in silico</em> CP prediction facilitated the design of split intein as well as circular permutants.</p> </div