Heteronuclear Single Quantum Coherence Experiments Research Articles

Multiplicity-edited heteronuclear single quantum coherence experiment modified for 19F-13C

The popular multiplicity-edited heteronuclear single quantum coherence experiment has been modified for 19F-13C experiments by insertion of broadband inversion and refocusing pulses in 19F. This experiment has particular utility in distinguishing CF3 from CF2X, and CF2 from CFX (X = O or halide). Examples are shown of 1,1,2-trichloro-1,2,2-trifluoroethane, a mixture of 1,1,2-trichloro-1,2,2-trifluoroethane and two perfluoroolefins, and a mixture of poly(hexafluoropropylene oxide) and perfluorohexyl iodide. Phase distortions are minimized by careful selection of the value of JCF for which delays are optimized; a slight overstatement of JCF yielded no distortions, while an understatement did. Large 19F homonuclear couplings do not report cleanly in-phase. A one-dimensional version of the experiment yielded analogous editing by phase.

Discovery of N-sulfonylated aminosalicylic acids as dual MCL-1/BCL-xL inhibitors.

The anti-apoptotic protein MCL-1, which is overexpressed in multiple cancers, is presently a focus for the development of targeted drugs in oncology. We previously discovered inhibitors of MCL-1 based on 1-sulfonylated 1,2,3,4-tetrahydroquinoline-6-carboxylic acids ("1,6-THQs"). However, with the nitrogen atom constrained in the bicyclic ring, we were unable to modify the alkyl portion of the tertiary sulfonamide functionality. Moreover, the introduction of additional functional groups onto the benzene ring portion of the THQ bicycle would not be trivial. Therefore, we elected to deconstruct the piperidine-type ring of the 6-carboxy-THQ lead to create a new 4-aminobenzoic acid scaffold. Given its simplicity, this permitted us to introduce diversity at the sulfonamide nitrogen, as well as vary the positions and substituents of the benzene ring. One of our most potent MCL-1 inhibitors, 6e-OH, exhibited a K i of 0.778 μM. Heteronuclear single quantum coherence experiments suggested 6e-OH bound in the canonical BH3-binding groove, with significant perturbations of R263, which forms a salt bridge with MCL-1's pro-apoptotic binding partners, as well as residues in the p2 pocket. Selectivity studies indicated that our compounds are dual inhibitors of MCL-1 and BCL-xL, with 17cd the most potent dual inhibitor: K i = 0.629 μM (MCL-1), 1.67 μM (BCL-xL). Whilst selective inhibitors may be more desirable in certain instances, polypharmacological agents whose additional target(s) address other pathways associated with the disease state, or serve to counter resistance mechanisms to the primary target, may prove particularly effective therapeutics. Since selective MCL-1 inhibition may be thwarted by overexpression of sister anti-apoptotic proteins, including BCL-xL and BCL-2, we believe our work lays a solid foundation towards the development of multi-targeting anti-cancer drugs.

HSQC‐NMR‐based profiling approaches for raffinose family oligosaccharides in pulses

AbstractBackground and ObjectivesDue to insufficient resolution, 1H nuclear magnetic resonance (NMR) spectroscopy‐based methods are limited to quantify carbohydrates. In the past, heteronuclear single quantum coherence (HSQC)‐based methods were demonstrated to be superior as the second dimension greatly improves resolution. However, whether these experiments are also suitable to determine structurally similar oligosaccharides such as raffinose family oligosaccharides (RFO) still needs to be demonstrated.FindingsBy optimizing NMR parameters, well resolved signals for the analysis of glucose, fructose, galactose, sucrose and the RFO raffinose, stachyose, and verbascose were identified. Application of fast HSQC methods in combination with nonuniform sampling enables analyses of sucrose and RFO in pulses (blue lupin seeds, red lentils, kidney beans) within 24 min. If the analytes are present at levels greater than 0.5 g/100 g, HSQC‐based methods provide data equivalent to an anion‐exchange chromatography‐based reference method.ConclusionsHigh resolution fast HSQC‐based approaches are suitable tools to analyze complex carbohydrate mixtures as demonstrated for RFO in different pulses.Significance and NoveltyFast HSQC experiments were applied for the first time to analyze structurally similar oligosaccharides. In the future, this approach will be a most valuable tool to analyze complex mixtures of carbohydrates in food products.

Quantification of Isomaltulose in Food Products by Using Heteronuclear Single Quantum Coherence NMR-Experiments.

