Solid-Phase Peptide Synthesis

Erickson, Bruce W.; Merrifield, R. B.

doi:10.1016/b978-0-12-516302-6.50008-6

Cited by 147 publications

(79 citation statements)

References 876 publications

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“…The peptides were synthesized by standard tert-butyloxycarbonyl (t-Boc) solidphase techniques developed by Merrifield (26) on an Applied Biosystems Peptide Synthesizer model 430A (Foster City, CA), using MBHA resin (0.74 mmol of NH 2 /g of resin) (Bachem, Torrance, CA) on a 0.5 mmol scale. The synthesis methodology is similar to that described by Sereda et al (27), except that activation and coupling were performed in situ and described as follows.…”

Section: Methodsmentioning

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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Section: Methodsmentioning

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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“…An OH denotes a peptide with a free a-carboxyl group at the C-terminus, whereas an NH2 denotes a peptide with an amide at the a-carboxyl group of the C-terminus. Peptide synthesis was performed following the general procedure for solid-phase synthesis described by Erickson and Merrifield (1976) with modifications made by Parker and Hodges (1985) and Hodges et al (1 981) on either a Beckman model 990 (Fullerton, California) or an Applied Biosystems model 430A (Foster City, California) peptide synthesizer. Syntheses of peptides with a C-terminal lysine and a free a-carboxyl group were started with tBoc lysine-OCH2-Pam resin (Applied Biosystems; 1% cross-linked, 0.67 mmol Lyslg).…”

Section: Peptide Synthesismentioning

confidence: 99%

Antigen‐antibody interactions: Elucidation of the epitope and strain‐specificity of a monoclonal antibody directed against the pilin protein adherence binding domain of Pseudomonas aeruginosa strain K

et al. 1992

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The C-terminal region of Pseudomonas aeruginosa strain K (PAK) pilin comprises both an epitope for the strainspecific monoclonal antibody PK99H, which blocks pilus-mediated adherence, and the adherence binding domain for buccal and tracheal epithelial cells. The PK99H epitope was located in sequence 134-140 (Asp-Glu-Gln-PheIle-Pro-Lys) by using a single alanine replacement analysis on the 17-residue synthetic peptide corresponding to the PAK C-terminal sequence 128-144. Indeed, a 7-residue peptide corresponding to this sequence was shown to have a similar binding affinity to that of the native conformationally constrained (disulfide bridged) 17-residue peptide. This epitope was found to contain two critical residues (PheI3' and LysI4O) and one nonessential residue ( G l r~'~~) .Interestingly, the peptide, Phe-Ile-Pro-Lys, which constitutes the four most important side chains for antibody binding did not bind to PK99H. It was of interest to investigate the structural basis of the strainspecificity of PK99H utilizing naturally occurring pilin sequences. Therefore, all different residues found in the sequence corresponding to the PK99H epitope of the four other strains (PAO, CD4, K122-4, and KB7) were substituted one at a time in the PAK sequence and the changes in binding affinity of these analogs to the antibody PK99H were determined by competitive ELISA. The strain-specificity of PK99H for strains PAO, K122-4, and KB7 can be explained by the accumulated sequence changes in these strains, and at least two amino acid changes were required to explain the strain-specificity of PK99H. Similarly, cross-reactivity of PK99H with CD4 can be explained by the fact that there was only one side chain responsible for decreasing binding affinity compared to the PAK sequence.

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“…Peptides HGLGHGH, GKEQGHTRRHDWGHEKQRK, HGLGHGHEQQHGLGHGH, and HNLGHGHKHR-RDQGHGHQRG were synthesized by the solid-phase method (Erickson & Merrifield, 1976) using automatic instrumentation (Applied Biosynthesis Peptide Synthesizer Model 430A) and purified by HPLC (Waters) on C-18 columns. When each peptide was eluted with a linear acetonitrile gradient containing TFA, over 90% emerged in a single peak.…”

Section: Synthetic Peptidesmentioning

confidence: 99%

The sequence HGLGHGHEQQHGLGHGH in the light chain of high molecular weight kininogen serves as a primary structural feature for zinc‐dependent binding to an anionic surface

Dela

Colman

1992

Protein Science

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The histidine-glycine-rich region of the light chain of cleaved high molecular weight kininogen (HK) is thought to be responsible for binding to negatively charged surfaces and initiation of the intrinsic coagulation, fibrinolytic, and kinin-forming systems. However, the specifically required amino acid sequences have not been delineated. An IgG fraction of a monoclonal antibody (MAb) CllCl to the HK light chain was shown to inhibit by 66% the coagulant activity and by 57% the binding of HK to the anionic surface of kaolin at a concentration of 1.5 pM and 27 pM, respectively. Proteolytic fragments of HK were produced by successive digestion with human plasma kallikrein and factor XIa (FXIa). Those polypeptides that bound tightly (Kd = 0.77 nM) to a C11C1 affinity column were eluted at pH 3.0 and purified by membrane filtration. On 15% SDS polyacrylamide electrophoresis, the approximate M, was 7.3 kDa (range 6.2-8.1 kDa). Based on N-terminal sequencing, this polypeptide (I2), which extends from the histidine residue 459 to a lysine at position 505, 509, 51 1, 512, 515, or 520, inhibits by 50% the coagulant activity expressed by HK at a concentration of 22 pM. The synthetic peptide HGLGHGH representing the N-terminal of the l2 fragment was synthesized, tested, and found at 4 mM to inhibit the procoagulant activity of HK 50%. A synthetic heptadecapeptide, HGLGHGHEQQHGLGHGH (residues 459-475) included within the l2 fragment, and with the ability to bind zinc, inhibited 50% of the HK coagulant activity at a concentration of 325 pM in the absence and presence of added Zn2+ (30 pM). The specific binding of 12'I-HK to a negatively charged surface (kaolin) was inhibited 50% by unlabeled HK (5 pM). HGLGHGH, at a concentration of 7.0 mM, inhibited the binding to kaolin by 50%. The heptadecapeptide inhibited the specific binding of I2'I-HK to kaolin by 50%, at a concentration of 2.3 mM, in the absence of Zn2+.In contrast, when Zn2+ was added, the concentration to achieve 50% inhibition decreased to 630 pM, indicating that Zn2+ was required to attain a favorable conformation for binding. Moreover, the I, fragment was found to inhibit 50% of the I2'I-HK binding to kaolin at a concentration of 380 pM. These results suggest that residues contained within the 1, fragment, notably HGLGHGHEQQHGLGHGH, serves as a primary structural feature for binding to a negatively charged surface.

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Solid-Phase Peptide Synthesis

Cited by 147 publications

References 876 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

Antigen‐antibody interactions: Elucidation of the epitope and strain‐specificity of a monoclonal antibody directed against the pilin protein adherence binding domain of Pseudomonas aeruginosa strain K

The sequence HGLGHGHEQQHGLGHGH in the light chain of high molecular weight kininogen serves as a primary structural feature for zinc‐dependent binding to an anionic surface

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