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Energetics Of Protein-Carbohydrate Recognition

Electronic Theses of Indian Institute of Science

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Title Energetics Of Protein-Carbohydrate Recognition
 
Creator Swaminathan, C P
 
Subject Protein Chemistry
Carbohydrates
Proteins
Molecular Recognition
Molecular Mimicry
Lectins
Epitope Recognition
Sugar Recognition
Leguminoceae
D-galactopyranoside
Concanavalin
Sugars
Galactopyranosides
Galectin
 
Description The work embodied in this thesis pertains to an attempt to understand better, the molecular basis of protein-carbohydrate recognition. For this purpose a systematic study was undertaken, not only of the energetics of lectin-sugar interactions, which serve as molecular recognition prototype of protein-carbohydrate interactions, but also of the complex effects of solvent water molecules surrounding both the species in solution state. The systems chosen for investigation include the specific recognition of sugars by lectins from diverse families, leguminosae and moraceae. The following salient aspects of the molecular recognition process constitute the focus of this thesis:

• Effect of site specifically modified, deoxy-, fluorodeoxy-, or methoxy- substituted
D-galactopyranoside binding to lectins. Isothermal titration calorimetric (ITC)
investigations of the binding of these sugars to a model lectin permitted the correct
prediction of the architecture of the primary binding site in the absence of x-ray
crystal or NMR structure of the combining site (Ref. 7). The study provided the
only unambiguous means of a site specific mapping of the hydrogen-bond donor-
acceptor relationship of the monosaccharide within the primary combining site of
the lectin.
• Novel features of lectin-sugar recognition. Molecular interactions and forces
contributing to the stabilization of the saccharides in the primary combining site of
lectins. Binding of site specifically modified fluoro- substituted D-
galactopyranosides to WBA I led to the demonstration of the involvement of C-
F««»H-0 hydrogen bonds in stabilizing the saccharide within the combining site
of lectin (Ref. 7). Implication of the novel C-H«**O hydrogen bonds at the
specificity determining C-4 position in enabling the methoxy- substituted D-
galactopyranoside to be stabilized within the primary binding site of galactose
specific lectins WBA I and jacalin.
• Development of a novel coupled osmotic-thermodynamic approach for
investigating the role of water molecules in determining the specificity of lectin-
sugar interactions. The results obtained led to the first direct demonstration of a
differential uptake of water molecules accompanying the specific process of
recognition of sugars by lectins (Ref 2)
• On the origin of enthalpy-entropy compensation, a ubiquitous phenomenon accompanying the thermodynamics of several ligand binding reactions in aqueous solutions in general and the molecular recognition involving all known lectin-sugar interactions, in particular. The results provide the first unequivocal solution state proof of water reorganization as the source of enthalpy-entropy compensation (Ref 3). A new diagnostic test of a true osmotic effect in molecular recognition phenomena was proposed (Ref. 2) and validated (Ref. 3).

As an introduction, Chapter 1 is a comprehensive review of literature that touches upon the diverse properties of lectins and our present understanding of their multifarious roles and applications, which has led to their christening, perhaps appropriately, as molecules that mediate the 'social' functions of cells and tissues. Although a challenge it is still, to decipher the "glycocode", it is apparent that the fundamental basis of the recognition function of lectin-sugar interactions is the initial specific binding of the saccharide molecule by the globular proteinaceous lectin molecule. It is imperative, therefore, that an incisive investigation of the origin of specificity of the binding reaction as well as the solvent effects influencing both the interacting species be undertaken for a better understanding of the complete molecular recognition process. Towards this end is introduced in Chapter 1 our present understanding of the results on lectin-sugar interactions from two complementary approaches viz structural, including X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, as well as thermodvnamic ones, which have provided important information on the architecture of the combining sites, the dynamic modes of saccharide recognition and forces involved therein. Despite a detailed knowledge available from such methods, a structure-energetics correlation has persisted as a current challenge of the field. Towards achieving this goal, studies on the energetics of the recognition of sugars by lectins were undertaken, with an aim to better understand the origin of specificity of lectin-sugar interactions. This thesis attempts to provide new insights on some of the possible lacunae precluding structure-energetics correlation and suggests ways to overcome them.