Isomaltulose is a commonly used sweetener in sports nutrition and in products intended for consumption by diabetics. Because previously established chromatographic methods for quantification of isomaltulose suffer from long analysis times (60–210 min), faster quantitative approaches are required. Here, an HSQC (heteronuclear single quantum coherence) experiment with reduced interscan delay was established in order to quantify isomaltulose next to potential additional sugars such as d-glucose, d-fructose, d-galactose, sucrose, lactose, and maltose in 53 min. By using HSQC coupled to non-uniform sampling (NUS) as well as ASAP-HSQC (acceleration by sharing adjacent polarization), analysis times were reduced to a few minutes. Application of NUS-HSQC with reduced interscan delay takes 27 min, resulting in accurate and precise data. In principle, application of ASAP-HSQC approaches (with analysis times as low as 6 min) can be used; however, precision data may not suffice all applications.

Application of accelerated heteronuclear single quantum coherence experiments to the rapid quantification of monosaccharides and disaccharides in dairy products.

Monosaccharides and disaccharides are important dietary components, but if insufficiently metabolized by some population subgroups, they are also linked to disease patterns. Thus, the correct analytical identification, quantification, and labeling of these food components are crucial to inform and potentially protect consumers. Enzymatic assays and high-performance anion-exchange chromatography with pulsed amperometric detection are established methods for the quantification of monosaccharides and disaccharides that, however, require long measuring times (60-180 min). Accelerated methods for the identification and quantification of the nutritionally relevant monosaccharides and disaccharides d-glucose, d-galactose, d-fructose, sucrose, lactose, and maltose were therefore developed. To realize this goal, the NMR experiments HSQC (heteronuclear single quantum coherence) and acceleration by sharing adjacent polarization (ASAP)-HSQC were applied. Measurement times were reduced to 27 and 6 min, respectively, by optimizing the interscan delay and applying non-uniform sampling. The optimized methods were used to quantify d-glucose, d-galactose, d-fructose, sucrose, and lactose in various dairy products. Results of the HSQC and ASAP-HSQC methods are equivalent to the results of the reference methods in terms of both precision and accuracy, demonstrating that these methods can be used to correctly analyze nutritionally relevant monosaccharides and disaccharides in short times.

Probing multiple enzymatic methylation events in real time with NMR spectroscopy

Post-translational modification (PTM) of proteins is of critical importance to the regulation of many cellular processes in eukaryotic organisms. One of the most well-studied protein PTMs is methylation, wherein an enzyme catalyzes the transfer of a methyl group from a cofactor to a lysine or arginine side chain. Lysine methylation is especially abundant in the histone tails and is an important marker for denoting active or repressed genes. Given their relevance to transcriptional regulation, the study of methyltransferase function through in vitro experiments is an important stepping stone toward understanding the complex mechanisms of regulated gene expression. To date, most methyltransferase characterization strategies rely on the use of radioactive cofactors, detection of a methyl transfer byproduct, or discontinuous-type assays. Although such methods are suitable for some applications, information about multiple methylation events and kinetic intermediates is often lost. Herein, we describe the use of two-dimensional NMR to monitor mono-, di-, and trimethylation in a single reaction tube. To do so, we incorporated 13C into the donor methyl group of the enzyme cofactor S-adenosyl methionine. In this way, we may study enzymatic methylation by monitoring the appearance of distinct resonances corresponding to mono-, di-, or trimethyl lysine without the need to isotopically enrich the substrate. To demonstrate the capabilities of this method, we evaluated the activity of three lysine methyltransferases, Set7, MWRAD2 (MLL1 complex), and PRDM9, toward the histone H3 tail. We monitored mono- or multimethylation of histone H3 tail at lysine 4 through sequential short two-dimensional heteronuclear single quantum coherence experiments and fit the resulting progress curves to first-order kinetic models. In summary, NMR detection of PTMs in one-pot, real-time reaction using facile cofactor isotopic enrichment shows promise as a method toward understanding the intricate mechanisms of methyltransferases and other enzymes.

NMR Analysis of Carboxylate Isotopomers of 13C-Metabolites by Chemoselective Derivatization with 15N-Cholamine.

