[13] Kinetic analysis of macromolecular interactions using surface plasmon resonance biosensors

Morton, Thomas A.; Myszka, David G.

doi:10.1016/s0076-6879(98)95044-3

Cited by 271 publications

(221 citation statements)

References 20 publications

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“…1990 [18,19]. Since then, there has been a steadily growing interest in using this powerful technique for real time determination of rate and affinity constants for the interaction of different biomolecules, e.g.…”

Section: Discussionmentioning

confidence: 99%

“…The Biacore 3000 instrument used in the present study has a low mass detection limit of ~180 Da [18]. Since this is close to the size range of the lipids of interest (fatty acids ~280 Da; cholesterol ~390 Da), we initially considered tethering the lipid ligand to a sensor chip via some linker extension and flowing SCP-2 over this surface for binding measurements.…”

Section: Discussionmentioning

confidence: 99%

“…In addressing this issue, we have developed a novel strategy for examining lipid-transfer protein interactions which eliminates the need for indicator groups. In this approach, a transfer protein is expressed with a selectively placed biotinylated peptide appendage, allowing it to be uniformly attached to a streptavidin chip for surface plasmon resonance (SPR)-based measurement [18][19][20] of lipid binding. Among the advantages of this approach is its very high sensitivity, which allows unsaturated fatty acids and certain other lipids to be studied below their critical micellar concentrations (CMC).…”

Section: Introductionmentioning

confidence: 99%

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Lipid transfer protein binding of unmodified natural lipids as assessed by surface plasmon resonance methodology

Kernstock

Girotti

2007

Analytical Biochemistry

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A new approach for analyzing lipid-lipid transfer protein interactions is described. The transfer protein is genetically engineered for expression with a C-terminal biotinylated peptide extension (AviTag ® ). This allows protein anchoring to a streptavidin-coated chip for surface plasmon resonance (SPR)-based assessment of lipid binding. Sterol carrier protein-2 (SCP-2), involved in the intracellular trafficking of cholesterol, fatty acids and other lipids, was selected as the prototype. Biotinylated SCP-2 (bSCP-2) was expressed in E. coli, purified to homogeneity by mutated streptavidin (SoftLink ® ) affinity chromatography, and confirmed by mass spectrometry to contain one biotin group at the expected position. Intermembrane [ 14 C]cholesterol transfer was strongly enhanced by bSCP-2, demonstrating that it was functional. Using bSCP-2 immobilized on a Biacore streptavidin chip, we determined on-and off-rate constants along with equilibrium dissociation constants for the following analytes: oleic acid, linoleic acid, cholesterol, and fluorophore (NBD)-derivatized cholesterol. The dissociation constant for NBD-cholesterol was similar to that determined by fluorescence titration for SCP-2 in solution, thus validating the SPR approach. This method can be readily adapted to other transfer proteins and has several advantages over existing techniques for measuring lipid binding, including (i) ability to study lipids in their natural states, i.e. without relatively large reporter groups; and (ii) ability to measure on-and off-rate constants as well as equilibrium constants.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Lipid transfer protein binding of unmodified natural lipids as assessed by surface plasmon resonance methodology

Kernstock

Girotti

2007

Analytical Biochemistry

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“…For this, Morton and Myszka have developed the computer program CLAMP. [32] In a global analysis, this program fits the SPR curves from several protein concentrations simultaneously, returning the kinetic parameters of the binding model. Changes in H/D exchange have been associated with conformational changes, [14,[33][34][35] and this inspired us to examine a two-step model with an initial binding event followed by a structural change of the complex from AB1 to AB2 (model 2, Scheme 1).…”

Section: Evaluation Of Binding Models By Spr Real-time Association Kimentioning

confidence: 99%

“…[32] Global analyses were performed by fitting several curves from different protein concentrations simultaneously, with use of a defined binding model. A diffusion step of the protein from bulk solution to the sensor surface was included (k tr ) in the binding models.…”

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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Biosensor analysis of the interleukin-2 receptor complex

Liparoto

Ciardelli

1999

J. Mol. Recognit.

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Surface plasmon resonance (SPR) biosensor technology has been a significant addition to the evolution and refinement of methods to study macromolecular interactions. Prior to the advent of SPR, we employed a variety of biochemical and biological techniques to study the interleukin-2/interleukin-2 receptor system (IL-2/IL-2R). By combining site-directed mutagenesis, equilibrium and kinetic radioligand binding, and competitive biological assays, we and others had begun to understand many aspects of the structure-activity relationships of the IL-2/IL-2R system. Due to the complexity of the IL-2R, cell-based assays proved limited in their ability to provide quantitative information on the binding characteristics of subclasses of the IL-2 receptor. SPR technology promised to be a new and powerful approach to the quantitative analysis of complex receptor systems. To demonstrate the feasibility of this technology, we employed Biacore analysis to investigate the ligand binding characteristics of novel, pre-assembled, IL-2R coiled-coil complexes. The results of these studies, although limited by instrumentation and data analysis, clearly established the utility of this method. Subsequently, by incorporating advancements in both of these areas, we have been able to carry out detailed kinetic analyses of the binding properties of individual IL-2R subunits as well as heteromeric complexes on the surface of a biosensor. Therefore, SPR biosensor analysis combined with other established analytical methods has proven to be a powerful tool for the analysis of complex hematopoietic receptor systems. Published in 1999 by John Wiley & Sons, Ltd.

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[13] Kinetic analysis of macromolecular interactions using surface plasmon resonance biosensors

Cited by 271 publications

References 20 publications

Lipid transfer protein binding of unmodified natural lipids as assessed by surface plasmon resonance methodology

Lipid transfer protein binding of unmodified natural lipids as assessed by surface plasmon resonance methodology

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

Biosensor analysis of the interleukin-2 receptor complex

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