PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (33)
 

Clipboard (0)
None

Select a Filter Below

Year of Publication
Document Types
1.  Unique structural, dynamical, and functional properties of K11-linked polyubiquitin chains 
Structure (London, England : 1993)  2013;21(7):1168-1181.
Summary
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.
doi:10.1016/j.str.2013.04.029
PMCID: PMC3802530  PMID: 23823328
2.  Nonenzymatic assembly of branched polyubiquitin chains for structural and biochemical studies 
Bioorganic & medicinal chemistry  2013;21(12):3421-3429.
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.
doi:10.1016/j.bmc.2013.02.052
PMCID: PMC3665622  PMID: 23557636
ubiquitin; polyubiquitin; branched chain; isopeptide bond; unnatural amino acids; deubiquitination
3.  Mixed-linkage ubiquitin chains send mixed messages 
Summary
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.
doi:10.1016/j.str.2013.02.019
PMCID: PMC3654000  PMID: 23562397
4.  Analyses of the effects of all ubiquitin point mutants on yeast growth rate 
Journal of molecular biology  2013;425(8):1363-1377.
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.
doi:10.1016/j.jmb.2013.01.032
PMCID: PMC3615125  PMID: 23376099
fitness; structure; function; thermodynamic stability; sequence
5.  Proteomic Identification and Analysis of K63-linked Ubiquitin Conjugates 
Analytical chemistry  2012;84(22):10121-10128.
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.
doi:10.1021/ac302675y
PMCID: PMC3509807  PMID: 23101881
6.  Structural and biochemical studies of the open state of Lys48-linked diubiquitin 
Biochimica et biophysica acta  2012;1823(11):2046-2056.
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.
doi:10.1016/j.bbamcr.2012.04.003
PMCID: PMC3429157  PMID: 22542781
ubiquitin; Lys48-linked diubiquitin; polyubiquitin; ubiquitin-associated domain; Lysine-48 linkage selectivity
7.  A Hierarchical Algorithm for Fast Debye Summation with Applications to Small Angle Scattering 
Journal of computational chemistry  2012;33(25):1981-1996.
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.
doi:10.1002/jcc.23025
PMCID: PMC3425727  PMID: 22707386
8.  Segmental Isotopic Labelling of Ubiquitin Chains to Unravel Monomer-Specific Molecular Behavior ** 
doi:10.1002/anie.201104649
PMCID: PMC3427647  PMID: 21957015
Lys33 linked chain; segmental isotope labelling; semi-synthesis; ubiquitin chains; ubiquitination
9.  Nonenzymatic assembly of natural polyubiquitin chains of any linkage composition and isotopic labeling scheme 
Journal of the American Chemical Society  2011;133(44):17855-17868.
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.
doi:10.1021/ja207220g
PMCID: PMC3226840  PMID: 21962295
10.  Long-Lived States to Monitor Protein Unfolding by Proton NMR 
Chemphyschem  2011;12(15):2729-2734.
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.
doi:10.1002/cphc.201100365
PMCID: PMC3368952  PMID: 21882334
NMR; Proteins; Mutation; Ubiquitin; Long-Lived States; Singlet States
11.  Fast Approximations of the Rotational Diffusion Tensor and their Application to Structural Assembly of Molecular Complexes 
Proteins  2011;79(7):2268-2281.
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.
doi:10.1002/prot.23053
PMCID: PMC3115445  PMID: 21604302
rigid-body docking; protein complexes; residual dipolar couplings; diffusion-guided molecular assembly; ellipsoid model; ELM; PATI
12.  Density functional calculations of backbone 15N shielding tensors in beta-sheet and turn residues of protein G 
Journal of biomolecular NMR  2011;50(1):19-33.
