The use of nanoparticles (NPs) in biomedical applications requires an in-depth understanding of the mechanisms by which NPs interact with biomolecules. NPs associating with proteins may interfere with protein-protein interactions and affect cellular communication pathways, however the impact of NPs on biomolecular recognition remains poorly characterized. In this respect, particularly relevant is the study of NP-induced functional perturbations of proteins implicated in the regulation of key biochemical pathways. Ubiquitin (Ub) is a prototypical protein post-translational modifier playing a central role in numerous essential biological processes. To contribute to an understanding of the interactions between this universally distributed biomacromolecule and NPs, we investigated the adsorption of polyhydroxylated fullerene to monomeric Ub and to a minimal polyubiquitin chain in vitro at atomic resolution. Site-resolved chemical shift and intensity perturbations of Ub’s NMR signals, together with 15N spin relaxation rate changes, exchange saturation transfer effects, and fluorescence quenching data were consistent with the reversible formation of soluble aggregates incorporating fullerenol clusters. Specific interaction epitopes were identified, coincident with functional recognition sites in monomeric and lysine48-linked dimeric Ub. Fullerenol appeared to target the open state of the dynamic structure of dimeric Ub according to a conformational selection mechanism. Importantly, protein-NP association prevented enzyme-catalyzed synthesis of polyubiquitin chains. Our findings provide experiment-based insight into protein/fullerenol recognition, with implications in functional biomolecular communication, including regulatory protein turnover, and for the opportunity of therapeutic intervention in Ub-dependent cellular pathways.
Ddi1 belongs to a family of shuttle proteins targeting polyubiquitinated substrates for proteasomal degradation. Unlike the other proteasomal shuttles, Rad23 and Dsk2, Ddi1 remains an enigma: its function is not fully understood and structural properties are poorly characterized. We determined the structure and binding properties of the ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains of Ddi1 from Saccharomyces cerevisiae. We found that, while Ddi1UBA forms a characteristic UBA:ubiquitin complex, Ddi1UBL has entirely uncharacteristic binding preferences. Despite having a ubiquitin-like fold, Ddi1UBL does not interact with typical UBL-receptors but, unexpectedly, binds ubiquitin, forming a unique interface mediated by hydrophobic contacts and by salt-bridges between oppositely-charged residues of Ddi1UBL and ubiquitin. In stark contrast with ubiquitin and other UBLs, the β-sheet surface of Ddi1UBL is negatively charged and, therefore, is recognized in a completely different way. The dual functionality of Ddi1UBL, capable of binding both ubiquitin and proteasome, suggests a novel mechanism for Ddi1 as a proteasomal shuttle.
Multiple studies demonstrate that ubiquitination of proteins codes for regulation of cell differentiation, apoptosis, endocytosis, and many other cellular functions. There is great interest in, and considerable effort being given to defining the relationships between the structures of polyubiquitin modifications and the fates of the modified proteins. Does each ubiquitin modification achieve a specific effect, much like phosphorylation, or is ubiquitin like glycosylation, where there is heterogeneity and redundancy in the signal? The sensitive analytical tools needed to address such questions readily are not yet mature. To lay the foundation for mass spectrometry-based studies of the ubiquitin code we have assembled seven isomeric diubiquitins with all-native sequences and isopeptide linkages. Using these compounds as standards enables the development and testing of a new mass spectrometry-based strategy tailored specifically to characterize the number and sites of isopeptide linkages in polyubiquitin chains. Here we report the use of Asp-selective acid cleavage, separation by reverse phase HPLC, and characterization by tandem mass spectrometry to distinguish and characterize all seven isomeric lysine-linked ubiquitin dimers.
cleavage has been characterized at modified
K48 residues in polyubiquitins. In particular, the tryptic products
of all seven of the lysine-linked dimers of ubiquitin and of three
trimers—linear Ub–48Ub–48Ub, linear Ub–63Ub–63Ub, and
the branched trimer [Ub]2–6,48Ub—have
been analyzed. In addition to the peptide products expected under
commonly used tryptic conditions, we observe that peptides are formed
with an unexpected ε-glycinylglycinyl-Lys carboxyl terminus
when the site of linkage is Lys48. Trypsin from three different commercial
sources exhibited this aberration. Initial cleavage at R74 is proposed
in a distal ubiquitin to produce a glycinylglycinyl-lysine residue
which is bound by trypsin.
