Linked Life Data

16964534

Source:http://linkedlifedata.com/resource/pubmed/id/16964534

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
2006-9-26
pubmed:abstractText
Interactions of proteins with small molecules or other macromolecules play key roles in many biological processes and in drug action, and NMR is an excellent tool for their structural characterization. Frequently, however, line broadening due to intermediate exchange completely eliminates the signals needed for measuring specific intermolecular NOEs. This limits the use of NMR for detailed structural studies in such kinetic situations. Here we show that an optimally chosen excess of ligand over protein can reduce the extent of line broadening for both the ligand and the protein. This makes observation of ligand resonances possible but reduces the size of the measurable NOEs due to the residual line broadening and the non-stoichiometric concentrations. Because the solubility of small molecule drug leads are often limited to high micromolar concentrations, protein concentrations are restricted to even lower values in the low micromolar range. At these non-stoichiometric concentrations and in the presence of significant residual line broadening, conventional NOESY experiments very often are not sensitive enough to observe intermolecular NOEs since the signals inverted by the NOESY preparation pulse sequence relax prior to significant NOE build up. Thus, we employ methods related to driven NOE spectroscopy to investigate protein-ligand interactions in the intermediate exchange regime. In this approach, individual protein resonances are selectively irradiated for up to five seconds to build up measurable NOEs at the ligand resonances. To enable saturation of individual protein resonances we prepare deuterated protein samples selectively protonated at a few sites so that the 1D (1)H spectrum of the protein is resolved well enough to permit irradiation of individual protein signals, which do not overlap with the ligand spectrum. This approach is suitable for measuring a sufficiently large number of protein-ligand NOEs that allow calculation of initial complex structures, suitable for structure-based optimization of primary drug leads obtained from high-throughput screening. The method was applied to measure individual intermolecular NOEs between the anti-apoptotic protein Bcl-xL at 25 microM and a "first generation" small-molecule ligand, for which the spectrum is entirely broadened at stoichiometric concentrations. This approach is general and can also be used to characterize protein-protein or protein-nucleic-acid complexes.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Sep
pubmed:issn
0925-2738
pubmed:author
pubmed:issnType
Print
pubmed:volume
36
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
1-11
pubmed:dateRevised
2007-12-3
pubmed:meshHeading
pubmed:year
2006
pubmed:articleTitle
Identification of individual protein-ligand NOEs in the limit of intermediate exchange.
pubmed:affiliation
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
pubmed:publicationType
Journal Article, Research Support, N.I.H., Extramural