Wen-Jin Winston Wu - Academia.edu (original) (raw)

Papers by Wen-Jin Winston Wu

Research paper thumbnail of Rapid data acquisition of 3D triple resonance experiments via projection reconstruction NMR (PR-NMR)

Research paper thumbnail of Synthesis of Heterobimetallic Phosphido-Bridged W-Mo Complexes with Chelating dppe and dppm Ligands

Journal of the Chinese Chemical Society, 1995

... of Heterobimetallic Phosphido-Bridged W-Mo Complexes with Chelating dppe and dppm Ligands Wen... more ... of Heterobimetallic Phosphido-Bridged W-Mo Complexes with Chelating dppe and dppm Ligands Wen-Jin Wu ( Щ^Щ ), Jiun-Yi Hsu ( $ШШ )> Yuh-Shang Wen ( fëULî. ... Scheme I Ph2 P /\ Cp(CO)2W Mo(CO)5 1 Ph2 О Ph2 Г THF Л L = dppm, dppe Ph2 О Cp(CO) PPh2-(CH2)x ...

Research paper thumbnail of Direct NMR resonance assignments of the active site histidine residue inEscherichia coli thioesterase I/protease I/lysophospholipase L1

Magnetic Resonance in Chemistry, 2006

Owing to the hydrogen-bond interaction and rapid exchange rate with the bulk water, the transvers... more Owing to the hydrogen-bond interaction and rapid exchange rate with the bulk water, the transverse relaxation time for the N(delta1)-H proton of the catalytic histidine in Escherichia coli thioesterase I/protease I/lysophospholipase L1 (TEP-I) is rather short. Because of its catalytic importance, it is desirable to detect and assign this proton resonance. In this paper, we report the first direct NMR correlation between the short-lived N(delta1)-H proton and its covalently attached N(delta1)-nitrogen of the catalytic His157 residue in E. coli thioesterase/protease I. We have used gradient-enhanced jump-return spin-echo HMQC (GE-JR SE HMQC) to obtain a direct correlation between the short-lived N(delta1)-H proton and its covalently attached N(delta1)-nitrogen. The sensitivity of detection for the short-lived N(delta1)-H proton was enhanced substantially by improved water suppression, in particular, the suppression of radiation damping via pulsed field gradients.

Research paper thumbnail of How a Low-Fidelity DNA Polymerase Chooses Non-Watson–Crick from Watson–Crick Incorporation

Journal of the American Chemical Society, 2014

A dogma for DNA polymerase catalysis is that the enzyme binds DNA first, followed by MgdNTP. This... more A dogma for DNA polymerase catalysis is that the enzyme binds DNA first, followed by MgdNTP. This mechanism contributes to the selection of correct dNTP by Watson-Crick base pairing, but it cannot explain how low-fidelity DNA polymerases overcome Watson-Crick base pairing to catalyze non-Watson-Crick dNTP incorporation. DNA polymerase X from the deadly African swine fever virus (Pol X) is a half-sized repair polymerase that catalyzes efficient dG:dGTP incorporation in addition to correct repair. Here we report the use of solution structures of Pol X in the free, binary (Pol X:MgdGTP), and ternary (Pol X:DNA:MgdGTP with dG:dGTP non-Watson-Crick pairing) forms, along with functional analyses, to show that Pol X uses multiple unprecedented strategies to achieve the mutagenic dG:dGTP incorporation. Unlike high fidelity polymerases, Pol X can prebind purine MgdNTP tightly and undergo a specific conformational change in the absence of DNA. The prebound MgdGTP assumes an unusual syn conformation stabilized by partial ring stacking with His115. Upon binding of a gapped DNA, also with a unique mechanism involving primarily helix αE, the prebound syn-dGTP forms a Hoogsteen base pair with the template anti-dG. Interestingly, while Pol X prebinds MgdCTP weakly, the correct dG:dCTP ternary complex is readily formed in the presence of DNA. H115A mutation disrupted MgdGTP binding and dG:dGTP ternary complex formation but not dG:dCTP ternary complex formation. The results demonstrate the first solution structural view of DNA polymerase catalysis, a unique DNA binding mode, and a novel mechanism for non-Watson-Crick incorporation by a low-fidelity DNA polymerase.

Research paper thumbnail of Stereospecific 1 H and 13 C NMR Assignments of Crotonyl CoA and Hexadienoyl CoA:  Conformational Analysis and Comparison with Protein−CoA Complexes

Journal of the American Chemical Society, 1998

Stereospecific 1 H and 13 C resonance assignments of hexadienoyl-CoA and crotonyl-CoA have been d... more Stereospecific 1 H and 13 C resonance assignments of hexadienoyl-CoA and crotonyl-CoA have been determined. The two diastereotopic methyl groups at the C2′′ carbon were assigned via transferred nuclear Overhauser effect experiments on the complex of hexadienoyl-CoA with the enzyme enoyl-CoA hydratase. The two diastereotopic 1′′ protons were assigned using heteronuclear multiple-bond correlation experiments in conjunction with rotating frame nuclear Overhauser spectroscopy. This represents the first set of complete stereospecific assignments for any CoA derivative. The assignments allow a detailed quantitative conformational analysis of the uncomplexed form of the molecule. In particular, the relative population of the various rotamers about the C1′′-C2′′ and the C2′′-C3′′ bonds have been determined. The database of protein-bound CoA structures has been surveyed and used to compare the structure(s) of CoA in protein-CoA complexes with the conformational preferences of free CoA.

