Nico J. de Mol

Surface plasmon resonance: a general introduction.

Surface plasmon resonance (SPR) analysis is rather unique in that it allows assay of binding constants (affinity) and kinetic analysis of binding phenomena. This introductory chapter deals with some specific features that are relevant to many diverse applications. The role and impact of kinetics in biomolecular interactions is highlighted. A concise description of the physical principles of the SPR phenomenon is given from a practical point of view, such that some possibilities and limitations of the method can be rationalized, e.g., depth of the evanescent field. A specific condition that may come forward in kinetic analysis is mass transport limitation (MTL). A practical model is presented, which allows estimation of the extent of MTL. Based on this model it can be rationalized whether MTL can be avoided by experimental design. In this framework also rules are presented to convert SPR signals (RU or millidegree) to mass/surface unit. The chapter concludes with an overview of commercially available SPR equipment.

Replacement of the Intervening Amino Acid Sequence of a Syk-Binding Diphosphopeptide by a Nonpeptide Spacer with Preservation of High Affinity

Divalent peptidomimetic compounds can bind a signal transduction protein and provide an avenue towards uncovering compounds capable of influencing or interfering with protein–protein interactions in general. High-affinity binding was realized by linking two relatively weakly interacting monophosphorylated peptides with an oligoethylene glycol spacer (see structure) the length of which was tuned by design.

Cyclic phosphopeptides for interference with Grb2 SH2 domain signal transduction prepared by ring-closing metathesis and phosphorylation

Cyclic phosphopeptides were prepared using ring-closing metathesis followed by phosphorylation. These cyclic phosphopeptides were designed to interact with the SH2 domain of Grb2, which is a signal transduction protein of importance as a target for antiproliferative drug development. Binding of these peptides to the Grb2 SH2 domain was evaluated by a surface plasmon resonance assay. High affinity binding to the Grb2 SH2 domain was maintained upon macrocyclization, thus indicating that this method can be used to assemble high affinity cyclic phosphopeptides that interfere with signal transduction cascades.

Surface Plasmon Resonance

Surface plasmon resonance (SPR) analysis is rather unique in that it allows assay of binding constants (affinity) and kinetic analysis of binding phenomena. This introductory chapter deals with some specific features that are relevant to many diverse applications. The role and impact of kinetics in biomolecular interactions is highlighted. A concise description of the physical principles of the SPR phenomenon is given from a practical point of view, such that some possibilities and limitations of the method can be rationalized, e.g., depth of the evanescent field. A specific condition that may come forward in kinetic analysis is mass transport limitation (MTL). A practical model is presented, which allows estimation of the extent of MTL. Based on this model it can be rationalized whether MTL can be avoided by experimental design. In this framework also rules are presented to convert SPR signals (RU or millidegree) to mass/surface unit. The chapter concludes with an overview of commercially available SPR equipment.

Protein flexibility and ligand rigidity : A thermodynamic and kinetic study of itam- based ligand binding to syk tandem SH2

The Syk tandem Src homology 2 domain (Syk tSH2) constitutes a flexible protein module involved in the regulation of Syk kinase activity. The Syk tSH2 domain is assumed to function by adapting the distance between its two SH2 domains upon bivalent binding to diphosphotyrosine ligands. A thermodynamic and kinetic analysis of ligand binding was performed by using surface plasmon resonance (SPR). Furthermore, the effect of binding on the Syk tSH2 structural dynamics was probed by hydrogen/deuterium exchange and electrospray mass spectrometry (ESI‐MS). Two ligands were studied: 1, a flexible peptide derived from the tSH2 recognition ITAM sequence at the γ chain of the FcεRI‐receptor, and 2, a ligand in which the amino acids between the two SH2 binding motifs in ligand 1 have been replaced by a rigid linker of comparable length. Both ligands display comparable affinity for Syk tSH2 at 25 °C, yet a major difference in thermodynamics is observed. Upon binding of the rigid ligand, 2, the expected entropy advantage is not realized. On the contrary, 2 binds with a considerably higher entropy price of ∼9 kcal mol−1, which is attributed to a further decrease in protein flexibility upon binding to this rigid ligand. The significant reduction in deuterium incorporation in the Syk tSH2 protein upon binding of either 1 or 2, as monitored by ESI‐MS, indicates a major reduction in protein dynamics upon binding. The results are consistent with a two‐step binding model: after an initial binding step, a rapid structural change of the protein occurs, followed by a second binding step. Such a bivalent binding model allows high affinity and fast dissociation kinetics, which are very important in transient signal‐transduction processes.