A frame-shift mutation in the transcript of the ubiquitin-B gene leads to a C-terminally extended ubiquitin, UBB+1. UBB+1 has been considered to inhibit proteasomes, and as such to be the underlying cause for toxic protein buildup correlated with certain neuropathological conditions. We demonstrated that expression of extended ubiquitin variants led to accumulation of heterogeneously-linked polyubiquitin conjugates indicating a pervasive effect on ubiquitin-dependent turnover. 20S proteasomes selectively proteolysed ubiquitin extensions, yet no evidence for inhibition of 26S holoenzymes was found. However, among susceptible targets for inhibition was Ubp6, the primary enzyme responsible for disassembly of lysine-48 linkages at 26S proteasomes. Processing of lysine-48 and lysine-63 linkages by other deubiquitinating enzymes (DUBs) was also inhibited. Disruption of ubiquitin-dependent degradation by extended ubiquitin variants may therefore be attributed to their inhibitory effect on select DUBs, thus shifting research efforts related to protein accumulation in neurodegenerative processes from proteasomes to DUBs.
ubiquitination; rubylation; ubiquitin chains; nonenzymatic assembly; deubiquitinases
26S proteasome, a major regulatory protease in eukaryotes, consists of a 20S proteolytic core particle (CP) capped by a 19S regulatory particle (RP). The 19S RP is divisible into base and lid sub-complexes. Even within the lid, subunits have been demarcated into two modules: module 1 (Rpn5, Rpn6, Rpn8, Rpn9 and Rpn11), which interacts with both CP and base sub-complexes and module 2 (Rpn3, Rpn7, Rpn12 and Rpn15) that is attached mainly to module 1. We now show that suppression of RPN11 expression halted lid assembly yet enabled the base and 20S CP to pre-assemble and form a base-CP. A key role for Regulatory particle non-ATPase 11 (Rpn11) in bridging lid module 1 and module 2 subunits together is inferred from observing defective proteasomes in rpn11–m1, a mutant expressing a truncated form of Rpn11 and displaying mitochondrial phenotypes. An incomplete lid made up of five module 1 subunits attached to base-CP was identified in proteasomes isolated from this mutant. Re-introducing the C-terminal portion of Rpn11 enabled recruitment of missing module 2 subunits. In vitro, module 1 was reconstituted stepwise, initiated by Rpn11–Rpn8 heterodimerization. Upon recruitment of Rpn6, the module 1 intermediate was competent to lock into base-CP and reconstitute an incomplete 26S proteasome. Thus, base-CP can serve as a platform for gradual incorporation of lid, along a proteasome assembly pathway. Identification of proteasome intermediates and reconstitution of minimal functional units should clarify aspects of the inner workings of this machine and how multiple catalytic processes are synchronized within the 26S proteasome holoenzymes.
Defective proteasome 19S regulatory particles (RPs) were identified in rpn11f–m1, a proteasomal mutant with mitochondrial phenotypes. The Rpn11 subunit initiates assembly of a five-subunit lid module competent to integrate into pre-assembled base-20S core particle (CP), with subsequent recruitment of remaining lid subunits.
26S proteasome; 19S regulatory particle; 20S core particle; lid; base; MPN; PCI; rpn11-m1; CP, 20S core particle; DUB, deubiquitinase; polyUb, polyubiquitin; RP, 19S regulatory particle; TPP, trans proteomic pipeline; Ub, ubiquitin; WCE, whole cell extract; WT, wild-type
Mutations at solvent inaccessible core positions in proteins can impact function through many biophysical mechanisms including alterations to thermodynamic stability and protein dynamics. As these properties of proteins are difficult to investigate, the impacts of core mutations on protein function are poorly understood for most systems. Here, we determined the effects of alanine mutations at all 15 core positions in ubiquitin on function in yeast. The majority (13 of 15) of alanine substitutions supported yeast growth as the sole ubiquitin. The two null mutants (I30A and L43A) were both less stable to temperature-induced unfolding in vitro than wild-type, but were well folded at physiological temperatures. Heteronuclear NMR studies indicated that the L43A mutation reduces temperature stability while retaining a ground-state structure similar to wild-type. This structure enables L43A to bind to common ubiquitin receptors in vitro. Many of the core alanine ubiquitin mutants, including one of the null variants (I30A), exhibited an increased accumulation of high molecular weight species, suggesting that these mutants caused a defect in the processing of ubiquitin-substrate conjugates. In contrast, L43A exhibited a unique accumulation pattern with reduced levels of high molecular weight species and undetectable levels of free ubiquitin. When conjugation to other proteins was blocked, L43A ubiquitin accumulated as free ubiquitin in yeast. Based on these findings we speculate that ubiquitin's stability to unfolding may be required for efficient recycling during proteasome-mediated substrate degradation.
