1H-n.m.r. studies of the isolated activation segment from pig procarboxypeptidase A

The isolated activation segment (asA) from pig pancreatic procarboxypeptidase A was studied by 1H-n.m.r. spectroscopy over a wide range of solution conditions. Isolated asA shows many characteristics of compactly folded globular proteins, such as the observation of perturbed positions for resonances from methyl groups, alpha-carbon atoms, histidine residues and the tyrosine residue. The single tyrosine residue (Tyr-70) exhibits a very high pKa, and both histidine and tyrosine residues show slow chemical modification (deuteration and iodination). In contrast, asA shows rapid NH exchange. Analysis of the spectra by pH titration and nuclear Overhauser effects revealed several residue interactions. Quantitative analysis of deuterium and tritium exchange allowed the assignment of the histidine C-2-H resonances to their respective residues in the sequence. His-66, the closest to the sites of proteolytic attack in the proenzyme, is shown to be the most accessible to solvent in procarboxypeptidase A. It was also shown that asA is thermally very stable [‘melting’ temperature (Tm) 88 degrees C] and requires a high urea concentration for denaturation (6.25 M, at pH 7.5). Evidence is presented for some degree of conformational flexibility in the premelting range, a feature that could be ascribed to the preponderance of helical secondary structure and to the lack of disulphide bridges. The free solution structure of asA is probably unchanged when it binds to carboxypeptidase A.

Download Full-text

The Solution Structure of Yeast tRNAPheas Studied by Nuclear Overhauser Effects in NMR

Journal of Biomolecular Structure and Dynamics ◽

10.1080/07391102.1983.10507434 ◽

1983 ◽

Vol 1 (1) ◽

pp. 183-207 ◽

Cited By ~ 29

Author(s):

C. W. Hilbers ◽

A. Heerschap ◽

C. A.G. Haasnoot ◽

J. A.L.I. Walters

Keyword(s):

Solution Structure ◽

Nuclear Overhauser ◽

Nuclear Overhauser Effects

Download Full-text

Structural characterization and exposure of aromatic residues in epidermal growth factor from the rat

Biochemical Journal ◽

10.1042/bj2390013 ◽

1986 ◽

Vol 239 (1) ◽

pp. 13-18 ◽

Cited By ~ 12

Author(s):

K H Mayo ◽

P Schaudies ◽

C R Savage ◽

A De Marco ◽

R Kaptein

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Structural Domain ◽

Solvent Exposure ◽

Ph Titration ◽

Aromatic Residues ◽

Tyrosine Residues ◽

Titration Data ◽

Epidermal Growth ◽

Nuclear Overhauser

Aromatic amino acid residues in epidermal growth factor (EGF) isolated from the rat have been investigated by proton n.m.r. and nuclear Overhauser methods at 500 MHz and by photochemically induced dynamic nuclear polarization (photo-c.i.d.n.p.) experiments at 360 MHz. Rat EGF contains six aromatic residues, i.e. one histidine and five tyrosine residues. pH titration data allow identification of the histidine imidazole ring protons, whereas two-dimensional n.m.r. correlated spectroscopy establishes connectivities between tyrosine ring (2,6) and (3,5) proton resonances. Photo-c.i.d.n.p. data give evidence for solvent exposure of the one histidine and the five tyrosine residues in rat EGF. Nuclear Overhauser experiments and pH titration data suggest proximity relationships among four of the tyrosine residues and the histidine residue. These data indicate the presence of a clustered, aromatic, structural domain on the protein surface and may provide a clue to the understanding of the functional structure of EGF.

Download Full-text

An Azo Coupling-Based Chemoproteomic Approach to Systematically Profile the Tyrosine Reactivity in the Human Proteome

10.26434/chemrxiv.14556180 ◽

2021 ◽

Author(s):

Fangxu Sun ◽

Suttipong Suttapitugsakul ◽

Ronghu Wu

Keyword(s):

Large Scale ◽

Diazonium Salt ◽

Human Proteome ◽

Binding Property ◽

Tyrosine Residue ◽

Azo Coupling ◽

Mcf7 Cells ◽

Bioorthogonal Chemistry ◽

Tyrosine Residues ◽

Wide Range

<p>The tyrosine residue of proteins participates in a wide range of activities including enzymatic catalysis, protein-protein interaction, and protein-ligand binding. However, the functional annotation of the tyrosine residues on a large scale is still very challenging. Here, we report a novel method integrating azo coupling, bioorthogonal chemistry, and multiplexed proteomics to globally investigate the tyrosine reactivity in the human proteome. Based on the azo-coupling reaction between aryl diazonium salt and the tyrosine residue, the two different probes were evaluated, and the probe with the best performance was employed to specifically target the tyrosine residues. After the reaction, tagged tyrosine containing peptides were selectively enriched using bioorthogonal chemistry, and a small tag on the peptides from the cleavage perfectly fits for site-specific analysis by MS. Coupling with multiplexed proteomics, we quantified over 5,000 tyrosine sites in MCF7 cells and these quantified sites displayed a wide range of reactivity. The tyrosine residues with high reactivity were found on functionally and structurally diverse proteins, including those with the catalytic activity and binding property. This method can be extensively applied to advance our understanding of protein functions and facilitate the development of covalent drugs to regulate protein activity.</p>