Research paper thumbnail of Stereospecificity of the Reaction Catalyzed by Enoyl-CoA Hydratase

Journal of the American Chemical Society, 2000

Enoyl-CoA hydratase catalyzes the stereospecific hydration of α, β-unsaturated acyl-CoA thioleste... more Enoyl-CoA hydratase catalyzes the stereospecific hydration of α, β-unsaturated acyl-CoA thiolesters. Hydration of trans-2-crotonyl-CoA to 3 (S)-hydroxybutyryl-CoA proceeds via the syn addition of water and thus the pro-2 R proton of 3 (S)-hydroxybutyryl-CoA is derived ...

Research paper thumbnail of Conformational Heterogeneity about Pipecolic Acid Peptide Bonds:  Conformational, Thermodynamic, and Kinetic Aspects

The Journal of Organic Chemistry, 1998

Cis-trans isomerization about pipecolic peptide bonds has been studied using a set of designed te... more Cis-trans isomerization about pipecolic peptide bonds has been studied using a set of designed tetrapeptides of the sequence acetyl-Gly-X-Pip-Gly-carboxamide (GXPipG), where X) A, F, Y, W, or cyclohexylalanine (Cha). The substitution of a pipecolic residue for a proline leads to (1) a significant increase of the population of the cis conformer, (2) a reduction in the Van't Hoff enthalpy for isomerism, and (3) acceleration of the rates of isomerization. GAPipG and GChaPipG are unstructured, and isomerization is controlled by local steric effects between the R and positions of the pipecolic ring and the R position of the preceding residue. The fraction of the cis isomer is the same for these two peptides, indicating that the bulk of the preceding amino acid does not play a significant role in influencing the equilibrium. In the trans form of the aromatic containing peptides, an i to i + 2 aromatic-amide interaction is observed, and in the cis form a stabilizing interaction between the aromatic residue and the succeeding pipecolic ring is observed. The sign of the Van't Hoff enthalpy indicates that the cis conformer is enthalpically favored for the aromatic containing peptides. The rate of isomerization for the aromatic containing peptides are reduced relative to GAPipG.

Research paper thumbnail of PWWP Module of Human Hepatoma-derived Growth Factor Forms a Domain-swapped Dimer with Much Higher Affinity for Heparin

Journal of Molecular Biology, 2007

Hepatoma-derived growth factor (hHDGF)-related proteins (HRPs) comprise a new growth factor famil... more Hepatoma-derived growth factor (hHDGF)-related proteins (HRPs) comprise a new growth factor family sharing a highly conserved and ordered Nterminal PWWP module (residues 1-100, previously referred to as a HATH domain) and a variable disordered C-terminal domain. We have shown that the PWWP module is responsible for heparin binding and have solved its structure in solution. Here, we show that under physiological conditions, both the PWWP module and hHDGF can form dimers. Surface plasmon resonance (SPR) studies revealed that the PWWP dimer binds to heparin with affinity that is two orders of magnitude higher (K d = 13 nM) than that of the monomeric PWWP module (K d = 1.2 μM). The monomer-dimer equilibrium properties and NMR structural data together suggest that the PWWP dimer is formed through a domain-swapping mechanism. The domain-swapped PWWP dimer structures were calculated on the basis of the NMR data. The results show that the two PWWP protomers exchange their N-terminal hairpin to form a domain-swapped dimer. The two monomers in a dimer are linked by the long flexible L2 loops, a feature supported by NMR relaxation data for the monomer and dimer. The enhanced heparin-binding affinity of the dimer can be rationalized in the framework of the dimer structure.

Research paper thumbnail of Conformational analysis of a set of peptides corresponding to the entire primary sequence of the N-terminal domain of the ribosomal protein L9: evidence for stable native-like secondary structure in the unfolded state

Journal of Molecular Biology, 1999

Research paper thumbnail of Mixing apparatus for preparing NMR samples under pressure