thermodynamic stability; structural dynamics; protein function; proteasome; ubiquitin recycling
Structure refinement protocols based on small-angle X-ray scattering iteratively adjust the posited positions of atoms in a structure via a gradient-based constrained optimization to ensure that experimental scattering profile data are matched. This requires computation of the profile and its gradient with respect to atomic coordinates at every step. The fast algorithms proposed in this article reduce these to a cost that is linear in the number of atoms for any specified accuracy.
The need for fast approximate algorithms for Debye summation arises in computations performed in crystallography, small/wide-angle X-ray scattering and small-angle neutron scattering. When integrated into structure refinement protocols these algorithms can provide significant speed up over direct all-atom-to-all-atom computation. However, these protocols often employ an iterative gradient-based optimization procedure, which then requires derivatives of the profile with respect to atomic coordinates. This article presents an accurate, O(N) cost algorithm for the computation of scattering profile derivatives. The results reported here show orders of magnitude improvement in computational efficiency, while maintaining the prescribed accuracy. This opens the possibility to efficiently integrate small-angle scattering data into the structure determination and refinement of macromolecular systems.
small-angle scattering; wide-angle scattering; Jacobian; gradients
Motions are essential for protein function, and knowledge of protein dynamics is a key to our understanding the mechanisms underlying protein folding and stability, ligand recognition, allostery, and catalysis. In the last two decades, NMR relaxation measurements have become a powerful tool for characterizing backbone and side chain dynamics in complex biological macromolecules like proteins and nucleic acids. Accurate analysis of the experimental data in terms of motional parameters is an essential prerequisite for developing physical models of motions in order to paint an adequate picture of protein dynamics. Here I describe in detail how to use the software package DYNAMICS that was developed for accurate characterization of the overall tumbling and local dynamics in a protein from nuclear spin-relaxation rates measured by NMR. Step by step instructions are provided and illustrated through analysis of 15N relaxation data for protein G.
Relaxation; protein dynamics; order parameter; spectral density; dipolar coupling; chemical shift anisotropy; CSA; overall tumbling; rotational diffusion tensor; monomer-dimer equilibrium
Understanding of the effects of the backbone cyclization on the structure and dynamics of a protein is essential for using protein topology engineering to alter protein stability and function. Here we have determined, for the first time, the structure and dynamics of the linear and various circular constructs of the N-SH3 domain from protein c-Crk. These constructs differ in the length and amino acid composition of the cyclization region. The backbone cyclization was carried out using intein-mediated intramolecular chemical ligation between the juxtaposed N- and the C-termini. The structure and backbone dynamics studies were performed using solution NMR. Our data suggest that the backbone cyclization has little effect on the overall three-dimensional structure of the SH3 domain: besides the termini, only minor structural changes were found in the proximity of the cyclization region. In contrast to the structure, backbone dynamics are significantly affected by the cyclization. On the subnanosecond time scale, the backbone of all circular constructs on average appears more rigid than that of the linear SH3 domain; this effect is observed over the entire backbone and is not limited to the cyclization site. The backbone mobility of the circular constructs becomes less restricted with increasing length of the circularization loop. In addition, significant conformational exchange motions (on the sub-millisecond time scale) were found in the N-Src loop and in the adjacent β-strands in all circular constructs studied in this work. These effects of backbone cyclization on protein dynamics have potential implications for the stability of the protein fold and for ligand binding.