A substantial fraction of common metabolites contains carboxyl functional groups. Their 13C isotopomer analysis by nuclear magnetic resonance (NMR) is hampered by the low sensitivity of the 13C nucleus, the slow longitudinal relaxation for the lack of an attached proton, and the relatively low chemical shift dispersion of carboxylates. Chemoselective (CS) derivatization is a means of tagging compounds in a complex mixture via a specific functional group. 15N1-cholamine has been shown to be a useful CS agent for carboxylates, producing a peptide bond that can be detected via 15N-attached H with high sensitivity in heteronuclear single quantum coherence experiments. Here, we report an improved method of derivatization and show how 13C-enrichment at the carboxylate and/or the adjacent carbon can be determined via one- and two-bond coupling of the carbons adjacent to the cholamine 15N atom in the derivatives. We have applied this method for the determination of 13C isotopomer distribution in the extracts of A549 cell culture and liver tissue from a patient-derived xenograft mouse.

2D NMR-Based Metabolomics with HSQC/TOCSY NOAH Supersequences.

Sensitivity-improved versions of two-dimensional (2D) 13C-1H HSQC (heteronuclear single quantum coherence) and HSQC-TOCSY (HSQC-total correlation spectroscopy) NMR experiments optimized for small biological molecules and their complex mixtures encountered in metabolomics are presented that preserve the magnetization of 1H spins not directly attached to 13C spins. This allows (i) the application of rapid acquisition techniques to substantially shorten measurement time and (ii) their incorporation into supersequences (NOAH-NMR by ordered acquisition using 1H detection) for the compact acquisition of multiple 2D NMR data sets with significant gains in sensitivity, resolution, and/or time. The new pulse sequences, which are demonstrated for both metabolite model mixtures and mouse urine, offer an attractive approach for the efficient measurement of multiple 2D NMR spectra (HSQCsi and/or HSQCsi-TOCSY and TOCSY) of metabolomics samples in a single experiment for the accurate and comprehensive identification and quantitation of metabolites. These new methods bring to bear the advantages of 2D NMR to metabolomics studies with larger cohorts of samples.

Synthesis and structural elucidation of a series of isoflavones-based on FPR antagonists

Synthesis and structural elucidation of a series of isoflavones-based on FPR antagonists

Comparison of the Effects of Different 19F π Pulses on the Sensitivity and Phaseability of the 19F-13C HSQC

The 19F-13C heteronuclear single quantum coherence (HSQC) experiment is vital for the structural elucidation of polyfluorinated organic species, yet its sensitivity and phaseability are limited by difficulties in uniform excitation of the widely disperse 19F spectral window. Adiabatic pulses of different types have previously been employed to generate effective π pulses for inversion and refocussing, but a systematic comparison of various adiabatic and other inversion pulses has not been published. In this work, it was observed that the use of a broadband inversion pulse (BIP) during the t 1 evolution period resulted in properly phaseable spectra for experiments optimized to detect 1 J CF, in contrast to CHIRP or WURST adiabatic pulses. For the INEPT and reverse-INEPT transfer segments of the HSQC, optimal sensitivity for resonances distant from the transmitter frequency was afforded by optimized universal rotation (BURBOP) or CHIRP pulses. In HSQC experiments with delays optimized for two-bond correlations, only the use of BURBOP pulses in INEPT and reverse-INEPT sequences afforded spectra cleanly phaseable across the F 2 and F 1 spectral windows. This observation is supported by off-resonance pulsed field gradient spin-echo experiments.

Characterization and quantification of microstructures of a fluorinated terpolymer by both homonuclear and heteronuclear two-dimensional NMR spectroscopy.

Fluoropolymers are usually insoluble in organic solvents. Insolubility of fluoropolymers limits basic characterization such as microstructural investigations. In the family of fluoropolymers, terpolymer of tetrafluorethylene (TFE), hexafluoropropylene (HFP), and vinylidene fluoride (VDF), named THV is one of the newest members. There are nine grades of THV available. Among the nine grades, THV-221 G is an ideal model polymer for basic characterization purposes. THV-221 G is soluble in solvents such as acetone and ethyl acetate. In the current report, both homonuclear and heteronuclear 2D NMR experiments were employed in solution on THV-221 G. The homonuclear gradient correlation spectroscopy NMR measurement revealed that THV has two adjacent TFE units in addition to TFE-HFP sequence orders. The fraction of the microstructures is quantified by the analysis of 1D solution (19)F NMR spectrum. Further, the gradient heteronuclear single quantum coherence experiment helped with the clarification of chemical environments of the units TFE, HFP, and VDF. The 1D solution (13)C NMR spectrum was helpful in clarifying sequence assignments of VDF. It is concluded that THV is a random polymer with a limited fraction of TFE-TFE and TFE-HFP sequence orders in addition to head-to-tail polymerization of VDF unit.