Journal of Magnetic Resonance, 2003

The size limit for protein NMR spectroscopy in solution arises in large part from line broadening... more The size limit for protein NMR spectroscopy in solution arises in large part from line broadening caused by slow molecular tumbling. One way to alleviate this problem is to increase the effective tumbling rate by reducing the viscosity of the solvent. Because proteins generally require an aqueous environment to remain folded, one approach has been to encapsulate hydrated proteins in reverse micelles formed by a detergent and to dissolve the encapsulated protein in a low-viscosity fluid. The high volatility of suitable low-viscosity fluids requires that the samples be prepared and maintained under pressure. We describe a novel apparatus used for the preparation of such samples. The apparatus includes a chamber for mixing the detergent with the low-viscosity solvent, a second chamber for mixing this with hydrated protein, and a 5-mm (o.d.) zirconium oxide NMR sample tube with shut-off valves designed to contain pressures on the order of 10 bar, sufficient for liquid propane. Liquids are moved from one location to another by introducing minor pressure differentials between two pressurization vessels. We discuss the operation of this apparatus and illustrate this with data on a 30-kDa protein complex (chymotrypsin:turkey ovomucoid third domain) encapsulated in reverse micelles of the detergent, sodium bis (2-ethylhexyl) sulfosuccinate, aerosol-ot (AOT), dissolved in liquid propane.

Research paper thumbnail of Modular organization of SARS coronavirus nucleocapsid protein

Journal of Biomedical Science, 2006

The SARS-CoV nucleocapsid (N) protein is a major antigen in severe acute respiratory syndrome. It... more The SARS-CoV nucleocapsid (N) protein is a major antigen in severe acute respiratory syndrome. It binds to the viral RNA genome and forms the ribonucleoprotein core. The SARS-CoV N protein has also been suggested to be involved in other important functions in the viral life cycle. Here we show that the N protein consists of two non-interacting structural domains, the N-terminal RNA-binding domain (RBD) (residues 45-181) and the C-terminal dimerization domain (residues 248-365) (DD), surrounded by flexible linkers. The C-terminal domain exists exclusively as a dimer in solution. The flexible linkers are intrinsically disordered and represent potential interaction sites with other protein and protein-RNA partners. Bioinformatics reveal that other coronavirus N proteins could share the same modular organization. This study provides information on the domain structure partition of SARS-CoV N protein and insights into the differing roles of structured and disordered regions in coronavirus nucleocapsid proteins.

Research paper thumbnail of The dimer interface of the SARS coronavirus nucleocapsid protein adapts a porcine respiratory and reproductive syndrome virus-like structure

FEBS Letters, 2005

We have employed NMR to investigate the structure of SARS coronavirus nucleocapsid protein dimer.... more We have employed NMR to investigate the structure of SARS coronavirus nucleocapsid protein dimer. We found that the secondary structure of the dimerization domain consists of five alpha helices and a beta-hairpin. The dimer interface consists of a continuous four-stranded beta-sheet superposed by two long alpha helices, reminiscent of that found in the nucleocapsid protein of porcine respiratory and reproductive syndrome virus. Extensive hydrogen bond formation between the two hairpins and hydrophobic interactions between the beta-sheet and the alpha helices render the interface highly stable. Sequence alignment suggests that other coronavirus may share the same structural topology.

Research paper thumbnail of Local control of peptide conformation: Stabilization ofcis proline peptide bonds by aromatic proline interactions

Biopolymers, 1998

In the native state of proteins there is a marked tendency for an aromatic amino acid to precede ... more In the native state of proteins there is a marked tendency for an aromatic amino acid to precede a cis proline. There are also significant differences between the three aromatic amino acids with Tyr exhibiting a noticeably higher propensity than Phe or Trp to precede a cis proline residue. In order to study the role that local interactions play in these conformation preferences, a set of tetrapeptides of the general sequence acetyl-Gly-X-Pro-Gly-carboxamide (GXPG), where X = Tyr, Phe, Trp, Ala, or cyclohexyl alanine, were synthesized and studied by nmr. Analysis of the nmr data shows that none of the peptides adopt a specific backbone structure. Ring current shifts, the equilibrium constant, the Van't Hoff enthalpy, and the measured rate of cis-trans isomerization all indicate that the cis proline conformer is stabilized by favorable interactions between the aromatic ring and the proline residue. Analysis of the side chain conformation of the aromatic residue and analysis of the chemical shifts of the pyrrolidine ring protons shows that the aromatic side chain adopts a preferred conformation in the cis form. The distribution of rotamers and the effect of an aromatic residue on the cis-trans equilibrium indicate that the preferred conformation is populated to approximately 62% for the Phe containing peptide, 67% for the Tyr containing peptide, and between 75 and 80% for the Trp containing peptide. The interaction is unaffected by the addition of 8M urea. These local interactions favor an aromatic residue immediately preceding a cis proline, but they cannot explain the relative propensities for Phe-Pro, Tyr-Pro, and Trp-Pro cis peptide bonds observed in the native state of proteins. In the model peptides the percentage of the cis proline conformer is 21% GYPG while it is 17% for GFPG. This difference is considerably smaller than the almost three to one preponderance observed for cis Tyr-Pro peptide bonds vs cis Phe-Pro peptide bonds in the protein database.