circular SH3; c-Crk; backbone dynamics; protein cyclization; inteins
To facilitate rigorous analysis of molecular motions in proteins, DNA, and RNA, we present a new version of ROTDIF, a program for determining the overall rotational diffusion tensor from single-or multiple-field Nuclear Magnetic Resonance (NMR) relaxation data. We introduce four major features that expand the program’s versatility and usability. The first feature is the ability to analyze, separately or together, 13C and/or 15N relaxation data collected at a single or multiple fields. A significant improvement in the accuracy compared to direct analysis of R2/R1 ratios, especially critical for analysis of 13C relaxation data, is achieved by subtracting high-frequency contributions to relaxation rates. The second new feature is an improved method for computing the rotational diffusion tensor in the presence of biased errors, such as large conformational exchange contributions, that significantly enhances the accuracy of the computation. The third new feature is the integration of the domain alignment and docking module for relaxation-based structure determination of multi-domain systems. Finally, to improve accessibility to all the program features, we introduced a graphical user interface (GUI) that simplifies and speeds up the analysis of the data. Written in Java, the new ROTDIF can run on virtually any computer platform. In addition, the new ROTDIF achieves an order of magnitude speedup over the previous version by implementing a more efficient deterministic minimization algorithm. We not only demonstrate the improvement in accuracy and speed of the new algorithm for synthetic and experimental 13C and 15N relaxation data for several proteins and nucleic acids, but also show that careful analysis required especially for characterizing RNA dynamics allowed us to uncover subtle conformational changes in RNA as a function of temperature that were opaque to previous analysis.
rotational diffusion tensor; ELM; ELMDOCK; ROTDIF
Structural analysis of proteins and nucleic acids is complicated by their inherent flexibility, conferred, for example, by linkers between their contiguous domains. Therefore, the macromolecule needs to be represented by an ensemble of conformations instead of a single conformation. Determining this ensemble is challenging because the experimental data are a convoluted average of contributions from multiple conformations. As the number of the ensemble degrees of freedom generally greatly exceeds the number of independent observables, directly deconvolving experimental data into a representative ensemble is an ill-posed problem. Recent developments in sparse approximations and compressive sensing have demonstrated that useful information can be recovered from underdetermined (ill-posed) systems of linear equations by using sparsity regularization. Inspired by these advances, we designed Sparse Ensemble Selection (SES) method for recovering multiple conformations from a limited number of observations. SES is more general and accurate than previously published minimum-ensemble methods, and we use it to obtain representative conformational ensembles of Lys48-linked di-ubiquitin, characterized by the residual dipolar coupling data measured at several pH conditions. These representative ensembles are validated against NMR chemical shift perturbation data and compared to maximum-entropy results. The SES method reproduced and quantified the previously observed pH dependence of the major conformation of Lys48-linked di-ubiquitin, and revealed lesser-populated conformations that are pre-organized for binding known di-ubiquitin receptors, thus providing insights into possible mechanisms of receptor recognition by polyubiquitin. SES is applicable to any experimental observables that can be expressed as a weighted linear combination of data for individual states.
In Every Direction
Chemical synthesis of proteins allowed the synthesis of ubiquitin chains modified in the vicinity of the isopeptide peptide to examine their behavior with deubiquitinases and ubiquitin binding domains. Our results set the ground for the generation of unique probes for studying the interactions of these chains with various ubiquitin-interacting proteins.
Ubiquitin Chains; Deubiquitinases; Ubiquitination; Protein synthesis; Peptide ligation
K11-linked polyubiquitin chains play important signaling and regulatory roles in both degradative and non-proteolytic pathways in eukaryotes. To understand the structural basis of how these chains are recognized and distinguished from other polyubiquitins, we determined solution structures of K11-linked di-ubiquitin (K11-Ub2) in the absence and presence of salt. These structures reveal that K11-Ub2 adopts conformations distinct from those of K48-linked or K63-linked chains. Importantly, our solution NMR and SANS data are inconsistent with published crystal structures of K11-Ub2. We found that increasing salt concentration compacts K11-Ub2 and strengthens interactions between the two Ub units. Binding studies indicate that K11-Ub2 interacts with ubiquitin-receptor proteins from both proteasomal and non-proteasomal pathways, but with intermediate affinity and different binding modes than either K48-linked or K63-linked di-ubiquitin. Our data support the hypothesis that polyubiquitin chains of different linkages possess unique conformational and dynamical properties allowing them to be recognized differently by downstream receptor proteins.
