Reply from Fauchère and Pliska

Fauchere, J.-L.; Pliška, V.

doi:10.1016/0968-0004(86)90266-5

Cited by 169 publications

(255 citation statements)

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“…The binding of calcium modifies the electrophoretic mobility of rCLSP and induces conformational changes which results in the exposure of a hydrophobic region. This hydrophobic region binds more strongly to the HIC matrix than the hydrophobic region of CaM, as revealed by the elution profile with an ethylene glycol gradient and confirmed by its computer calculated hydrophobicity (data not shown) (26).…”

Section: Discussionmentioning

confidence: 99%

Identification and Cloning of a New Calmodulin-like Protein from Human Epidermis

Méhul

Bernard

Simonetti

et al. 2000

Journal of Biological Chemistry

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After separating by two-dimensional gel electrophoresis an extract of total proteins from human stratum corneum, two spots were extracted and analyzed for their peptide sequence. The resulting internal protein sequences provided evidence for the identification of a new calcium-binding protein. Cloning of the corresponding full-length cDNA was achieved by reverse transcriptase-polymerase chain reaction using two keratinocyte libraries, one from proliferating cultured keratinocytes and one from differentiated keratinocytes of reconstructed human epidermis. The cDNA had an open reading frame encoding a new calcium-binding protein of 146 amino acids, a member of the calmodulin family. We named this new protein calmodulin-like skin protein (CLSP), since reverse transcriptase-polymerase chain reaction studies of CLSP expression in 10 different human tissues revealed that this protein was particularly abundant in the epidermis where its expression is directly related to keratinocyte differentiation. Expression of the cloned cDNA in Escherichia coli yielded a recombinant protein which allowed its further characterization. rCLSP is able to bind calcium, and similarly to calmodulin, exposes thereafter hydrophobic parts which most likely interact with target proteins. Epidermal proteins retained by CaM affinity column are quantitatively and qualitatively distinct from those of the rCLSP column. Sequencing of a rCLSP affinity purified protein revealed 100% identity with transglutaminase 3, a key enzyme in terminal differentiation, indicating an important role of CLSP in this process.

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Section: Discussionmentioning

confidence: 99%

Identification and Cloning of a New Calmodulin-like Protein from Human Epidermis

Méhul

Bernard

Simonetti

et al. 2000

Journal of Biological Chemistry

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“…If such interactions are absent, the increase in stability of apoMb I overpacking variants may be related to the increased nonpolar surface area introduced by the substitutions. To examine the relationship between apoMb I stability and nonpolar surface area of position 131 variants, we plotted ⌬G IU°v alues versus free energies of transfer from water to octanol (⌬G tr°) for substituting side chains (27). A striking correlation between these two quantities is observed, with r ϭ 0.98 (Fig.…”

Section: Discussionmentioning

confidence: 99%

Nonspecific hydrophobic interactions stabilize an equilibrium intermediate of apomyoglobin at a key position within the AGH region

Bertagna

Barrick

2004

Proc. Natl. Acad. Sci. U.S.A.

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Acid-induced unfolding of apomyoglobin (apoMb) proceeds in a multistate process involving at least one equilibrium intermediate (I) at pH 4.2. The structure of the I form has been investigated thoroughly, with significant effort devoted to identifying potentially stabilizing native contacts. Here, we test whether rigid side-chain packing interactions like those in holomyoglobin persist at a buried position, Met-131, within the low-pH apoMb intermediate. We have measured the urea-induced unfolding transitions of overpacking, underpacking, and polar substitutions of Met-131 to determine the effect on the stability of the native and intermediate states of apoMb. Whereas underpacking substitutions should destabilize the I form irrespective of the degree of native side-chainpacking interactions, we anticipate that overpacking replacements might show opposite effects in a tightly packed environment, compared with a region lacking native side-chain packing interactions. We observe that, whereas underpacking and polar substitutions destabilize the I form, overpacking substitutions are stabilizing, implying that I is structurally plastic. We also report a strong correlation between the I state unfolding free energies and side-chain transfer free energies from water to octanol. Our results suggest that, whereas side-chain hydrophobicity is important for the stability of the I form, specific side-chain packing interactions are not.