Research paper thumbnail of Folding of the Multidomain Ribosomal Protein L9:  The Two Domains Fold Independently with Remarkably Different Rates †

Biochemistry, 1999

The folding and unfolding behavior of the multidomain ribosomal protein L9 from Bacillus stearoth... more The folding and unfolding behavior of the multidomain ribosomal protein L9 from Bacillus stearothermophilus was studied by a novel combination of stopped-flow fluorescence and nuclear magnetic resonance (NMR) spectroscopy. One-dimensional 1 H spectra acquired at various temperatures show that the C-terminal domain unfolds at a lower temperature than the N-terminal domain (T m) 67°C for the C-terminal domain, 80°C for the N-terminal domain). NMR line-shape analysis was used to determine the folding and unfolding rates for the N-terminal domain. At 72°C, the folding rate constant equals 2980 s-1 and the unfolding rate constant equals 640 s-1. For the C-terminal domain, saturation transfer experiments performed at 69°C were used to determine the folding rate constant, 3.3 s-1 , and the unfolding rate constant, 9.0 s-1. Stopped-flow fluorescence experiments detected two resolved phases: a fast phase for the N-terminal domain and a slow phase for the C-terminal domain. The folding and unfolding rate constants determined by stopped-flow fluorescence are 760 s-1 and 0.36 s-1 , respectively, for the N-terminal domain at 25°C and 3.0 s-1 and 0.0025 s-1 for the C-terminal domain. The Chevron plots for both domains show a V-shaped curve that is indicative of two-state folding. The measured folding rate constants for the N-terminal domain in the intact protein are very similar to the values determined for the isolated N-terminal domain, demonstrating that the folding kinetics of this domain is not affected by the rest of the protein. The remarkably different rate constants between the N-and C-terminal domains suggest that the two domains can fold and unfold independently. The folding behavior of L9 argues that extremely rapid folding is not necessarily functionally important.

Research paper thumbnail of Calcium Binding Peptides from α-Lactalbumin:  Implications for Protein Folding and Stability †

Biochemistry, 1997

The calcium binding protein R-lactalbumin folds via a molten globule intermediate. Calcium does n... more The calcium binding protein R-lactalbumin folds via a molten globule intermediate. Calcium does not bind strongly to the unfolded protein or the molten globule, but does bind to the transition state between the molten globule and the native protein. Of interest are the structures formed in the transition state that promote calcium binding. To study the importance of local secondary structure on calcium binding, we have synthesized two peptides corresponding to the calcium binding site that include the flanking C-helix and 3 10-helix. The first peptide, elbow-A, consists of residues 72-100 from bovine R-lactalbumin, but with Cys 73, Cys 77, and Cys 91 replaced by alanines. In the second peptide, denoted elbow, the cysteines at position 73 and 91 are included and the nativelike disulfide bond is formed. Both peptides are monomeric and unstructured in aqueous solution and bind calcium weakly with apparent K d 's on the order of 10-2 M. In 50% trifluoroethanol (v/v), the peptides are 45% helical as judged by CD. NMR studies performed on elbow and elbow-A in TFE indicate that the helical structure is confined to the C-helix. In this solvent system elbow binds calcium one-to-one with a K d of 50 µM. Removing the disulfide bond reduces, but does not eliminate calcium binding (K d) 170 µM in 50% TFE). These results suggest that formation of the C-helix promotes calcium binding and may be a key determinant of calcium binding in the transition state.

Research paper thumbnail of Structure of Hexadienoyl-CoA Bound to Enoyl-CoA Hydratase Determined by Transferred Nuclear Overhauser Effect Measurements:  Mechanistic Predictions Based on the X-ray Structure of 4-(Chlorobenzoyl)-CoA Dehalogenase †

Biochemistry, 1997

... function as a general acid/base. Enoyl-CoA hydratase (EC 4.2.1.17) catalyzes the syn addition... more ... function as a general acid/base. Enoyl-CoA hydratase (EC 4.2.1.17) catalyzes the syn addition of water across the C C bond of α,β-unsaturated fatty acid CoA thioesters (Willadsen & Eggerer, 1975). On the basis of kinetic isotope ...

Research paper thumbnail of RIBRA-an error-tolerant algorithm for the NMR backbone assignment problem

We develop an iterative relaxation algorithm, called RIBRA, for NMR protein backbone assignment. ... more We develop an iterative relaxation algorithm, called RIBRA, for NMR protein backbone assignment. RIBRA applies nearest neighbor and weighted maximum independent set algorithms to solve the problem. To deal with noisy NMR spectral data, RIBRA is executed in an iterative fashion based on the quality of spectral peaks. We first produce spin system pairs using the spectral data without missing peaks, then the data group with one missing peak, and finally, the data group with two missing peaks. We test RIBRA on two real NMR datasets: hbSBD and hbLBD, and perfect BMRB data (with 902 proteins) and four synthetic BMRB data which simulate four kinds of errors. The accuracy of RIBRA on hbSBD and hbLBD are 91.4% and 83.6%, respectively. The average accuracy of RIBRA on perfect BMRB datasets is 98.28%, and 98.28%, 95.61%, 98.16% and 96.28% on four kinds of synthetic datasets, respectively.