Dual effect of the anti-allergic astemizole on Ca2+ fluxes in rat basophilic leukemia (RBL-2H3) cells : Release of Ca2+ from intracellular stores and inhibition of Ca2+ release-activated Ca2+ influx

The antiallergic drugs astemizole and norastemizole inhibit exocytosis in mast cells, which might be relevant for their therapeutic action. From previous studies, it appeared that the drugs inhibited 45Ca2+ influx. Here, we present a more detailed study on the effects of astemizole and norastemizole on Ca2+ fluxes. Fura-2-loaded rat basophilic leukemia (RBL-2H3) cells were activated through the high-affinity receptor for IgE (FcepsilonRI) with antigen or by the endoplasmatic reticulum ATPase inhibitor thapsigargin, bypassing direct FcepsilonRI-related events. It appeared that astemizole (>15 microM), in contrast to norastemizole, showed a dual effect on intracellular calcium concentration ([Ca2+]i): a rise in intracellular calcium concentration was induced, which originated in the release of intracellular Ca2+ stores, whereas Ca2+ influx via store-operated Ca2+ (SOC) channels was inhibited. Ca2+ influx was further characterized using Ba2+ influx, whereas processes in the absence of Ca2+ influx were studied using Ni2+ or EGTA. It was concluded that the drugs most likely affect the store-operated Ca2+ channels in RBL cells directly. The two effects of astemizole on Ca2+ fluxes had opposing influences on exocytosis, thereby accounting for the biphasic effect of increasing astemizole concentration on mediator release in RBL cells.

Surface plasmon resonance for proteomics.

Surface plasmon resonance (SPR) is a well-established label-free technique to detect mass changes near an SPR surface. For 20 years the benefits of SPR have been proven in biomolecular interaction analysis, including measurements of affinity and kinetics. The emergence of proteomics and a need for high throughput analysis drives the development of SPR systems capable of analyzing microarrays. The use of SPR imaging (also known as SPR microscopy) makes it possible to use multiplexed arrays to follow binding reactions. As SPR only analyzes the binding process, but not the identity of captured molecules on the SPR surface, technologies have been developed to integrate SPR with mass spectrometric (MS) analysis. Such approaches involve the recovery of analytes from the SPR surface and subsequent MALDI-TOF MS analysis, or LC-MS/MS after tryptic digestion of recovered proteins. An approach compatible with SPR arrays is on-chip MALDI-TOF MS, from arrayed spots on an SPR surface. This review describes some exciting developments in the application of SPR to proteomics, using instruments which are on the market already, or are expected to be available in the years to come.Surface plasmon resonance (SPR) is a well-established label-free technique to detect mass changes near an SPR surface. For 20 years the benefits of SPR have been proven in biomolecular interaction analysis, including measurements of affinity and kinetics. The emergence of proteomics and a need for high throughput analysis drives the development of SPR systems capable of analyzing microarrays. The use of SPR imaging (also known as SPR microscopy) makes it possible to use multiplexed arrays to follow binding reactions. As SPR only analyzes the binding process, but not the identity of captured molecules on the SPR surface, technologies have been developed to integrate SPR with mass spectrometric (MS) analysis. Such approaches involve the recovery of analytes from the SPR surface and subsequent MALDI-TOF MS analysis, or LC-MS/MS after tryptic digestion of recovered proteins. An approach compatible with SPR arrays is on-chip MALDI-TOF MS, from arrayed spots on an SPR surface. This review describes some exciting developments in the application of SPR to proteomics, using instruments which are on the market already, or are expected to be available in the years to come.

Experimental and Calculated Shift in pKa upon Binding of Phosphotyrosine Peptide to the SH2 Domain of p56lck

The pH dependence of the affinity of a 11-mer phosphotyrosine (pY) peptide (EPQpYEEIPIYL-NH2) for the SH2 domain of the tyrosine kinase p56(lck) was investigated with surface plasmon resonance (SPR). From SPR competition experiments the affinity in solution was obtained. The pH dependence of the affinity in solution can be well described by a proton linkage model with a single pK(a) shift upon binding, from 6.1 to 4.7. This shift is ascribed to the transition from the -2 to the -1 ionisation state of the tyrosine phosphate group. Based on the X-ray structure for the complex with Lck SH2, a pK(a) value of 5.3 for the bound pY peptide was computed, modelling the solvated protein as a system of point charges in a continuum. With the phosphate in the -2 state the binding energy is 1.8 kcal/mol more favourable than for the -1 state, corresponding to a 20-fold higher affinity. A proper charge is relevant in the design of potential therapeutic Lck SH2 ligands with mimics for the metabolically unstable tyrosine phosphate group.

Chapter 5:Kinetic and Thermodynamic Analysis of Ligand–Receptor Interactions: SPR Applications in Drug Development

Increasing evidence can be found that describing receptor ligand interactions in terms of a “lock-and-key” model is no longer adequate. Receptors can be regarded as part of a “molecular machinery”, in which ligand binding forms a trigger to activate or deactivate the machinery. According to this vie...

Amino propynyl benzoic acid building block in rigid spacers of divalent ligands binding to the syk SH2 domains with equally high affinity as the natural ligand

The construction of rigid spacers composed of amino propynyl benzoic acid building blocks is described. These spacers were used to link two phosphopeptide ligand sites towards obtaining divalent ligands with a high affinity for Syk tandem SH2 domains, which are important in signal transduction. The spacer containing two of those rigid building blocks led to a ligand which was as active as the natural ligand, indicating that this building block can be used in the design and synthesis of high affinity divalent constructs that can successfully interfere with crucial protein-protein interactions.