Study of cytochrome c-DNA interaction--evaluation of binding sites on the redox protein.

Artificial assemblies consisting of the cationic cytochrome c (cyt c) and double-stranded DNA are interesting for the field of biohybrid systems because of the high electro-activity of the incorporated redox protein. However, little is known about the interactions between these two biomolecules. Here, the complex of reduced cyt c and a 41 base pair oligonucleotide was characterized in solution as a function of pH and ionic strength. Persistent cyt c-DNA agglomerates were observed by UV-vis and DLS (dynamic light scattering) at pH 5.0 and low ionic strength. The strength of the interaction was attenuated by raising the pH or the ionic strength. At pH 7.0 agglomerates were not formed, allowing interaction analysis by NMR spectroscopy. Using TROSY (transverse relaxation-optimized spectroscopy)-HSQC (heteronuclear single quantum coherence) experiments it was possible to identify the DNA binding site on the cyt c surface. Numerous residues surrounding the exposed heme edge of cyt c were involved in transient binding to DNA under these conditions. This result was supported by SEC (size exclusion chromatography) experiments at pH 7.0 showing that the interaction is sufficient for co-elution of cyt c and DNA.

Thermodynamic and Structural Analysis of Human NFU Conformational Chemistry

Human NFU has been implicated in the formation of inorganic sulfide required for cellular iron-sulfur cluster biosynthesis. The protein contains a well-structured N-terminal domain and a C-terminal domain with molten globule characteristics that also contains a thioredoxin-like pair of redox active Cys residues that promote persulfide reductase activity. Recent reports have highlighted the existence of structural flexibility in the ISU/IscU-type scaffold proteins that mediate Fe-S cluster assembly, which is also likely to serve an important role in the pathway to Fe-S cluster maturation. We have previously reported similar structural mobility for the C-terminal domain of human NFU, a protein that has been implicated in the production of sulfide for cluster synthesis, while homologous proteins have also been suggested to serve as Fe-S cluster carriers. Herein we quantitatively characterize the structural stability of the two domains of human NFU and in particular the functional C-terminal domain. The results of differential scanning calorimetry and variable temperature circular dichroism (VTCD) studies have been used to analyze the temperature-dependent structural melting profiles of the N- and C-terminal domains, relative to both full-length NFU and an equimolar ratio of the N- and C-terminal domains, and correlated with structural information derived from NMR data. Calorimetry results indicate that the C-terminal NFU domain undergoes a significant structural stabilization following interaction with the N-terminal domain, which resulted in a novel and distinctive transition melting profile (Tm(sec) = 58.1 ± 0.4 °C, ΔHv(sec) = 60.4 ± 5.3 kcal/mol, Tm(ter) = 49.3 ± 0.3 °C, ΔHv(ter) = 71.8 ± 5.8 kcal/mol). VTCD experiments also revealed a secondary structure transition at 59.2 °C in agreement with calorimetry results. The degree of stabilization was found to be more significant in the full-length NFU, as the C-terminal domain transitions were recorded at higher temperatures (Tm(sec) = 63.3 ± 3.4 °C, ΔHv(sec) = 41.8 ± 8.2 kcal/mol). The interactions between the two domains demonstrated the hallmarks of a hydrophobic character, as increased ionic strength decreased the degree of stabilization of the C-terminal domain. An increase of 2% in α-helix content further supports interaction between the two domains, leading to greater secondary structure stabilization. Heteronuclear single-quantum coherence experiments indicate that the C-terminal domain adopts an alternate tertiary conformation following binding to the N-terminal domain. The structural rigidity of the N-terminal domain leads to an alternative conformation of the C-terminal domain, suggesting that such an interaction, although weaker than that of the covalently attached native NFU, is important for the structural chemistry of the native full-length protein. The results also emphasize the likely general importance of such structural flexibility in select proteins mediating metal cofactor biosynthesis.

