Kinetic Analysis of the Mass Transport Limited Interaction between the Tyrosine Kinase lck SH2 Domain and a Phosphorylated Peptide Studied by a New Cuvette-Based Surface Plasmon Resonance Instrument

Mol, Nico J. de; Plomp, E.; Fischer, Marcel J.E.; Ruijtenbeek, Rob

doi:10.1006/abio.1999.4464

Cited by 62 publications

(66 citation statements)

References 33 publications

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“…Due to the heterogeneous SPR format and the poorly defined surface density of the immobilized antibody, SPR cannot make direct measurement of k on . 14 However, k on can be calculated via the relationship: 19 Finally, the SPR immobilization procedure can be time-consuming, labor-intensive, and expensive. 20 Kinetic capillary electrophoresis (KCE) is a powerful alternate technique for making direct measurements of k on and k off .…”

Section: * S Supporting Informationmentioning

confidence: 99%

Binding Kinetic Rates Measured via Electrophoretic Band Crossing in a Pseudohomogeneous Format

Kapil

Herr

2014

Anal. Chem.

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With relevance spanning from immunohistochemistry to immunoassays and therapeutics, antibody reagents play critical roles in the life sciences, clinical chemistry, and clinical medicine. Nevertheless, nonspecific interactions and performance reproducibility remain problematic. Consequently, scalable and efficient analytical tools for informed selection of reliable antibody reagents would have wide impact. Therefore, we introduce a kinetic polyacrylamide gel electrophoresis (KPAGE) microfluidic assay that directly measures antibody−antigen association and dissociation rate constants, k on and k off .To study antibody−antigen association, an antigen zone is electrophoresed through a zone of immobilized antibody. Upon crossing, the interaction yields a zone of immobilized immunocomplex. To quantify k on, we assess immunocomplex formation for a range of antigen−antibody interaction times. Here, interaction time is controlled by the velocity of the electromigrating antigen zone, which is determined by the strength of the applied electric field. All species are fluorescently labeled. To quantify k off , an immobilized zone of immunocomplex is subjected to in situ buffer dilution, while measuring the decay in immunocomplex concentration. Two approaches for antibody immobilization are detailed: (i) size-exclusion-based antibody immobilization via a molecular weight cutoff (MWCO) filter fabricated using polyacrylamide gel and (ii) covalent antibody immobilization realized using a photoactive benzophenone methacrylamide polyacrylamide gel. We determine k on and k off for prostate-specific antigen (PSA) and compare to gold-standard values. The KPAGE assay completes in 90 min, requiring 45 ng of often-limited antibody material, thus offering a quantitative antibody screening platform relevant to important but difficult to characterize interaction kinetics.

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Section: * S Supporting Informationmentioning

confidence: 99%

Binding Kinetic Rates Measured via Electrophoretic Band Crossing in a Pseudohomogeneous Format

Kapil

Herr

2014

Anal. Chem.

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“…[20] SPR binding experiments: SPR experiments were performed with an IBIS II instrument (Eco Chemie, Utrecht, The Netherlands), essentially as previously described. [44] In brief, the Ahx-extended g-ITAM peptide was covalently coupled to a Biacore CM5 sensor chip (Biacore AB, Uppsala, Sweden) through the free amino group by EDC/ NHS chemistry. The Ahx moiety was added to provide a spacer between the dextrane surface of the sensor chip and the binding peptide, to avoid steric hindrance upon binding of the tSH2 domain.…”

Section: Synthesis Of Fceri G-itam-based Syk Tsh2 Ligandsmentioning

confidence: 99%

“…Before fitting, a small correction of the free protein concentration for depletion due to binding to the sensor surface in the cuvette system was applied. [44] The equilibrium constant in solution (K S ) was obtained from SPR competition experiments.…”

Section: Synthesis Of Fceri G-itam-based Syk Tsh2 Ligandsmentioning

confidence: 99%

Protein Flexibility and Ligand Rigidity: A Thermodynamic and Kinetic Study of ITAM‐Based Ligand Binding to Syk Tandem SH2

et al. 2005

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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.