Research paper thumbnail of Rapid data acquisition of 3D triple resonance experiments via projection reconstruction NMR (PR-NMR)

Research paper thumbnail of Synthesis of Heterobimetallic Phosphido-Bridged W-Mo Complexes with Chelating dppe and dppm Ligands

Journal of the Chinese Chemical Society, 1995

... of Heterobimetallic Phosphido-Bridged W-Mo Complexes with Chelating dppe and dppm Ligands Wen... more ... of Heterobimetallic Phosphido-Bridged W-Mo Complexes with Chelating dppe and dppm Ligands Wen-Jin Wu ( Щ^Щ ), Jiun-Yi Hsu ( $ШШ )> Yuh-Shang Wen ( fëULî. ... Scheme I Ph2 P /\ Cp(CO)2W Mo(CO)5 1 Ph2 О Ph2 Г THF Л L = dppm, dppe Ph2 О Cp(CO) PPh2-(CH2)x ...

Research paper thumbnail of Direct NMR resonance assignments of the active site histidine residue inEscherichia coli thioesterase I/protease I/lysophospholipase L1

Magnetic Resonance in Chemistry, 2006

Owing to the hydrogen-bond interaction and rapid exchange rate with the bulk water, the transvers... more Owing to the hydrogen-bond interaction and rapid exchange rate with the bulk water, the transverse relaxation time for the N(delta1)-H proton of the catalytic histidine in Escherichia coli thioesterase I/protease I/lysophospholipase L1 (TEP-I) is rather short. Because of its catalytic importance, it is desirable to detect and assign this proton resonance. In this paper, we report the first direct NMR correlation between the short-lived N(delta1)-H proton and its covalently attached N(delta1)-nitrogen of the catalytic His157 residue in E. coli thioesterase/protease I. We have used gradient-enhanced jump-return spin-echo HMQC (GE-JR SE HMQC) to obtain a direct correlation between the short-lived N(delta1)-H proton and its covalently attached N(delta1)-nitrogen. The sensitivity of detection for the short-lived N(delta1)-H proton was enhanced substantially by improved water suppression, in particular, the suppression of radiation damping via pulsed field gradients.

Research paper thumbnail of How a Low-Fidelity DNA Polymerase Chooses Non-Watson–Crick from Watson–Crick Incorporation

Journal of the American Chemical Society, 2014

A dogma for DNA polymerase catalysis is that the enzyme binds DNA first, followed by MgdNTP. This... more A dogma for DNA polymerase catalysis is that the enzyme binds DNA first, followed by MgdNTP. This mechanism contributes to the selection of correct dNTP by Watson-Crick base pairing, but it cannot explain how low-fidelity DNA polymerases overcome Watson-Crick base pairing to catalyze non-Watson-Crick dNTP incorporation. DNA polymerase X from the deadly African swine fever virus (Pol X) is a half-sized repair polymerase that catalyzes efficient dG:dGTP incorporation in addition to correct repair. Here we report the use of solution structures of Pol X in the free, binary (Pol X:MgdGTP), and ternary (Pol X:DNA:MgdGTP with dG:dGTP non-Watson-Crick pairing) forms, along with functional analyses, to show that Pol X uses multiple unprecedented strategies to achieve the mutagenic dG:dGTP incorporation. Unlike high fidelity polymerases, Pol X can prebind purine MgdNTP tightly and undergo a specific conformational change in the absence of DNA. The prebound MgdGTP assumes an unusual syn conformation stabilized by partial ring stacking with His115. Upon binding of a gapped DNA, also with a unique mechanism involving primarily helix αE, the prebound syn-dGTP forms a Hoogsteen base pair with the template anti-dG. Interestingly, while Pol X prebinds MgdCTP weakly, the correct dG:dCTP ternary complex is readily formed in the presence of DNA. H115A mutation disrupted MgdGTP binding and dG:dGTP ternary complex formation but not dG:dCTP ternary complex formation. The results demonstrate the first solution structural view of DNA polymerase catalysis, a unique DNA binding mode, and a novel mechanism for non-Watson-Crick incorporation by a low-fidelity DNA polymerase.

Research paper thumbnail of Stereospecific 1 H and 13 C NMR Assignments of Crotonyl CoA and Hexadienoyl CoA:  Conformational Analysis and Comparison with Protein−CoA Complexes

Journal of the American Chemical Society, 1998

Stereospecific 1 H and 13 C resonance assignments of hexadienoyl-CoA and crotonyl-CoA have been d... more Stereospecific 1 H and 13 C resonance assignments of hexadienoyl-CoA and crotonyl-CoA have been determined. The two diastereotopic methyl groups at the C2′′ carbon were assigned via transferred nuclear Overhauser effect experiments on the complex of hexadienoyl-CoA with the enzyme enoyl-CoA hydratase. The two diastereotopic 1′′ protons were assigned using heteronuclear multiple-bond correlation experiments in conjunction with rotating frame nuclear Overhauser spectroscopy. This represents the first set of complete stereospecific assignments for any CoA derivative. The assignments allow a detailed quantitative conformational analysis of the uncomplexed form of the molecule. In particular, the relative population of the various rotamers about the C1′′-C2′′ and the C2′′-C3′′ bonds have been determined. The database of protein-bound CoA structures has been surveyed and used to compare the structure(s) of CoA in protein-CoA complexes with the conformational preferences of free CoA.