Influence of Allosteric Regulators on Individual Steps in the Reaction Catalyzed by Mycobacterium tuberculosis 2-Hydroxy-3-oxoadipate Synthase

Allosteric regulation often controls key branch points in metabolic processes. Mycobacterium tuberculosis 2-hydroxy-3-oxoadipate synthase (HOAS), a thiamin diphosphate (ThDP)-dependent enzyme, produces 2-hydroxy-3-oxoadipate using 2-ketoglutarate and glyoxylate. The proposed chemical mechanism in analogy with other ThDP-dependent carboligases involves multiple ThDP-bound covalent intermediates. Acetyl coenzyme A is an activator, and GarA, a forkhead association domain-containing protein known to regulate glutamate metabolism, is an allosteric inhibitor of HOAS. Steady state kinetics using assays to study the first half and the full catalytic cycle suggested that the regulators act at different steps in the overall mechanism. To explore the modes of regulation and to test the effects on individual catalytic steps, we performed circular dichroism (CD) studies using a non-decarboxylatable 2-ketoglutarate analog and determined the distribution of ThDP-bound covalent intermediates during the steady state of the HOAS reaction using one-dimensional (1)H gradient carbon heteronuclear single quantum coherence NMR. The results suggest that acetyl coenzyme A acts as a mixed V and K type activator and predominantly affects the predecarboxylation steps. GarA does not inhibit the formation of the predecarboxylation analog and does not affect the accumulation of the postdecarboxylation covalent intermediate derived from 2-ketoglutarate; however, it decreases the abundance of the product ThDP adduct in the HOAS pathway. Thus, the two regulators act on different halves of the catalytic cycle in an unusual regulatory regime.

The H/D-Exchange Kinetics of the Escherichia coli Co-Chaperonin GroES Studied by 2D NMR and DMSO-Quenched Exchange Methods

We studied hydrogen/deuterium-exchange reactions of peptide amide protons of GroES using two different techniques: (1) two-dimensional 1H–15N transverse-optimized NMR spectroscopy and (2) the dimethylsulfoxide-quenched hydrogen-exchange method combined with conventional 1H–15N heteronuclear single quantum coherence spectroscopy. By using these techniques together with direct heteronuclear single quantum coherence experiments, we quantitatively evaluated the exchange rates for 33 out of the 94 peptide amide protons of GroES and their protection factors, and for the remaining 61 residues, we obtained the lower limits of the exchange rates. The protection factors of the most highly protected amide protons were on the order of 106–107, and the values were comparable in magnitude to those observed in typical small globular proteins, but the number of the highly protected amide protons with a protection factor larger than 106 was only 10, significantly smaller than the numbers reported for the small globular proteins, indicating that significant portions of free heptameric GroES are flexible and natively unfolded. The highly protected amino acid residues with a protection factor larger than 105 were mainly located in three β-strands that form the hydrophobic core of GroES, while the residues in a mobile loop (residues 17–34) were not highly protected. The protection factors of the most highly protected amide protons were orders of magnitude larger than the value expected from the equilibrium unfolding parameters previously reported, strongly suggesting that the equilibrium unfolding of GroES is more complicated than a simple two-state or three-state mechanism and may involve more than a single intermediate.

In-Cell NMR Spectroscopy–In vivo Monitoring of the Structure, Dynamics, Folding, and Interactions of Proteins at Atomic Resolution

Thallapuranam K Suresh Kumar, Ryan Thurman and Srinivas Jayanthi-In-Cell NMR Spectroscopy–<em>In vivo</em> Monitoring of the Structure, Dynamics, Folding, and Interactions of Proteins at Atomic Resolution

Two-Dimensional NMR Evidence for Cleavage of Lignin and Xylan Substituents in Wheat Straw Through Hydrothermal Pretreatment and Enzymatic Hydrolysis

Solution-state two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy of plant cell walls is a powerful tool for characterizing changes in cell wall chemistry during the hydrothermal pretreatment process of wheat straw for second-generation bioethanol production. One-bond 13C–1H NMR correlation spectroscopy, via an heteronuclear single quantum coherence experiment, revealed substantial lignin β-aryl ether cleavage, deacetylation via cleavage of the natural acetates at the 2-O- and 3-O-positions of xylan, and uronic acid depletion via cleavage of the (1 → 2)-linked 4-O-methyl-α-d-glucuronic acid of xylan. In the polysaccharide anomeric region, decreases in the minor β-d-mannopyranosyl, and α-l-arabinofuranosyl units were observed in the NMR spectra from hydrothermally pretreated wheat straw. The aromatic region indicated only minor changes to the aromatic structures during the process (e.g., further deacylation revealed by the depletion in ferulate and p-coumarate structures). Supplementary chemical analyses showed that the hydrothermal pretreatment increased the cellulose and lignin concentration with partial removal of extractives and hemicelluloses. The subsequent enzymatic hydrolysis incurred further deacetylation of the xylan, leaving approximately 10 % of acetate intact based on the weight of original wheat straw.