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“…Further, inclusion of specific proteins or molecules that may be used to bind to the released ligands or un-occupied binding sites and thus make them unavailable for rebinding is another technique targeted specifically at the rebinding problem. This technique has been used successfully for measuring the interaction of the SH2 domain of lck with a phosphotyrosine peptide [de Mol et al 2000]. However, in the absence of quantitative information on the affinity of these agents for binding to either the ligand or the receptor, it is difficult to estimate the general reliability of these methods.…”

Section: (I) Introductionmentioning

confidence: 99%

Ligand rebinding: self-consistent mean-field theory and numerical simulations applied to surface plasmon resonance studies

et al. 2005

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Rebinding of dissociated ligands from cell surface proteins can confound quantitative measurements of dissociation rates important for characterizing the affinity of binding interactions. This can be true also for in vitro techniques such as surface plasmon resonance (SPR). We present experimental results using SPR for the interaction of insulin-like growth factor-I (IGF-I) with one of its binding proteins, IGF binding protein-3 (IGFBP-3), and show that the dissociation, even with the addition of soluble heparin in the dissociation phase, does not exhibit the expected exponential decay characteristic of a 1:1 binding reaction. We thus consider the effect of (multiple) rebinding events and, within a self-consistent mean-field approximation, we derive the complete mathematical form for the fraction of bound ligands as a function of time. We show that, except for very low association rate and surface coverage, this function is non-exponential at all times, indicating that multiple rebinding events strongly influence dissociation even at early times. We compare the mean-field results with numerical simulations and find good agreement, although deviations are measurable in certain cases. Our analysis of the IGF-I-IGFBP-3 data indicates that rebinding is prominent for this system and that the theoretical predictions fit the experimental data well. Our results provide a means for analyzing SPR biosensor data where rebinding is problematic and a methodology to do so is presented.1 Present Address: Max Planck Institut für Physik komplexer Systeme, Nöthnitzer Stra e 38, 01187 Dresden, Germany. (i) IntroductionSignal transduction via transmembrane receptor proteins is initiated by extracellular binding with specific proteins known as growth factors. These interactions tend to be of high affinity and, in many systems, are regulated by binding proteins present in the extracellular environment. Insulin-like growth factor-I (IGF-I) constitutes one prominent example of such a growth factor. Cell signaling is transmitted by direct interaction with the IGF-I receptor but this binding can be impacted by solution and cell-associated IGF binding proteins (IGFBPs), of which there are at least six. Quantification of the interactions of IGF-I with IGFBPs is critical if one is to understand how changes in expression and secretion will impact IGF-I signaling. Surface plasmon resonance (SPR) is one technique amenable to such measurements. SPR is an optical sensor technique that has the advantage of being able to take real-time measurements using low concentrations of unlabeled biologicals [reviewed in Cooper 2003].Quantification of IGF-I interactions with both cell surface receptors and IGFBPs using SPR has been performed as a means of evaluating and predicting the competition between these molecules for IGF-I. Studies have used immobilized IGF-I [Wong et al. 1999;Dubaquie and Lowman 1999;Galanis et al. 2001;Fong et al. 2002; Vorwerk et al. 2002], IGF-I receptor [Jansson et al. 1997], or IGFBPs [Heding et al. 1996Jansson et al. ...

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Kinetic Analysis of the Mass Transport Limited Interaction between the Tyrosine Kinase lck SH2 Domain and a Phosphorylated Peptide Studied by a New Cuvette-Based Surface Plasmon Resonance Instrument

Cited by 62 publications

References 33 publications

Binding Kinetic Rates Measured via Electrophoretic Band Crossing in a Pseudohomogeneous Format

Binding Kinetic Rates Measured via Electrophoretic Band Crossing in a Pseudohomogeneous Format

Protein Flexibility and Ligand Rigidity: A Thermodynamic and Kinetic Study of ITAM‐Based Ligand Binding to Syk Tandem SH2

Ligand rebinding: self-consistent mean-field theory and numerical simulations applied to surface plasmon resonance studies

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