Research paper thumbnail of Stereospecificity of the Reaction Catalyzed by Enoyl-CoA Hydratase

Journal of the American Chemical Society, 2000

Enoyl-CoA hydratase catalyzes the stereospecific hydration of α, β-unsaturated acyl-CoA thioleste... more Enoyl-CoA hydratase catalyzes the stereospecific hydration of α, β-unsaturated acyl-CoA thiolesters. Hydration of trans-2-crotonyl-CoA to 3 (S)-hydroxybutyryl-CoA proceeds via the syn addition of water and thus the pro-2 R proton of 3 (S)-hydroxybutyryl-CoA is derived ...

Research paper thumbnail of Conformational Heterogeneity about Pipecolic Acid Peptide Bonds:  Conformational, Thermodynamic, and Kinetic Aspects

The Journal of Organic Chemistry, 1998

Cis-trans isomerization about pipecolic peptide bonds has been studied using a set of designed te... more Cis-trans isomerization about pipecolic peptide bonds has been studied using a set of designed tetrapeptides of the sequence acetyl-Gly-X-Pip-Gly-carboxamide (GXPipG), where X) A, F, Y, W, or cyclohexylalanine (Cha). The substitution of a pipecolic residue for a proline leads to (1) a significant increase of the population of the cis conformer, (2) a reduction in the Van't Hoff enthalpy for isomerism, and (3) acceleration of the rates of isomerization. GAPipG and GChaPipG are unstructured, and isomerization is controlled by local steric effects between the R and positions of the pipecolic ring and the R position of the preceding residue. The fraction of the cis isomer is the same for these two peptides, indicating that the bulk of the preceding amino acid does not play a significant role in influencing the equilibrium. In the trans form of the aromatic containing peptides, an i to i + 2 aromatic-amide interaction is observed, and in the cis form a stabilizing interaction between the aromatic residue and the succeeding pipecolic ring is observed. The sign of the Van't Hoff enthalpy indicates that the cis conformer is enthalpically favored for the aromatic containing peptides. The rate of isomerization for the aromatic containing peptides are reduced relative to GAPipG.

Research paper thumbnail of PWWP Module of Human Hepatoma-derived Growth Factor Forms a Domain-swapped Dimer with Much Higher Affinity for Heparin

Journal of Molecular Biology, 2007

Hepatoma-derived growth factor (hHDGF)-related proteins (HRPs) comprise a new growth factor famil... more Hepatoma-derived growth factor (hHDGF)-related proteins (HRPs) comprise a new growth factor family sharing a highly conserved and ordered Nterminal PWWP module (residues 1-100, previously referred to as a HATH domain) and a variable disordered C-terminal domain. We have shown that the PWWP module is responsible for heparin binding and have solved its structure in solution. Here, we show that under physiological conditions, both the PWWP module and hHDGF can form dimers. Surface plasmon resonance (SPR) studies revealed that the PWWP dimer binds to heparin with affinity that is two orders of magnitude higher (K d = 13 nM) than that of the monomeric PWWP module (K d = 1.2 μM). The monomer-dimer equilibrium properties and NMR structural data together suggest that the PWWP dimer is formed through a domain-swapping mechanism. The domain-swapped PWWP dimer structures were calculated on the basis of the NMR data. The results show that the two PWWP protomers exchange their N-terminal hairpin to form a domain-swapped dimer. The two monomers in a dimer are linked by the long flexible L2 loops, a feature supported by NMR relaxation data for the monomer and dimer. The enhanced heparin-binding affinity of the dimer can be rationalized in the framework of the dimer structure.

Research paper thumbnail of Conformational analysis of a set of peptides corresponding to the entire primary sequence of the N-terminal domain of the ribosomal protein L9: evidence for stable native-like secondary structure in the unfolded state

Journal of Molecular Biology, 1999

Research paper thumbnail of Mixing apparatus for preparing NMR samples under pressure