Polymeric chains of a small protein ubiquitin are involved in regulation of nearly all vital processes in eukaryotic cells. Elucidating the signaling properties of polyubiquitin requires the ability to make these chains in vitro. In recent years, chemical and chemical-biology tools have been developed that produce fully natural isopeptide-linked polyUb chains with no need for linkage-specific ubiquitin-conjugating enzymes. These methods produced unbranched chains (in which no more than one lysine per ubiquitin is conjugated to another ubiquitin). Here we report a nonenzymatic method for the assembly of fully natural isopeptide-linked branched polyubiquitin chains. This method is based on the use of mutually orthogonal removable protecting groups (e.g., Boc- and Alloc-) on lysines combined with an Ag-catalyzed condensation reaction between a C-terminal thioester on one ubiquitin and a specific ε-amine on another ubiquitin, and involves genetic incorporation of more than one Lys(Boc) at the desired linkage positions in the ubiquitin sequence. We demonstrate our method by making a fully natural branched tri-ubiquitin containing isopeptide linkages via Lys11 and Lys33, and a 15N-enriched proximal ubiquitin, which enabled monomer-specific structural and dynamical studies by NMR. Furthermore, we assayed disassembly of branched and unbranched tri-ubiquitins as well as control di-ubiquitins by the yeast proteasome-associated deubiquitinase Ubp6. Our results show that Ubp6 can recognize and disassemble a branched polyubiquitin, wherein cleavage preferences for individual linkages are retained. Our spectroscopic and functional data suggest that, at least for the chains studied here, the isopeptide linkages are effectively independent of each other. Together with our method for nonenzymatic assembly of unbranched polyubiquitin, these developments now provide tools for making fully natural polyubiquitin chains of essentially any type of linkage and length.
ubiquitin; polyubiquitin; branched chain; isopeptide bond; unnatural amino acids; deubiquitination
Research on ubiquitin (Ub) signaling has focused primarily on homogeneously-linked polyUb. Although polyUb containing different linkages within the same chain exist, their structures and signaling properties are unknown. These mixed-linkage chains could be unbranched (i.e., no more than one lysine- or methionine-linkage per Ub) or branched. Here we examined the structure, dynamics, receptor selectivity, and disasssembly of branched and unbranched tri-Ub containing both K48 and K63-linkages. Each linkage was virtually indistinguishable from its counterpart in homogeneously-linked polyUb. Linkage-selective receptors from hHR23A and Rap80 preferentially bound to the K48- or K63-linkages in the branched trimer. Linkage-selective deubiquitinases specifically cleaved their cognate Ub-Ub linkages in mixed-linkage chains, and the 26S proteasome recognized and processed branched tri-Ub. We conclude that mixed-linkage chains retain the distinctive signaling properties of their K48- and K63-components, and that these multiple signals can be recognized by multiple linkage-specific receptors. Finally, we propose a new, comprehensive notation for Ub and Ub-like polymers.
The amino acid sequence of a protein governs its function. We used bulk competition and focused deep sequencing to investigate the effects of all ubiquitin point mutants on yeast growth rate. Many aspects of ubiquitin function have been carefully studied, which enabled interpretation of our growth analyses in light of a rich structural, biophysical and biochemical knowledge base. In one highly sensitive cluster on the surface of ubiquitin almost every amino acid substitution caused growth defects. In contrast, the opposite face tolerated virtually all possible substitutions. Surface locations between these two faces exhibited intermediate mutational tolerance. The sensitive face corresponds to the known interface for many binding partners. Across all surface positions, we observe a strong correlation between burial at structurally characterized interfaces and the number of amino acid substitutions compatible with robust growth. This result indicates that binding is a dominant determinant of ubiquitin function. In the solvent inaccessible core of ubiquitin all positions tolerated a limited number of substitutions, with hydrophobic amino acids especially interchangeable. Some mutations null for yeast growth were previously shown to populate folded conformations indicating that for these mutants subtle changes to conformation caused functional defects. The most sensitive region to mutation within the core was located near the C-terminus that is a focal binding site for many critical binding partners. These results indicate that core mutations may frequently cause functional defects through subtle disturbances to structure or dynamics.