Summary
We performed density functional calculations of backbone 15N shielding tensors in the regions of beta-sheet and turns of protein G. The calculations were carried out for all twenty-four beta-sheet residues and eight beta-turn residues in the protein GB3 and the results were compared with the available experimental data from solid-state and solution NMR measurements. Together with the alpha-helix data, our calculations cover 39 out of the 55 residues (or 71%) in GB3. The applicability of several computational models developed previously (Cai, Fushman, Kosov, J. Biomol NMR 2009, 45:245-253) to compute 15N shielding tensors of alpha-helical residues is assessed. We show that the proposed quantum chemical computational model is capable of predicting isotropic 15N chemical shifts for an entire protein that are in good correlation with experimental data. However, the individual components of the predicted 15N shielding tensor agree with experiment less well: the computed values show much larger spread than the experimental data, and there is a profound difference in the behavior of the tensor components for alpha-helix/turns and beta-sheet residues. We discuss possible reasons for this.
doi:10.1007/s10858-011-9474-8
PMCID: PMC3085593  PMID: 21305337
nitrogen-15 shielding tensor; ab initio calculation; density-functional calculation; beta-sheet; turns; protein G
13.  Controlled enzymatic synthesis of natural-linkage, defined-length polyubiquitin chains using lysines with removable protecting groups† 
E2 enzymes catalyze the ATP-dependent polymerization of polyubiquitin chains which function as molecular signals in the regulation of numerous cellular processes. Here we present a method that uses genetically encoded unnatural amino acids to halt and re-start ubiquitin polymerization providing access to natural-linkage, precision-length ubiquitin chains that can be used for biochemical, structural, and dynamics studies.
doi:10.1039/c0cc04868b
PMCID: PMC3190230  PMID: 21212884
14.  Structure and recognition of polyubiquitin chains of different lengths and linkage 
The polyubiquitin signal is post-translationally attached to a large number of proteins, often directing formation of macromolecular complexes resulting in the translocation, assembly or degradation of the attached protein. Recent structural and functional studies reveal general mechanisms by which different architectures and length of the signal are distinguished.
doi:10.3410/B3-26
PMCID: PMC3229271  PMID: 22162729
15.  Condensed E. coli Cultures for Highly Efficient Production of Proteins Containing Unnatural Amino Acids 
Current biosynthetic methods for producing proteins containing site-specifically incorporated unnatural amino acids are inefficient because the majority of the amino acid goes unused. Here we present a universal approach to improve the efficiency of such processes using condensed E. coli cultures.
doi:10.1016/j.bmcl.2010.08.049
PMCID: PMC2941765  PMID: 20805030
unnatural amino acid; tRNA; ubiquitin; aminoacyl-tRNA synthetase
16.  Reversible Post-Translational Carboxylation Modulates The Enzymatic Activity Of N-Acetyl-L-Ornithine Transcarbamylase† 
Biochemistry  2010;49(32):6887-6895.
N-acetyl-L-ornithine transcarbamylase (AOTCase), rather than ornithine transcarbamylase (OTCase), is the essential carbamylase enzyme in the arginine biosynthesis of several plant and human pathogens. The specificity of this unique enzyme provides a potential target for controlling the spread of these pathogens. Recently, several crystal structures of AOTCase from Xanthomonas campestris (xc) have been determined. In these structures, an unexplained electron density at the tip of Lys302 side-chain was observed. Using 13C NMR spectroscopy, we show herein that Lys302 is post-translationally carboxylated. The structure of wild-type AOTCase complexed with the bisubstrate analogue, Nδ-(phosphonoacetyl)-Nα-acetyl-L-ornithine (PALAO), indicates that the carboxyl group on Lys302 forms a strong hydrogen bonding network with surrounding active site residues, Lys252, Ser253, His293, and Glu92 from the adjacent subunit either directly or via a water molecule. Furthermore, the carboxyl group is involved in binding N-acetyl-L-ornithine via a water molecule. Activity assays with the wild-type enzyme and several mutants demonstrate that the post translational modification of lysine 302 has an important role in catalysis.