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“…Cystine has been shown to be fairly hydrophobic, and thus, most disulfide bonds in the native protein are buried in the protein structure (1)(2)(3)(4). To suppress the modification of the hydrophobicity of the protein, it would be better to mutate the buried hydrophobic amino acids.…”

Section: Discussionmentioning

confidence: 99%

“…Cystine is hydrophobic, and thus, most of naturally occurring disulfide bonds are buried in the protein (1)(2)(3)(4). Therefore, the introduction of an engineered disulfide bond into the hydrophobic core better maintains the biophysical properties of the target protein.…”

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confidence: 99%

Stabilization of an Immunoglobulin Fold Domain by an Engineered Disulfide Bond at the Buried Hydrophobic Region

Hagihara

Mine

Uegaki

2007

Journal of Biological Chemistry

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We report for the first time the stabilization of an immunoglobulin fold domain by an engineered disulfide bond. In the llama single-domain antibody, which has human chorionic gonadotropin as its specific antigen, Ala 49 and Ile 70 are buried in the structure. A mutant with an artificial disulfide bond at this position showed a 10°C higher midpoint temperature of thermal unfolding than that without the extra disulfide bond. The modified domains exhibited an antigen binding affinity comparable with that of the wild-type domain. Ala 49 and Ile 70 are conserved in camel and llama single-domain antibody frameworks. Therefore, domains against different antigens are expected to be stabilized by the engineered disulfide bond examined here. In addition to the effect of the loop constraints in the unfolded state, thermodynamic analysis indicated that internal interaction and hydration also control the stability of domains with disulfide bonds. The change in physical properties resulting from mutation often causes unpredictable and destabilizing effects on these interactions. The introduction of a hydrophobic cystine into the hydrophobic region maintains the hydrophobicity of the protein and is expected to minimize the unfavorable mutational effects.One of the major objectives of antibody engineering is to stabilize the three-dimensional structure of the antibody. Disulfide bonds often significantly stabilize the structure of native proteins. Thus, the introduction of artificial disulfide bonds is recognized as a useful protein engineering technique to increase conformational stability. Although this technique has been applied to many proteins, there are no reports of engineered disulfide bonds in an immunoglobulin fold framework.Cystine is hydrophobic, and thus, most of naturally occurring disulfide bonds are buried in the protein (1-4). Therefore, the introduction of an engineered disulfide bond into the hydrophobic core better maintains the biophysical properties of the target protein. There are several examples of artificial disulfide bonds that can replace a pair of buried hydrophobic residues, the accessible surface areas of which were Ͻ20% (5-11). The introduction of a disulfide bond into the buried hydrophobic core of human carbonic anhydrase (A23C/L203C) markedly stabilizes this enzyme; the midpoint temperature of thermal unfolding (T m ) of the mutant is 10°C higher than that of the wild-type protein (6). The engineered disulfide bonds in alkaline protease AprP (G199C/F236C) (11), xylanase (V98C/ A152C) (7), and manganese peroxidase (A48C/A63C) (10) mutants increase their tolerance against heat inactivation. On the other hand, subtilisin BPNЈ mutants (V26C/A232C and A29C/M119C) exhibit similar or slightly lower stability to irreversible thermal inactivation (8). Tolerance against heat denaturation is not directly correlated with conformational stability. Only the mutational effect on human carbonic anhydrase II (6) was examined in a reversible system. Little information is available about the thermodynamic effe...

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Reply from Fauchère and Pliska

Cited by 169 publications

References 1 publication

Identification and Cloning of a New Calmodulin-like Protein from Human Epidermis

Identification and Cloning of a New Calmodulin-like Protein from Human Epidermis

Nonspecific hydrophobic interactions stabilize an equilibrium intermediate of apomyoglobin at a key position within the AGH region

Stabilization of an Immunoglobulin Fold Domain by an Engineered Disulfide Bond at the Buried Hydrophobic Region

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