Download Full-text

Study of ion pair solution structure using nuclear overhauser effects: interionic 1H{1H} and 11B{1H} NOES in the (CH3CH2CH2CH2)4N+,BH4- ion pair

Journal of the American Chemical Society ◽

10.1021/ja00174a042 ◽

1990 ◽

Vol 112 (18) ◽

pp. 6714-6715 ◽

Cited By ~ 21

Author(s):

Thomas C. Pochapsky ◽

Patricia M. Stone

Keyword(s):

Solution Structure ◽

Ion Pair ◽

Nuclear Overhauser ◽

Nuclear Overhauser Effects

Download Full-text

High-Resolution Solution Structure of a Designed Peptide Bound to Lipopolysaccharide: Transferred Nuclear Overhauser Effects, Micelle Selectivity, and Anti-Endotoxic Activity†,‡

Biochemistry ◽

10.1021/bi6025159 ◽

2007 ◽

Vol 46 (20) ◽

pp. 5864-5874 ◽

Cited By ~ 34

Author(s):

Surajit Bhattacharjya ◽

Prerna N. Domadia ◽

Anirban Bhunia ◽

Subbalakshmi Malladi ◽

Sunil A. David

Keyword(s):

High Resolution ◽

Solution Structure ◽

Nuclear Overhauser ◽

Nuclear Overhauser Effects ◽

Endotoxic Activity

Download Full-text

New theoretical methodology for elucidating the solution structure of peptides from NMR data. II. free energy of dominant microstates of Leu-enkephalin and population-weighted average nuclear Overhauser effects intensities

Biopolymers ◽

10.1002/(sici)1097-0282(199601)38:1<69::aid-bip6>3.0.co;2-u ◽

1996 ◽

Vol 38 (1) ◽

pp. 69-88 ◽

Cited By ~ 17

Author(s):

Eva Meirovitch ◽

Hagai Meirovitch

Keyword(s):

Free Energy ◽

Solution Structure ◽

Weighted Average ◽

Nmr Data ◽

Nuclear Overhauser ◽

Nuclear Overhauser Effects

Download Full-text

Interaction of the antitumour antibiotic luzopeptin with the hexanucleotide duplex d(5′-GCATGC)2. One-dimensional and two-dimensional n.m.r. studies

Biochemical Journal ◽

10.1042/bj2590433 ◽

1989 ◽

Vol 259 (2) ◽

pp. 433-441 ◽

Cited By ~ 18

Author(s):

M S Searle ◽

J G Hall ◽

W A Denny ◽

L P Wakelin

Keyword(s):

Base Pair ◽

Solution Structure ◽

Major Axis ◽

Base Pairs ◽

Carbocyclic Rings ◽

Nuclear Overhauser ◽

Nuclear Overhauser Effects ◽

Hoogsteen Base Pairing ◽

Dyad Symmetry ◽

Hydroxy Groups

1H- and 31P-n.m.r. spectroscopy were used to characterize the solution structure of the 1:1 complex formed between the antitumour antibiotic luzopeptin and the self-complementary hexanucleotide d(5'-GCATGC)2. Eighteen nuclear Overhauser effects between antibiotic and nucleotide protons, together with ring-current-induced perturbations to base-pair and quinoline 1H resonances, define the position and orientation of the bound drug molecule. Luzopeptin binds in the minor groove of the DNA with full retention of dyad symmetry, its quinoline chromophores intercalating at the 5'-CpA and 5'-TpG steps and its depsipeptide ring spanning the central two A.T base-pairs. The chromophores stack principally on the adenine base with their carbocyclic rings pointing towards the deoxyribose of the cytosine. There is no evidence for Hoogsteen base-pairing in the complex, all glycosidic bond angles and sugar puckers being typical of B-DNA as found for the free hexanucleotide. The ‘breathing’ motions of the A.T and internal G.C base-pairs are substantially slowed in the complex compared with the free DNA, and the observation that two phosphate resonances are shifted downfield by at least 0.5 p.p.m. in the 31P-n.m.r. spectrum of the complex suggests pronounced local helix unwinding at the intercalation sites. The data are consistent with a model of the complex in which luzopeptin bisintercalates with its depsipeptide essentially in the conformation found in the crystal of the free antibiotic [Arnold & Clardy (1981) J. Am. Chem. Soc. 103, 1243-1244]. We postulate only one conformational change within the peptide ring, which involves rotation of the pyridazine-glycine amide group linkage by 90 degrees towards the DNA surface. This manoeuvre breaks the glycine-to-glycine transannular hydrogen bonds and enables the glycine NH groups to bond to the thymine O-2 atoms of the sandwiched A.T base-pairs. It also shortens the major axis of the depsipeptide so that the interchromophore distance is more suitable for spanning two base-pairs. The model further implies that the carboxy and hydroxy groups of the L-beta-hydroxyvaline residue are appropriately positioned for hydrogen-bonding to the 2-amino group of guanine and the O-2 atom of cytosine of the adjacent G.C base-pair.

Download Full-text