doi:10.1021/bi1007386
PMCID: PMC2959125  PMID: 20695527
17.  Structural Assembly of Molecular Complexes Based on Residual Dipolar Couplings 
We present and evaluate a rigid-body molecular docking method, called PATIDOCK, that relies solely on the three-dimensional structure of the individual components and the experimentally derived residual dipolar couplings (RDC) for the complex. We show that, given an accurate ab initio predictor of the alignment tensor from a protein structure, it is possible to accurately assemble a protein-protein complex by utilizing the RDC’s sensitivity to molecular shape to guide the docking. The proposed docking method is robust against experimental errors in the RDCs and computationally efficient. We analyze the accuracy and efficiency of this method using experimental or synthetic RDC data for several proteins, as well as synthetic data for a large variety of protein-protein complexes. We also test our method on two protein systems for which the structure of the complex and steric-alignment data are available (Lys48-linked diubiquitin and a complex of ubiquitin and a ubiquitin-associated domain) and analyze the effect of flexible unstructured tails on the outcome of docking. The results demonstrate that it is fundamentally possible to assemble a protein-protein complex based solely on experimental RDC data and the prediction of the alignment tensor from three-dimensional structures. Thus, despite the purely angular nature of residual dipolar couplings, they can be converted into intermolecular distance/translational constraints. Additionally we show a method for combining RDCs with other experimental data, such as ambiguous constraints from interface mapping, to further improve structure characterization of the protein complexes.
doi:10.1021/ja100447p
PMCID: PMC2931813  PMID: 20550109
18.  Exploring linkage dependence of polyubiquitin conformations using molecular modeling 
Journal of molecular biology  2009;395(4):803.
Post-translational modification of proteins by covalent attachment of a small protein ubiquitin or a polymeric chain of ubiquitin molecules (called polyubiquitin) is involved in controlling a vast variety of processes in eukaryotic cells. The question of how different polyubiquitin signals are recognized is central to understanding the specificity of various types of polyubiquitination. In polyubiquitin, the monomers are linked to each other via an isopeptide bond between the C-terminal glycine of one ubiquitin and a lysine of the other. The functional outcome of polyubiquitination depends on the particular lysine involved in the chain formation and appears to rely on linkage-dependent conformation of polyubiquitin. Thus, K48-linked chains, a universal signal for proteasomal degradation, under physiological conditions adopt a closed conformation where functionally important residues L8, I44, and V70 are sequestered at the interface between the two adjacent ubiquitin monomers. By contrast, K63-linked chains, which act as a non-proteolytic, regulatory signal, adopt an extended conformation that lacks the hydrophobic inter-ubiquitin contact. Little is known about functional roles of the so-called “non-canonical” chains, linked via K6, K11, K27, K29, K33, or head-to-tail; and no structural information on these chains is available, except for the crystal structure of the head-to-tail linked diubiquitin. In this study, we use molecular modeling to examine whether any of the non-canonical chains can adopt a closed conformation similar to that in K48-linked polyubiquitin. Our results show that the eight possible di-ubiquitin chains can be divided into two groups: K6-, K11-, K27-, and K48-linked chains are predicted to form a closed conformation, whereas chains linked via K29, K33, K63, or head-to-tail are unable to form such a contact due to steric occlusion. These predictions are validated by the known structures of K48-, K63-, and head-to-tail linked chains. Our study also predicts structural models for di-ubiquitins linked via K6, K11, 3 and K27. Implications of these findings for linkage-selective recognition of the non-canonical polyubiquitin signals by various receptors are discussed.
doi:10.1016/j.jmb.2009.10.039
PMCID: PMC2813430  PMID: 19853612
polyubiquitin; non-canonical linkage; isopeptide linkage; head-to-tail; modeling
19.  Together, Rpn10 and Dsk2 can serve as a polyubiquitin chain-length sensor 
Molecular cell  2009;36(6):1018-1033.
Summary
As a signal for substrate targeting, polyubiquitin meets various layers of receptors upstream to the 26S proteasome. We obtained structural information on two receptors, Rpn10 and Dsk2, alone, and in complex with (poly)ubiquitin or with each other. A hierarchy of affinities emerges with Dsk2 binding monoubiquitin tighter than Rpn10 does, whereas Rpn10 prefers the ubiquitin-like domain of Dsk2 to monoubiquitin, with increasing affinities for longer polyubiquitin chains. We demonstrated the formation of ternary complexes of both receptors simultaneously with (poly)ubiquitin and found that, depending on the ubiquitin-chain length, the orientation of the resulting complex is entirely different, providing for alternate signals. Dynamic rearrangement provides a chain-length sensor, possibly explaining how accessibility of Dsk2 to the proteasome is limited unless it carries a properly-tagged cargo. We propose a mechanism for a malleable ubiquitin-signal that depends both on chain-length and combination of receptors to produce tetra-ubiquitin as an efficient signal threshold.
