Interpretation of Deviations from Pseudo-First-Order Kinetic Behavior in the Characterization of Ligand Binding by Biosensor Technology

O’Shannessy, Daniel J.; Winzor, Donald J.

doi:10.1006/abio.1996.0167

Cited by 237 publications

(156 citation statements)

References 8 publications

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“…Another surfacerelated artifact, referred to as the "parking" or "crowding" problem, corresponds to the masking of ligand molecules by previously formed complexes. This phenomenon, first described by O'Shannesy (48), is particularly critical when the molecular weight of the analyte is large compared to the ligand. Since the two binding partners have almost the same size and are relatively small, it is unlikely that this "parking problem" would occur during our experiments.…”

Section: Resultsmentioning

confidence: 98%

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Real-Time Monitoring of the Interactions of Two-Stranded de Novo Designed Coiled-Coils: Effect of Chain Length on the Kinetic and Thermodynamic Constants of Binding

et al. 2003

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We have de novo designed a heterodimeric coiled-coil formed by two peptides as a capture/ delivery system that can be used in applications such as affinity tag purification, immobilization in biosensors, etc. The two strands are designated as K coil (KVSALKE heptad sequence) and E coil (EVSALEK heptad sequence), where positively charged or negatively charged residues occupy positions e and g of the heptad repeat. In this study, for each E coil or K coil, three peptides were synthesized with lengths varying from three to five heptads. The effect of the chain length of each partner upon the kinetic and thermodynamic constants of interaction were determined using a surface plasmon resonance-based biosensor. Global fitting of the interactions revealed that the E5 coil interacted with the K5 coil according to a simple binding model. All the other interactions involving shorter coils were better described by a more complex kinetic model involving a rate-limiting reorganization of the coiled-coil structure. The affinities of these de novo designed coiled-coil interactions were found to range from 60 pM (E5/K5) to 30 µM (E3/K3). From these K d values, we were able to determine the free energy contribution of each heptad, depending on its relative position within the coiled-coils. We found that the free energy contribution of a heptad occupying a central position was 3-fold higher than that of a heptad at either end of the coiled-coil. The wide range of stabilities and affinities for the E/K coil system provides considerable flexibility for protein engineering and biotechnological applications.The R-helical coiled-coil is an oligomerization domain that is naturally present in a wide variety of proteins such as transcription factors, cytoskeletal proteins, motor proteins, and viral fusion proteins (1-3). The structural properties of coiled-coils have been extensively characterized from numerous high-resolution crystal and NMR structures (3-5). The formation of coiled-coils requires the wrapping of two or more amphipathic R-helices around each other in a lefthanded supercoil fashion; the helices may be aligned in either a parallel or antiparallel manner. The R-helices composing the coiled-coil structure are defined by a heptad repeat (denoted abcdefg) in which hydrophobic residues occupy the positions a and d and pack in a characteristic "knobs-intoholes" manner (6), forming the hydrophobic core ( Figure 1). Many studies (1,(7)(8)(9)(10)(11)(12) emphasized the role of charged residues (typically in the e and g positions) in controlling the specificity of oligomerization and opened up the way to the de novo design of heterodimeric coiled-coils (13-17). Their relatively small size, in addition to their well-defined structure, make coiled-coils a very attractive system for protein engineering and biotechnological applications (5, 18) such as the construction of miniaturized antibodies, the control of signaling protein domain oligomerization, and the specific targeting of GFP to cytoskeletal proteins (see ref 4 for ...

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Section: Resultsmentioning

confidence: 98%

“…The most obvious artifact is surface ligand heterogeneity; this can occur when more than one coupling site is present in the ligand molecule (46)(47)(48). The de novo designed E and K coils are relatively small peptides whose molecular weights vary from 2300 to 4100 Da, each of them containing multiple carboxyl and amino groups.…”

Section: Resultsmentioning

confidence: 99%

Real-Time Monitoring of the Interactions of Two-Stranded de Novo Designed Coiled-Coils: Effect of Chain Length on the Kinetic and Thermodynamic Constants of Binding

et al. 2003

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“…Furthermore, because equilibrium was not reached during the association phase, the direct use of Scatchard analysis to calculate the apparent equilibrium dissociation constant was not allowed. Instead, to calculate the equilibrium dissociation constant, association curves were extrapolated to infinite time using recip-rocal plots in order to estimate the equilibrium binding value R eq (43,44). Nonlinear regression to the standard hyperbolic expression (see "Materials and Methods"), also shown in Fig.…”

Section: Binding Of C-propeptide Trimers To Pcpe-interactionsmentioning

confidence: 99%

Interaction Properties of the Procollagen C-proteinase Enhancer Protein Shed Light on the Mechanism of Stimulation of BMP-1