Journal of Magnetic Resonance, 2003

The size limit for protein NMR spectroscopy in solution arises in large part from line broadening... more The size limit for protein NMR spectroscopy in solution arises in large part from line broadening caused by slow molecular tumbling. One way to alleviate this problem is to increase the effective tumbling rate by reducing the viscosity of the solvent. Because proteins generally require an aqueous environment to remain folded, one approach has been to encapsulate hydrated proteins in reverse micelles formed by a detergent and to dissolve the encapsulated protein in a low-viscosity fluid. The high volatility of suitable low-viscosity fluids requires that the samples be prepared and maintained under pressure. We describe a novel apparatus used for the preparation of such samples. The apparatus includes a chamber for mixing the detergent with the low-viscosity solvent, a second chamber for mixing this with hydrated protein, and a 5-mm (o.d.) zirconium oxide NMR sample tube with shut-off valves designed to contain pressures on the order of 10 bar, sufficient for liquid propane. Liquids are moved from one location to another by introducing minor pressure differentials between two pressurization vessels. We discuss the operation of this apparatus and illustrate this with data on a 30-kDa protein complex (chymotrypsin:turkey ovomucoid third domain) encapsulated in reverse micelles of the detergent, sodium bis (2-ethylhexyl) sulfosuccinate, aerosol-ot (AOT), dissolved in liquid propane.

Research paper thumbnail of Modular organization of SARS coronavirus nucleocapsid protein

Journal of Biomedical Science, 2006

The SARS-CoV nucleocapsid (N) protein is a major antigen in severe acute respiratory syndrome. It... more The SARS-CoV nucleocapsid (N) protein is a major antigen in severe acute respiratory syndrome. It binds to the viral RNA genome and forms the ribonucleoprotein core. The SARS-CoV N protein has also been suggested to be involved in other important functions in the viral life cycle. Here we show that the N protein consists of two non-interacting structural domains, the N-terminal RNA-binding domain (RBD) (residues 45-181) and the C-terminal dimerization domain (residues 248-365) (DD), surrounded by flexible linkers. The C-terminal domain exists exclusively as a dimer in solution. The flexible linkers are intrinsically disordered and represent potential interaction sites with other protein and protein-RNA partners. Bioinformatics reveal that other coronavirus N proteins could share the same modular organization. This study provides information on the domain structure partition of SARS-CoV N protein and insights into the differing roles of structured and disordered regions in coronavirus nucleocapsid proteins.

Research paper thumbnail of The dimer interface of the SARS coronavirus nucleocapsid protein adapts a porcine respiratory and reproductive syndrome virus-like structure

FEBS Letters, 2005

We have employed NMR to investigate the structure of SARS coronavirus nucleocapsid protein dimer.... more We have employed NMR to investigate the structure of SARS coronavirus nucleocapsid protein dimer. We found that the secondary structure of the dimerization domain consists of five alpha helices and a beta-hairpin. The dimer interface consists of a continuous four-stranded beta-sheet superposed by two long alpha helices, reminiscent of that found in the nucleocapsid protein of porcine respiratory and reproductive syndrome virus. Extensive hydrogen bond formation between the two hairpins and hydrophobic interactions between the beta-sheet and the alpha helices render the interface highly stable. Sequence alignment suggests that other coronavirus may share the same structural topology.

Research paper thumbnail of Local control of peptide conformation: Stabilization ofcis proline peptide bonds by aromatic proline interactions

Biopolymers, 1998

In the native state of proteins there is a marked tendency for an aromatic amino acid to precede ... more In the native state of proteins there is a marked tendency for an aromatic amino acid to precede a cis proline. There are also significant differences between the three aromatic amino acids with Tyr exhibiting a noticeably higher propensity than Phe or Trp to precede a cis proline residue. In order to study the role that local interactions play in these conformation preferences, a set of tetrapeptides of the general sequence acetyl-Gly-X-Pro-Gly-carboxamide (GXPG), where X = Tyr, Phe, Trp, Ala, or cyclohexyl alanine, were synthesized and studied by nmr. Analysis of the nmr data shows that none of the peptides adopt a specific backbone structure. Ring current shifts, the equilibrium constant, the Van't Hoff enthalpy, and the measured rate of cis-trans isomerization all indicate that the cis proline conformer is stabilized by favorable interactions between the aromatic ring and the proline residue. Analysis of the side chain conformation of the aromatic residue and analysis of the chemical shifts of the pyrrolidine ring protons shows that the aromatic side chain adopts a preferred conformation in the cis form. The distribution of rotamers and the effect of an aromatic residue on the cis-trans equilibrium indicate that the preferred conformation is populated to approximately 62% for the Phe containing peptide, 67% for the Tyr containing peptide, and between 75 and 80% for the Trp containing peptide. The interaction is unaffected by the addition of 8M urea. These local interactions favor an aromatic residue immediately preceding a cis proline, but they cannot explain the relative propensities for Phe-Pro, Tyr-Pro, and Trp-Pro cis peptide bonds observed in the native state of proteins. In the model peptides the percentage of the cis proline conformer is 21% GYPG while it is 17% for GFPG. This difference is considerably smaller than the almost three to one preponderance observed for cis Tyr-Pro peptide bonds vs cis Phe-Pro peptide bonds in the protein database.