doi:10.1016/j.molcel.2009.11.012
PMCID: PMC2807407  PMID: 20064467
20.  Improvement and Analysis of Computational Methods for Prediction of Residual Dipolar Couplings 
We describe a new, computationally effcient method for computing the molecular alignment tensor based on the molecular shape. The increase in speed is achieved by re-expressing the problem as one of numerical integration, rather than a simple uniform sampling (as in the PALES method), and by using a convex hull rather than a detailed representation of the surface of a molecule. This method is applicable to bicelles, PEG/hexanol, and other alignment media that can be modeled by steric restrictions introduced by a planar barrier. This method is used to further explore and compare various representations of protein shape by an equivalent ellipsoid. We also examine the accuracy of the alignment tensor and residual dipolar couplings (RDC) prediction using various ab initio methods. We separately quantify the inaccuracy in RDC prediction caused by the inaccuracy in the orientation and in the magnitude of the alignment tensor, concluding that orientation accuracy is much more important in accurate prediction of RDCs.
doi:10.1016/j.jmr.2009.07.028
PMCID: PMC2763024  PMID: 19700353
residual dipolar coupling; alignment tensor; ab initio prediction; PALES
21.  Density functional calculations of chemical shielding of backbone 15N in helical residues of protein G 
Journal of biomolecular NMR  2009;45(3):245-253.
Summary
We performed density functional calculations of backbone 15N chemical shielding tensors in selected helical residues of protein G. Here we describe a computationally efficient methodology to include most of the important effects in the calculation of chemical shieldings of backbone 15N. We analyzed the role of long-range intra-protein electrostatic interactions by comparing models with different complexity in vacuum and in charge field. Our results show that the dipole moment of the α-helix can cause significant deshielding of 15N; therefore, it needs to be considered when calculating 15N chemical shielding. We found that it is important to include interactions with the side chains that are close in space when the charged form for ionizable side chains is adopted in the calculation. We also illustrate how the ionization state of these side chains can affect the chemical shielding tensor elements. Chemical shielding calculations using a 8-residue fragment model in vacuum and adopting the charged form of ionizable side chains yield a generally good agreement with experimental data.
doi:10.1007/s10858-009-9358-3
PMCID: PMC2884268  PMID: 19644655
chemical shielding tensor; chemical shielding calculation; ab initio; nitrogen-15; density-functional calculation; protein G
22.  Perturbing the Ubiquitin Pathway Reveals How Mitosis Is Hijacked to Denucleate and Regulate Cell Proliferation and Differentiation In Vivo 
PLoS ONE  2010;5(10):e13331.
Background
The eye lens presents a unique opportunity to explore roles for specific molecules in cell proliferation, differentiation and development because cells remain in place throughout life and, like red blood cells and keratinocytes, they go through the most extreme differentiation, including removal of nuclei and cessation of protein synthesis. Ubiquitination controls many critical cellular processes, most of which require specific lysines on ubiquitin (Ub). Of the 7 lysines (K) least is known about effects of modification of K6.
Methodology and Principal Findings
We replaced K6 with tryptophan (W) because K6 is the most readily modified K and W is the most structurally similar residue to biotin. The backbone of K6W-Ub is indistinguishable from that of Wt-Ub. K6W-Ub is effectively conjugated and deconjugated but the conjugates are not degraded via the ubiquitin proteasome pathways (UPP). Expression of K6W-ubiquitin in the lens and lens cells results in accumulation of intracellular aggregates and also slows cell proliferation and the differentiation program, including expression of lens specific proteins, differentiation of epithelial cells into fibers, achieving proper fiber cell morphology, and removal of nuclei. The latter is critical for transparency, but the mechanism by which cell nuclei are removed has remained an age old enigma. This was also solved by expressing K6W-Ub. p27kip, a UPP substrate accumulates in lenses which express K6W-Ub. This precludes phosphorylation of nuclear lamin by the mitotic kinase, a prerequisite for disassembly of the nuclear membrane. Thus the nucleus remains intact and DNAseIIβ neither gains entry to the nucleus nor degrades the DNA. These results could not be obtained using chemical proteasome inhibitors that cannot be directed to specific tissues.