fitness; structure; function; thermodynamic stability; sequence
Post-translational modification of proteins by covalent attachment of ubiquitin or a polyubiquitin chain is involved in myriad of processes in eukaryotic cells. The particular outcome of ubiquitination is directed by the length of the ubiquitin conjugate and its linkage composition. Among seven possible isopeptide linkage sites in ubiquitin, K48 and K63 occur most commonly and act as distinct cellular signals. Strategies are reported here for analysis of linkage sites and complexity of K63-linked polyubiquitin chains, based on rapid chemical proteolysis at aspartate residues combined with immunoprecipitation and mass spectrometry. Rapid chemical proteolysis at aspartate residues results in K63-linked peptides with truncated branches, which enable identification and characterization of stretches of consecutive K63 linkages on generally available instruments. A characteristic cleavage pattern and a characteristic fragmentation pattern allow recognition of K63 oligomers in proteolytic mixtures. Engineered K63-linked polyubiquitin chains of defined lengths were used to evaluate and demonstrate the method. In-gel microwave-supported acid hydrolysis was used to observe peptides specific to K63-linked ubiquitin dimers and trimers. Acid hydrolysis in solution, used in conjunction with linkage-specific immunoprecipitation, allowed more complex K63-linked branches to be characterized. Finally a substrate protein, UbcH5b, was conjugated to mono-ubiquitin and to polyubiquitin chains containing only K63 linkages, and the sites of conjugation and chain lengths were characterized.
Ubiquitin (Ub) is a small protein highly conserved among eukaryotes and involved in practically all aspects of eukaryotic cell biology. Polymeric chains assembled of covalently-linked Ub monomers function as molecular signals in the regulation of a host of cellular processes. Our previous studies have shown that the predominant state of Lys48-linked di- and tetra-Ub chains at near-physiological conditions is a closed conformation, in which the Ub/Ub interface is formed by the hydrophobic surface residues of the adjacent Ub units. Because these very residues are involved in (poly)Ub interactions with the majority of Ub-binding proteins, their sequestration at the Ub/Ub interface renders the closed conformation of polyUb binding incompetent. Thus the existence of open conformation(s) and the interdomain motions opening and closing the Ub/Ub interface is critical for the recognition of Lys48-linked polyUb by its receptors. Knowledge of the conformational properties of polyUb signal is essential for our understanding of its specific recognition by various Ub-receptors. Despite their functional importance, open states of Lys48-linked chains are poorly characterized. Here we report a crystal structure of the open state of Lys48-linked di-Ub. Moreover, using NMR, we examined interactions of the open state of this chain (at pH4.5) with a Lys48-linkage-selective receptor, the UBA2 domain of a shuttle protein hHR23a. Our results show that di-Ub binds UBA2 in the same mode and with comparable affinity as the closed state. Our data suggest a mechanism for polyUb signal recognition, whereby Ub-binding proteins select specific conformations out of the available ensemble of polyUb chain conformations.
ubiquitin; Lys48-linked diubiquitin; polyubiquitin; ubiquitin-associated domain; Lysine-48 linkage selectivity
Debye summation, which involves the summation of sinc functions of distances between all pair of atoms in three dimensional space, arises in computations performed in crystallography, small/wide angle X-ray scattering (SAXS/WAXS) and small angle neutron scattering (SANS). Direct evaluation of Debye summation has quadratic complexity, which results in computational bottleneck when determining crystal properties, or running structure refinement protocols that involve SAXS or SANS, even for moderately sized molecules. We present a fast approximation algorithm that efficiently computes the summation to any prescribed accuracy ε in linear time. The algorithm is similar to the fast multipole method (FMM), and is based on a hierarchical spatial decomposition of the molecule coupled with local harmonic expansions and translation of these expansions. An even more efficient implementation is possible when the scattering profile is all that is required, as in small angle scattering reconstruction (SAS) of macromolecules. We examine the relationship of the proposed algorithm to existing approximate methods for profile computations, and show that these methods may result in inaccurate profile computations, unless an error bound derived in this paper is used. Our theoretical and computational results show orders of magnitude improvement in computation complexity over existing methods, while maintaining prescribed accuracy.