Research paper thumbnail of Folding of the Multidomain Ribosomal Protein L9:  The Two Domains Fold Independently with Remarkably Different Rates †

Biochemistry, 1999

The folding and unfolding behavior of the multidomain ribosomal protein L9 from Bacillus stearoth... more The folding and unfolding behavior of the multidomain ribosomal protein L9 from Bacillus stearothermophilus was studied by a novel combination of stopped-flow fluorescence and nuclear magnetic resonance (NMR) spectroscopy. One-dimensional 1 H spectra acquired at various temperatures show that the C-terminal domain unfolds at a lower temperature than the N-terminal domain (T m) 67°C for the C-terminal domain, 80°C for the N-terminal domain). NMR line-shape analysis was used to determine the folding and unfolding rates for the N-terminal domain. At 72°C, the folding rate constant equals 2980 s-1 and the unfolding rate constant equals 640 s-1. For the C-terminal domain, saturation transfer experiments performed at 69°C were used to determine the folding rate constant, 3.3 s-1 , and the unfolding rate constant, 9.0 s-1. Stopped-flow fluorescence experiments detected two resolved phases: a fast phase for the N-terminal domain and a slow phase for the C-terminal domain. The folding and unfolding rate constants determined by stopped-flow fluorescence are 760 s-1 and 0.36 s-1 , respectively, for the N-terminal domain at 25°C and 3.0 s-1 and 0.0025 s-1 for the C-terminal domain. The Chevron plots for both domains show a V-shaped curve that is indicative of two-state folding. The measured folding rate constants for the N-terminal domain in the intact protein are very similar to the values determined for the isolated N-terminal domain, demonstrating that the folding kinetics of this domain is not affected by the rest of the protein. The remarkably different rate constants between the N-and C-terminal domains suggest that the two domains can fold and unfold independently. The folding behavior of L9 argues that extremely rapid folding is not necessarily functionally important.

Research paper thumbnail of Calcium Binding Peptides from α-Lactalbumin:  Implications for Protein Folding and Stability †

Biochemistry, 1997

The calcium binding protein R-lactalbumin folds via a molten globule intermediate. Calcium does n... more The calcium binding protein R-lactalbumin folds via a molten globule intermediate. Calcium does not bind strongly to the unfolded protein or the molten globule, but does bind to the transition state between the molten globule and the native protein. Of interest are the structures formed in the transition state that promote calcium binding. To study the importance of local secondary structure on calcium binding, we have synthesized two peptides corresponding to the calcium binding site that include the flanking C-helix and 3 10-helix. The first peptide, elbow-A, consists of residues 72-100 from bovine R-lactalbumin, but with Cys 73, Cys 77, and Cys 91 replaced by alanines. In the second peptide, denoted elbow, the cysteines at position 73 and 91 are included and the nativelike disulfide bond is formed. Both peptides are monomeric and unstructured in aqueous solution and bind calcium weakly with apparent K d 's on the order of 10-2 M. In 50% trifluoroethanol (v/v), the peptides are 45% helical as judged by CD. NMR studies performed on elbow and elbow-A in TFE indicate that the helical structure is confined to the C-helix. In this solvent system elbow binds calcium one-to-one with a K d of 50 µM. Removing the disulfide bond reduces, but does not eliminate calcium binding (K d) 170 µM in 50% TFE). These results suggest that formation of the C-helix promotes calcium binding and may be a key determinant of calcium binding in the transition state.

Research paper thumbnail of Structure of Hexadienoyl-CoA Bound to Enoyl-CoA Hydratase Determined by Transferred Nuclear Overhauser Effect Measurements:  Mechanistic Predictions Based on the X-ray Structure of 4-(Chlorobenzoyl)-CoA Dehalogenase †

Biochemistry, 1997

... function as a general acid/base. Enoyl-CoA hydratase (EC 4.2.1.17) catalyzes the syn addition... more ... function as a general acid/base. Enoyl-CoA hydratase (EC 4.2.1.17) catalyzes the syn addition of water across the C C bond of α,β-unsaturated fatty acid CoA thioesters (Willadsen & Eggerer, 1975). On the basis of kinetic isotope ...

Research paper thumbnail of RIBRA-an error-tolerant algorithm for the NMR backbone assignment problem

We develop an iterative relaxation algorithm, called RIBRA, for NMR protein backbone assignment. ... more We develop an iterative relaxation algorithm, called RIBRA, for NMR protein backbone assignment. RIBRA applies nearest neighbor and weighted maximum independent set algorithms to solve the problem. To deal with noisy NMR spectral data, RIBRA is executed in an iterative fashion based on the quality of spectral peaks. We first produce spin system pairs using the spectral data without missing peaks, then the data group with one missing peak, and finally, the data group with two missing peaks. We test RIBRA on two real NMR datasets: hbSBD and hbLBD, and perfect BMRB data (with 902 proteins) and four synthetic BMRB data which simulate four kinds of errors. The accuracy of RIBRA on hbSBD and hbLBD are 91.4% and 83.6%, respectively. The average accuracy of RIBRA on perfect BMRB datasets is 98.28%, and 98.28%, 95.61%, 98.16% and 96.28% on four kinds of synthetic datasets, respectively.