Tupisteroide A–C, three new polyhydroxylated steroidal constituents from the roots of Tupistra chinensis

Three new steroidal compounds with polyhydroxy groups, tupisteroide A-C (1-3), were obtained from the roots of Tupistra chinensis, together with one known compound (4) that was isolated from this plant for the first time. The structures of tupisteroide A-C were determined on the basis of one- and two-dimensional NMR spectroscopy, including (1) H-(1) H Correlation Spectroscopy, Heteronuclear Multiple Bond Correlation, and Heteronuclear Single Quantum Coherence experiments. The isolated compounds were evaluated for their cytotoxic activities against A549, HepG2, and CaSki cancer cell lines in vitro. Among them, compounds 1, 2, and 4 did not show significant inhibitory activity, but compound 3 showed cytotoxicity against A549 cancer cell lines with IC(50) values of 25.0 μM.

Structures and Interaction Analyses of Integrin αMβ2 Cytoplasmic Tails

Integrins are heterodimeric (α and β subunits) signal transducer proteins involved in cell adhesions and migrations. The cytosolic tails of integrins are essential for transmitting bidirectional signaling and also implicated in maintaining the resting states of the receptors. In addition, cytosolic tails of integrins often undergo post-translation modifications like phosphorylation. However, the consequences of phosphorylation on the structures and interactions are not clear. The leukocyte-specific integrin αMβ2 is essential for myeloid cell adhesion, phagocytosis, and degranulation. In this work, we determined solution structures of the myristoylated cytosolic tail of αM and a Ser phosphorylated variant in dodecylphosphocholine micelles by NMR spectroscopy. Furthermore, the interactions between non-phosphorylated and phosphorylated αM tails with β2 tail were investigated by NMR and fluorescence resonance energy transfer (FRET). The three-dimensional structures of the 24-residue cytosolic tail of αM or phosphorylated αM are characterized by an N-terminal amphipathic helix and a loop at the C terminus. The residues at the loop are involved in packing interactions with the hydrophobic face of the helix. 15N-1H heteronuclear single quantum coherence experiments identified residues of αM and β2 tails that may be involved in the formation of a tail-tail heterocomplex. We further examined interactions between myristoylated β2 tail in dodecylphosphocholine micelles with dansylated αM tail peptides by FRET. These studies revealed enhanced interactions between αM or phosphorylated αM tails with β2 tail with Kd values ∼5.2±0.6 and ∼4.4±0.7 μm, respectively. Docked structures of tail-tail complexes delineated that the αM/β2 interface at the cytosolic region could be sustained by a network of polar interactions, ionic interactions, and/or hydrogen bonds.

E2 Conjugating Enzyme Selectivity and Requirements for Function of the E3 Ubiquitin Ligase CHIP

The transfer of ubiquitin (Ub) to a substrate protein requires a cascade of E1 activating, E2 conjugating, and E3 ligating enzymes. E3 Ub ligases containing U-box and RING domains bind both E2∼Ub conjugates and substrates to facilitate transfer of the Ub molecule. Although the overall mode of action of E3 ligases is well established, many of the mechanistic details that determine the outcome of ubiquitination are poorly understood. CHIP (carboxyl terminus of Hsc70-interacting protein) is a U-box E3 ligase that serves as a co-chaperone to heat shock proteins and is critical for the regulation of unfolded proteins in the cytosol. We have performed a systematic analysis of the interactions of CHIP with E2 conjugating enzymes and found that only a subset bind and function. Moreover, some E2 enzymes function in pairs to create products that neither create individually. Characterization of the products of these reactions showed that different E2 enzymes produce different ubiquitination products, i.e. that E2 determines the outcome of Ub transfer. Site-directed mutagenesis on the E2 enzymes Ube2D1 and Ube2L3 (UbcH5a and UbcH7) established that an SPA motif in loop 7 of E2 is required for binding to CHIP but is not sufficient for activation of the E2∼Ub conjugate and consequent ubiquitination activity. These data support the proposal that the E2 SPA motif provides specificity for binding to CHIP, whereas activation of the E2∼Ub conjugate is derived from other molecular determinants.