Lys33 linked chain; segmental isotope labelling; semi-synthesis; ubiquitin chains; ubiquitination
Polymeric chains made of a small protein ubiquitin act as molecular signals regulating a variety of cellular processes controlling essentially all aspects of eukaryotic biology. Uncovering the mechanisms that allow differently linked polyubiquitin chains to serve as distinct molecular signals requires the ability to make these chains with the native connectivity, defined length, linkage composition, and in sufficient quantities. This however has been a major impediment in the ubiquitin field. Here we present a robust, efficient, and widely accessible method for controlled iterative non-enzymatic assembly of polyubiquitin chains using recombinant ubiquitin monomers as the primary building blocks. This method uses silver-mediated condensation reaction between the C-terminal thioester of one ubiquitin and the ε-amine of a specific lysine on the other ubiquitin. We augment the non-enzymatic approaches developed recently by using removable orthogonal amine-protecting groups, Alloc and Boc. The use of bacterially expressed ubiquitins allows cost-effective isotopic enrichment of any individual monomer in the chain. We demonstrate that our method yields completely natural polyubiquitin chains (free of mutations and linked through native isopeptide bonds) of essentially any desired length, linkage composition, and isotopic labeling scheme, and in milligram quantities. Specifically, we successfully made Lys11-linked di-, tri-, and tetra-ubiquitins, Lys33-linked di-ubiquitin, and a mixed-linkage Lys33,Lys11-linked tri-ubiquitin. We also demonstrate the ability to obtain, by high-resolution NMR, residue-specific information on ubiquitin units at any desired position in such chains. This method opens up essentially endless possibilities for rigorous structural and functional studies of polyubiquitin signals.
The relaxation of long-lived states (LLS) corresponds to the slow return to statistical thermal equilibrium between symmetric and antisymmetric proton spin states. This process is remarkably sensitive to the presence of external spins and can be used to obtain information about partial unfolding of proteins. We detected the appearance of a destabilized conformer of ubiquitin when urea is added to the protein in its native state. This conformer shows increased mobility in the C-terminus, which significantly extends the lifetimes of proton LLS magnetisation in Ser-65. These changes could not be detected by conventional measurements of T1 and T2 relaxation times of protons, and would hardly be sensed by carbon-13 or nitrogen-15 relaxation measurements. Conformers with similar dynamic and structural features, as revealed by LLS relaxation times, could be observed, in the absence of urea, in two ubiquitin mutants, L67S and L69S.
NMR; Proteins; Mutation; Ubiquitin; Long-Lived States; Singlet States
We present and evaluate a rigid-body, deterministic, molecular docking method, called ELMDOCK, that relies solely on the three-dimensional structure of the individual components and the overall rotational diffusion tensor of the complex, obtained from nuclear spin-relaxation measurements. We also introduce a docking method, called ELMPATIDOCK, derived from ELMDOCK and based on the new concept of combining the shape-related restraints from rotational diffusion with those from residual dipolar couplings, along with ambiguous contact/interface-related restraints obtained from chemical shift perturbations. ELMDOCK and ELMPATIDOCK use two novel approximations of the molecular rotational diffusion tensor that allow computationally efficient docking. We show that these approximations are accurate enough to properly dock the two components of a complex without the need to recompute the diffusion tensor at each iteration step. We analyze the accuracy, robustness, and efficiency of these methods using synthetic relaxation data for a large variety of protein-protein complexes. We also test our method on three protein systems for which the structure of the complex and experimental relaxation data are available, and analyze the effect of flexible unstructured tails on the outcome of docking. Additionally, we describe a method for integrating the new approximation methods into the existing docking approaches that use the rotational diffusion tensor as a restraint. The results show that the proposed docking method is robust against experimental errors in the relaxation data or structural rearrangements upon complex formation and is computationally more efficient than current methods. The developed approximations are accurate enough to be used in structure refinement protocols.
rigid-body docking; protein complexes; residual dipolar couplings; diffusion-guided molecular assembly; ellipsoid model; ELM; PATI