Carbohydrate-protein interaction studies by laser photo CIDNP NMR methods |
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Authors: | Hans-Christian Siebert Robert Kaptein Jaap J Beintema Ukun M Soedjanaatmadja Christine S Wright Ann Rice Reinhard G Kleineidam Susanne Kruse Roland Schauer Petra JW Pouwels Johannis P Kamerling Hans-Joachim Gabius Johannes FG Vliegenthart |
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Institution: | (1) Institut fur Physiologische Chemie der Ludwig-Maximilians Universitat, Tierarztliche Fakultat, Veterinarstrae 13, 80539 Munchen, Germany;(2) Bijvoet Center for Biomolecular Research, University of Utrecht, PO Box 80075, 3508 TB Utrecht, The Netherlands;(3) Biochemisch Laboratorium and Bioson Research Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands;(4) Laboratorium Biokimia, JI. Singaperbangsa 2, Padjadjaran University, Bandung, 4093, Indonesia;(5) Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Box 614, MCV Station, Richmond, Virginia 23298, USA;(6) Biochemisches Institut der Christian-Albrechts-Universitat, Olshausentrae, 4024098 Kiel, Germany;(7) Bijvoet Center for Biomolecular Research, University of Utrecht, PO Box 80075, 3508 TB Utrecht, The Netherlands |
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Abstract: | The side chains of tyrosine, tryptophan and histidine are able to produce CIDNP (Chemically Induced Dynamic Nuclear Polarization)
signals after laser irradiation in the presence of a suitable radical pair-generating dye. Elicitation of such a response
in proteins implies surface accessibility of the respective groups to the light-absorbing dye. In principle, this technique
allows the monitoring of the effect of ligand binding to a receptor and of site-directed mutagenesis on conformational aspects
of any protein if CIDNP-reactive amino acids are involved. The application of this method in glycosciences can provide insights
into the protein-carbohydrate interaction process, as illustrated in this initial model study for several N-acetyl-glucosamine-binding
lectins of increasing structural complexity as well as for a wild type bacterial sialidase and its mutants. Experimentally,
the shape and intensity of CIDNP signals are determined in the absence and in the presence of specific glycoligands. When
the carbohydrate is bound, CIDNP signals of side chain protons of tyrosine, tryptophan or histidine residues can be broadened
and of reduced intensity. This is the case for hevein, pseudo-hevein, the four hevein domains-containing lectin wheat germ
agglutinin (WGA) and the cloned B-domain of WGA 1 (domB) representing one hevein domain. This response indicates either a
spatial protection by the ligand or a ligand-induced positioning of formerly surface-exposed side chains into the protein’s
interior part, thereby precluding interaction with the photo-activated dye. Some signals of protons from the reactive side
chains can even disappear when the lectin-ligand complexes are monitored. The ligand binding, however, can apparently also
induce a conformational change in a related lectin that causes the appearance of a new signal, as seen for Urtica dioica agglutinin
(UDA) which consists of two hevein domains. Additionally, the three CIDNP-reactive amino acids are used as sensors for the
detection of conformational changes caused by pH variations or by deliberate amino acid exchanges, as determined for the isolectins
hevein and pseudo-hevein as well as for the cloned small sialidase of Clostridium perfringens and two of its mutants. Therefore,
CIDNP has proven to be an excellent tool for protein-carbohydrate binding studies and can be established in glycosciences
as a third biophysical method beside X-ray-crystallography and high-resolution multidimensional NMR studies which provides
reliable information of certain structural aspects of carbohydrate-binding proteins in solution.
This revised version was published online in November 2006 with corrections to the Cover Date. |
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Keywords: | Chemically induced dynamic nuclear polarization (CIDNP) carbohydrate-protein interaction NMR |
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