Ricard‐Blum

Bernocco

Font

et al. 2002

Journal of Biological Chemistry

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Procollagen C-proteinase enhancer (PCPE) is an extracellular matrix glycoprotein that binds to the C-propeptide of procollagen I and can enhance the activities of procollagen C-proteinases up to 20-fold. To determine the molecular mechanism of PCPE activity, the interactions of the recombinant protein with the procollagen molecule as well as with its isolated C-propeptide domain were studied using surface plasmon resonance (BIAcore) technology. Binding required the presence of divalent metal cations such as calcium and manganese. By ligand blotting, calcium was found to bind to the C-propeptide domains of procollagens I and III but not to PCPE. By chemical cross-linking, the stoichiometry of the PCPE/C-propeptide interaction was found to be 1:1 in accordance with enzyme kinetic data. The use of a monoclonal antibody directed against the N-terminal region of the C-propeptide suggested that this region is probably not involved in binding to PCPE. Association and dissociation kinetics of the Cpropeptide domains of procollagens I and III on immobilized PCPE were rapid. Extrapolation to saturation equilibrium yielded apparent equilibrium dissociation constants in the range 150 -400 nM. In contrast, the association/dissociation kinetics of intact procollagen molecules on immobilized PCPE were relatively slow, corresponding to a dissociation constant of 1 nM. Finally, pN-collagen (i.e. procollagen devoid of the C-terminal propeptide domain) was also found to bind to immobilized PCPE, suggesting that PCPE binds to sites on either side of the procollagen cleavage site, thereby facilitating the action of procollagen C-proteinases.Bone morphogenetic protein-1 (BMP-1) 1 and other tolloidrelated metalloproteinases (1, 2), also known as procollagen C-proteinases (PCPs), have recently been shown to be involved in the control of a variety of morphogenetic events during development and tissue repair. These include: (i) the deposition of collagen fibrils in the extracellular matrix following the processing of procollagen propeptides (3-6); (ii) dorsoventral patterning (7-10) through the cleavage of the growth factor inhibitors chordin and SOG; (iii) collagen and elastin crosslinking by the processing of the inactive precursors of lysyl oxidases (11, 12); and (iv) adhesion/migration of epithelial cells by the cleavage of laminin 5 chains (13-15). The activities of PCPs on procollagen substrates may be stimulated up to 20-fold by another glycoprotein of the extracellular matrix, procollagen C-proteinase enhancer (PCPE) (16 -19), which lacks intrinsic proteinase activity. Similarly, in the case of chordin and SOG, the protein TSG or its homologues stimulates cleavage by tolloid proteinases (20, 21), thus raising the possibility that PCP processing of different substrates might be specifically regulated by distinct enhancer proteins.Both tolloid proteinases and PCPE are multidomain glycoproteins containing multiple copies of the so-called CUB domain (22), a protein module common to several extracellular and plasma membrane-associated...

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“…Don used his experiences gained from characterising solute partition in affinity chromatography to improve the quantification of antigen-antibody interactions by ELISA (Hogg et al 1987;Winzor 2011), as well as by the biosensor technologies that emerged in the early 1990s (Ward et al 1995;O'Shannessy and Winzor 1996;Winzor 1997, 1999;Hall et al 1997;Edwards et al 1998), methods in which the measured binding constant was previously based on 1:1 reaction stoichiometry, and referred to the solute-matrix interaction rather than that between reactants in the solution phase.…”

Section: Elisa and Biosensor Studies Of Interactionsmentioning

confidence: 99%

Foreword to ‘Quantitative and analytical relations in biochemistry’—a special issue in honour of Donald J. Winzor’s 80th birthday

Hall

Harding

2016

Biophys Rev

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The purpose of this special issue is to honour Professor Donald J. Winzor's long career as a researcher and scientific mentor, and to celebrate the milestone of his 80th birthday. Throughout his career, Don has been renowned for his development of clever approximations to difficult quantitative relations governing a range of biophysical measurements. The theme of this special issue, 'Quantitative and analytical relations in biochemistry', was chosen to reflect this aspect of Don's scientific approach.

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Interpretation of Deviations from Pseudo-First-Order Kinetic Behavior in the Characterization of Ligand Binding by Biosensor Technology

Cited by 237 publications

References 8 publications

Real-Time Monitoring of the Interactions of Two-Stranded de Novo Designed Coiled-Coils: Effect of Chain Length on the Kinetic and Thermodynamic Constants of Binding

Real-Time Monitoring of the Interactions of Two-Stranded de Novo Designed Coiled-Coils: Effect of Chain Length on the Kinetic and Thermodynamic Constants of Binding

Interaction Properties of the Procollagen C-proteinase Enhancer Protein Shed Light on the Mechanism of Stimulation of BMP-1

Foreword to ‘Quantitative and analytical relations in biochemistry’—a special issue in honour of Donald J. Winzor’s 80th birthday

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