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- PDB-8dj4: NMR Solution Structure of C-terminally amidated, Full-length Huma... -

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Basic information

Entry
Database: PDB / ID: 8dj4
TitleNMR Solution Structure of C-terminally amidated, Full-length Human Galanin
ComponentsGalanin
KeywordsNEUROPEPTIDE / synthetic
Function / homology
Function and homology information


galanin receptor binding / type 1 galanin receptor binding / type 2 galanin receptor binding / type 3 galanin receptor binding / galanin receptor activity / positive regulation of large conductance calcium-activated potassium channel activity / positive regulation of timing of catagen / positive regulation of cortisol secretion / regulation of glucocorticoid metabolic process / negative regulation of lymphocyte proliferation ...galanin receptor binding / type 1 galanin receptor binding / type 2 galanin receptor binding / type 3 galanin receptor binding / galanin receptor activity / positive regulation of large conductance calcium-activated potassium channel activity / positive regulation of timing of catagen / positive regulation of cortisol secretion / regulation of glucocorticoid metabolic process / negative regulation of lymphocyte proliferation / neuropeptide hormone activity / feeding behavior / insulin secretion / response to immobilization stress / neuropeptide signaling pathway / protein kinase A signaling / cAMP-mediated signaling / Peptide ligand-binding receptors / secretory granule / response to insulin / response to estrogen / G alpha (i) signalling events / response to xenobiotic stimulus / positive regulation of apoptotic process / neuronal cell body / positive regulation of transcription by RNA polymerase II / extracellular space / extracellular region
Similarity search - Function
Galanin / Galanin precursor / Galanin message associated peptide (GMAP) / Galanin / Galanin message associated peptide (GMAP) / Galanin signature. / Galanin
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodSOLUTION NMR / simulated annealing
AuthorsWilkinson, R.E. / Kraichely, K.N. / Buchanan, L.E. / Parnham, S. / Giuliano, M.W.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P20GM103499-20 United States
CitationJournal: Biochem.Biophys.Res.Commun. / Year: 2022
Title: The neuropeptide galanin adopts an irregular secondary structure.
Authors: Wilkinson, R.E. / Kraichely, K.N. / Hendy, C.M. / Buchanan, L.E. / Parnham, S. / Giuliano, M.W.
History
DepositionJun 30, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 24, 2022Provider: repository / Type: Initial release
Revision 1.1Sep 7, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Galanin


Theoretical massNumber of molelcules
Total (without water)3,1591
Polymers3,1591
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: assay for oligomerization, Peptide concentration was assessed over a fifty-fold range of concentrations in 1D 1H NMR spectra from 0.1 mM up to 5.0 mM. While all concentrations below and ...Evidence: assay for oligomerization, Peptide concentration was assessed over a fifty-fold range of concentrations in 1D 1H NMR spectra from 0.1 mM up to 5.0 mM. While all concentrations below and inclusive of 1.0 mM peptide were acceptable - free of dispersion and broadening effects across multiple weeks in solution - 0.5 mM was selected as the optimal choice for further analysis with optimal resolution.
TypeNameSymmetry operationNumber
identity operation1_5551
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)25 / 1000structures with the lowest energy
RepresentativeModel #1lowest energy

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Components

#1: Protein/peptide Galanin /


Mass: 3159.454 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: P22466
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
111isotropic11H 1D NMR Spectrum
121isotropic12D 1H-1H COSY
131isotropic12D 1H-1H TOCSY
141isotropic12D 1H-1H ROESY
NMR detailsText: The authors state the following regarding the geometric deviations with this ensemble: With most of the hydrogen bonds in the N-terminus of the peptide bifurcated, or even more, they are ...Text: The authors state the following regarding the geometric deviations with this ensemble: With most of the hydrogen bonds in the N-terminus of the peptide bifurcated, or even more, they are confident that the ensemble represents the time-averaged structure of a peptide that is rapidly interconverting turns of alpha, 310-, and pi-helix. Their shift assignments were largely unambiguous and can be found in BMRB entry 31032. The "irregular helix restraints" used (and described in the manuscript and its references) were employed to allow the ensemble to sample multiple arrangements during simulated annealing. By definition, this ensemble existing as an average will display a significant degree of geometric irregularity, particularly with regard the backbone and sidechain outliers as compared to statistical norms. Further, the clashscore level is quite high. This appears to be a consequence of the very high density of ROE-derived distance restraints in the N-terminus of the peptide, which were maximally relaxed to account for any uncertainty in their measurement throughout several rounds of validation and re-calculation. Tight sidechain packing and compression seems to be the dictated outcome of our data. Much of their study is concerned with compact hydrophobic packing in this sequence - this full-length peptide is indeed much more rigid across the stretch of residues it shares with fragment peptides described in entries 7S3O, 7S3Q, and 7S3R. Together with the compactness of the region of the sequence, the clashes appear to be a consequence of several averaged, coiling, helical conformers all existing on the NMR timescale. This is discussed at further length in the citation affiliated with this deposition

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Sample preparation

DetailsType: lyophilized powder
Contents: 0.5 mM hGal(1-30)NH2, 4.14 mM [U-2H] sodium acetate, 5.86 mM [U-2H] acetic acid, 95% H2O/5% D2O
Details: peptide was suspended and dissolved into deuterated acetate buffer, which had been prepared in 95:5 H2O:D2O to within 0.02 units of pH 4.6
Label: 1 / Solvent system: 95% H2O/5% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
0.5 mMhGal(1-30)NH2natural abundance1
4.14 mMsodium acetate[U-2H]1
5.86 mMacetic acid[U-2H]1
Sample conditionsIonic strength: sample was dissolved in 10 mM sodium acetate-d3/acetic acid-d4 buffer at pH 4.6; no additional additives were present. mM
Label: 1 / pH: 4.6 / PH err: 0.02 / Pressure: 1 atm / Temperature: 298 K

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NMR measurement

NMR spectrometerType: Bruker AVANCE III / Manufacturer: Bruker / Model: AVANCE III / Field strength: 600 MHz / Details: TCI H-C/N-D Cryoprobe

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Processing

NMR software
NameDeveloperClassification
CNSBrunger A. T. et.al.refinement
CNSBrunger, Adams, Clore, Gros, Nilges and Readstructure calculation
MestreLab (Mnova / MestReNova / MestReC)MestreLabchemical shift assignment
MestreLab (Mnova / MestReNova / MestReC)MestreLabpeak picking
RefinementMethod: simulated annealing / Software ordinal: 1
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 1000 / Conformers submitted total number: 25

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