Conclusions and Significance
K6W-Ub provides a novel, genetic means to study functions of the UPP because it can be targeted to specific cells and tissues. A fully functional UPP is required to execute most stages of lens differentiation, specifically removal of cell nuclei. In the absence of a functional UPP, small aggregate prone, cataractous lenses are formed.
doi:10.1371/journal.pone.0013331
PMCID: PMC2958118  PMID: 20975996
23.  Structure of the S5a:K48-linked diubiquitin complex and its interactions with Rpn13 
Molecular cell  2009;35(3):280-290.
Summary
Degradation by the proteasome typically requires substrate ubiquitination. Two ubiquitin receptors exist in the proteasome, S5a/Rpn10 and Rpn13. Whereas Rpn13 has only one ubiquitin-binding surface, S5a binds ubiquitin with two independent ubiquitin interacting motifs (UIMs). Here, we use NMR and analytical ultracentrifugation to define at atomic level resolution how S5a binds K48-linked diubiquitin, in which K48 of one ubiquitin subunit (the “proximal” one) is covalently bonded to G76 of the other (the “distal” subunit). We demonstrate that S5a’s UIMs bind the two subunits simultaneously with a preference for UIM2 binding to the proximal subunit while UIM1 binds to the distal one. In addition, NMR experiments reveal that Rpn13 and S5a bind K48-linked diubiquitin simultaneously with subunit specificity, and a model structure of S5a and Rpn13 bound to K48-linked polyubiquitin is provided. Altogether, our data demonstrate that S5a is highly adaptive and cooperative towards binding ubiquitin chains.
doi:10.1016/j.molcel.2009.06.010
PMCID: PMC2748877  PMID: 19683493
proteasome; ubiquitin; protein degradation; S5a; Rpn13
24.  Density functional calculations of 15N chemical shifts in solvated dipeptides 
Journal of biomolecular NMR  2008;41(2):77-88.
Summary
We performed density functional calculations to examine the effects of solvation, hydrogen bonding, backbone conformation, and the side chain on 15N chemical shielding in proteins. We used N-methylacetamide (NMA) and N-formyl-alanyl-X (with X being one of the 19 naturally occurring amino acids excluding proline) as model systems. In addition, calculations were performed for selected fragments from protein GB3. The conducting polarizable continuum model was employed to include the effect of solvent in the density functional calculations. Our calculations for NMA show that the augmentation of the polarizable continuum model with the explicit water molecules in the first solvation shell has a significant influence on isotropic 15N chemical shift but not as much on the chemical shift anisotropy. The difference in the isotropic chemical shift between the standard β-sheet and α-helical conformations ranges from 0.8 ppm to 6.2 ppm depending on the residue type, with the mean of 2.7 ppm. This is in good agreement with the experimental chemical shifts averaged over a database of 36 proteins containing >6100 amino acid residues. The orientation of the 15N chemical shielding tensor as well as its anisotropy and asymmetry are also in the range of values experimentally observed for peptides and proteins.
doi:10.1007/s10858-008-9241-7
PMCID: PMC2891059  PMID: 18484179
chemical shielding tensor; chemical shift calculation; dipeptides; solvent effect; nitrogen-15; density-functional calculation
25.  1H, 13C, and 15N resonance assignment of the ubiquitin-like domain from Dsk2p 
Biomolecular NMR assignments  2008;2(2):147-149.
The ubiquitin-like domain (UBL) of yeast protein Dsk2p is widely believed to recognize and bind to ubiquitin receptors on the proteasome and, as part of Dsk2p, to bridge polyubiquitinated substrates and proteasomal degradation machinery. Here we report NMR resonance assignment for 1H, 15N, and 13C nuclei in the backbone and side chains of the UBL domain of Dsk2p. This assignment will aid in NMR studies focused on understanding of Dsk2’s interactions with proteasomal receptors and its role as a polyubiquitin shuttle in the ubiquitin-dependent proteasomal degradation as well as other cellular pathways.
doi:10.1007/s12104-008-9107-7
PMCID: PMC2892233  PMID: 19636891
Dsk2p; ubiquitin-like domain; UBL; proteasome

Results 1-25 (33)