Chapter 2 deals with ITC investigation of the effect of deoxy-, fluorodeoxy-,
and methoxy- substitutions on the binding of monosaccharides to the primary combining site of the lectin WBA I isolated from the mature seeds of the leguminosae family member Psophocarpus tetragonolobus as well as the moraceae lectin jacalin. These studies provide valuable information on the hydrogen-bond donor-acceptor relationships within the combining site of the lectins wherein the sugar molecule is liganded with the amino-acid residues of the lectin. This study is relevant for understanding the origin of specificity of monosaccharide binding within the primary combining site of the lectins. It has recently become apparent that there is a predisposition in three-dimensional space, of the donor-acceptor pairs within the sugar binding site of the lectins. Hence there appears to be a stereochemical basis of distinguishing the recognition of the donor group vis-a-vis that of the acceptor group and that their spatial disposition determines the specificity of the saccharide recognition. Unambiguous assignment of which of the groups within the hydrogen bonded pairs is a donor and which one is the acceptor assumes greater importance. The ITC measurements of the binding of deoxy-, flurodeoxy-and methoxy-derivatives of D-galactopyranoside (oc-D-Gal) to the basic lectin from winged bean Psophocarpus tetragonolobus, WBA I revealed that each of the ligands bind to WBA I with the same stoichiometry of one per subunit (29 kDa) of WBA I. The binding enthalpies for various derivatives were essentially independent of temperature and showed complementary changes with respect to binding entropies. Replacement of the hydroxyl group by fluorine or hydrogen on C3 and C4 of the galactopyranoside eliminated their binding to the lectin, consistent with C3-OH and C4-OH acting as hydrogen bond donors. The affinity for C2 derivatives of galactose decreased in the order: GalNAc>2MeOGal>2FGal=Gal>2HGal which suggests that both polar and non-polar residues surround the C2 locus of galactose, consistent with the observed high affinity of WBA I towards GalNAc, where the acetamido group at C2 position is probably stabilized by both non-polar interactions with the methyl-group and polar interactions with the carbonyl group. The binding of C6 derivatives followed the order: Gal>6FGal>D-Fuc»6MeOGal=L-Ara indicating the presence of favourable polar interactions with a hydrogen bond donor in the vicinity. Based on these results
the hydrogen bond donor-acceptor relationship of the complexation of methyl-a-D-galactopyranoside with the primary combining site of WBA I was proposed (Ref. /), which was subsequently validated by the crystal structure of methyl-a-D-galactopyranoside complexed with WBA I. This chapter also describes the results from ITC studies on the binding of monosaccharides and disaccharides to the lectin jacalin isolated from the mature seeds of the moraceae family member Artocarpus integrifolia. The novel observation about the existence of C-F*«*H-0 and C-H**»O hydrogen bonds in lectin-sugar interactions is also discussed in this chapter.

Chapter 3 is a description of the detailed investigation on the role of water molecules in influencing the energetics of lectin-sugar recognition. A novel coupled osmotic-thermodynamic approach was developed to dissect the role of water molecules in determining the recognition of the sugars by lectins. For this purpose, the model system of mannotriose-concanavalin A was used because atomic level structural information on these complexes were available. The work described in this chapter, is the first solution state evidence for the role of water molecules in the specific interaction of carbohydrates with a legume lectin, concanavalin A (Con A) (Ref. 2). Sugar binding to Con A was accompanied by linear changes in the logarithm of binding constants as a function of neutral osmolyte strength, and were described by well defined negative slopes characteristic for each sugar. As these changes were independent of the chemical nature of the osmolyte used, the results were rationalized in terms of a true osmotic effect. It was demonstrated that the specific recognition of the branched trimannoside (3,6-di-0-(a-D-mannopyranosyl)~a-D-mannopyranoside), the individual dimannosidic arms (3-
 
Publisher Indian Institute of Science
 
Contributor Surolia, Avadhesha
 
Date 2006-04-28T09:16:57Z
2006-04-28T09:16:57Z
2006-04-28T09:16:57Z
2000-01
 
Type Electronic Thesis and Dissertation
 
Format 40272672 bytes
application/pdf
 
Identifier http://etd.iisc.ernet.in/handle/2005/210
null
 
Language en
 
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