Nonspecific Amine Immobilization of Ligand Can Be a Potential Source of Error in BIAcore Binding Experiments and May Reduce Binding Affinities

Kortt, Alexander A.; Oddie, Geoffrey W.; Iliades, Peter; Gruen, L.C.; Hudson, Peter J.

doi:10.1006/abio.1997.2333

Cited by 45 publications

(32 citation statements)

References 21 publications

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“…rium constants when comparing full-length and truncated PABP validates the two-site model for the full-length PABPPaip2 interaction and, importantly, indicates that binding of Paip2 to the two sites in PABP is noncooperative. Since Paip2 was used as ligand when binding to RRM1-4 and RRM2-3 and as an analyte when binding to full-length PABP, the consistency of the results also suggests that our immobilization strategy did not alter the affinities of the PABP binding sites or introduce any bias in the kinetic analysis due to heterogeneity which might be caused by protein immobilization (22). Two binding regions on Paip2 interact selectively with defined PABP fragments.…”

Section: Characterization Of Pabp Binding Sites In Paip2mentioning

confidence: 66%

Dual Interactions of the Translational Repressor Paip2 with Poly(A) Binding Protein

Khaleghpour

Kahvejian

Crescenzo

et al. 2001

Molecular and Cellular Biology

146

185

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Section: Characterization Of Pabp Binding Sites In Paip2mentioning

confidence: 66%

Dual Interactions of the Translational Repressor Paip2 with Poly(A) Binding Protein

Khaleghpour

Kahvejian

Crescenzo

et al. 2001

Molecular and Cellular Biology

146

185

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“…Based on these three measurements and the fact that surface plasmon resonance is frequently known to underestimate affinities (20), we conclude that the affinity of CP for CARMIL is in the submicromolar range. If we assume a dissociation constant of ϳ0.4 M and use cellular concentrations for CP and CARMIL of ϳ1 M (21) and ϳ2 M (6), respectively, we calculate that about 75% the cellular CP could be complexed with CARMIL, barring some form of regulation or compartmentalization, and excluding other proteins that also bind CP (e.g.…”

Section: Fig 5 Sedimentation Equilibrium Analyses and Rsemmentioning

confidence: 81%

“…After formation of the boundary by slow acceleration to 15,000 rpm, the speed was decreased to 3,000 rpm for CARMIL and 5,000 rpm for CP, while maintaining the temperature at 20°C. Repetitive interference scans were taken 20 For sedimentation equilibrium runs in the XL-A, liquid columns of 0.080 or 0.110 ml of CARMIL in buffer A (with 0.015 ml more dialysate buffer A in the reference channel) and a speed of 7,500 rpm were used. Scans at 280 nm were collected at 2-h intervals in step mode (0.001-cm steps) with 11 or 13 averages/scan.…”

Section: Methodsmentioning

confidence: 99%

CARMIL Is a Bona Fide Capping Protein Interactant

Remmert

Olszewski

Bowers

et al. 2004

Journal of Biological Chemistry

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CARMIL, also known as Acan 125, is a multidomain protein that was originally identified on the basis of its interaction with the Src homology 3 (SH3) domain of type I myosins from Acanthamoeba. In a subsequent study of CARMIL from Dictyostelium, pull-down assays indicated that the protein also bound capping protein and the Arp2/3 complex. Here we present biochemical evidence that Acanthamoeba CARMIL interacts tightly with capping protein. In biochemical preparations, CARMIL copurified extensively with two polypeptides that were shown by microsequencing to be the ␣-and ␤-subunits of Acanthamoeba capping protein. The complex between CARMIL and capping protein, which is readily demonstratable by chemical cross-linking, can be completely dissociated by size exclusion chromatography at pH 5.4. Analytical ultracentrifugation, surface plasmon resonance and SH3 domain pull-down assays indicate that the dissociation constant of capping protein for CARMIL is ϳ0.4 M or lower. Using CARMIL fusion proteins, the binding site for capping protein was shown to reside within the carboxyl-terminal, ϳ200 residue, proline-rich domain of CARMIL. Finally, chemical cross-linking, analytical ultracentrifugation, and rotary shadowed electron microscopy revealed that CARMIL is asymmetric and that it exists in a monomer 7 dimer equilibrium with an association constant of 1.0 ؋ 10 6 M ؊1 . Together, these results indicate that CARMIL selfassociates and interacts with capping protein with affinities that, given the cellular concentrations of the proteins (ϳ1 and 2 M for capping protein and CARMIL, respectively), indicate that both activities should be physiologically relevant.In 1995, Zot (1) and colleagues identified a ϳ125-kDa protein from Acanthamoeba on the basis of its ability to bind to the isolated Src homology 3 (SH3) 1 domain of Acanthamoeba myosin IC (1). This protein, which they called Acan 125, coimmunoprecipitated with myosin IC and appeared to colocalize with the myosin in cellular surface projections involved in pinocytosis. The subsequent cloning of the Acanthamoeba gene for Acan 125 (2) revealed a multidomain protein dominated by a central, ϳ460-residue leucine-rich repeat (LRR) domain. LRRs are ϳ29-residue sequences that contain a loose consensus dominated by leucines, form amphipathic ␣-␤-structural units and mediate protein-protein interactions, either by serving as the ligand binding sites themselves or by increasing the affinity and/or specificity of binding at a separate site (3). The second most striking structural feature of Acan 125 is its ϳ200-residue, proline-rich COOH-terminal domain. Consistent with the fact that SH3 domains mediate protein-protein interactions by binding to proline-rich target sequences containing the core element PXXP (4), Xu et al. (2) showed that a fusion protein containing the COOH-terminal 344 residues of Acan 125 bound to the isolated SH3 domain of myosin IC and that this interaction was abrogated by an 18-residue deletion spanning two PXXP motifs fitting the consensus for SH3 domain tar...

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“…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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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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Nonspecific Amine Immobilization of Ligand Can Be a Potential Source of Error in BIAcore Binding Experiments and May Reduce Binding Affinities

Cited by 45 publications

References 21 publications

Dual Interactions of the Translational Repressor Paip2 with Poly(A) Binding Protein

Dual Interactions of the Translational Repressor Paip2 with Poly(A) Binding Protein

CARMIL Is a Bona Fide Capping Protein Interactant

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

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