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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Interaction Homology model a b s t r a c tWe previously observed highly rapid and robust response of murine olfactory receptor S6 (mOR-S6) with chimeric Ga 15_olf , compared to Ga 15 . To identify residues responsible for this difference in response, mutations of the cytosolic helix 8 were analyzed in a heterologous functional expression system. The N-terminal hydrophobic core between helix 8 and TM1-2 of mOR-S6 is important for activation of both Ga 15_olf and Ga 15 . Point mutation of a helix 8 N-terminal acidic residue eliminated the differences in response dynamics via Ga. This result suggests that an N-terminal acidic residue of helix 8 is responsible for rapid response via Ga 15_olf .

G protein-coupled receptors (GPCRs) transduce various extracellular signals, such as neurotransmitters, hormones, light, and odorous chemicals, into intracellular signals via G protein activation during neurological, cardiovascular, sensory and reproductive signaling. Common and unique features of interactions between GPCRs and specific G proteins are important for structure-based design of drugs in order to treat GPCR-related diseases. Atomic resolution structures of GPCR complexes with G proteins have revealed shared and extensive interactions between the conserved DRY motif and other residues in transmembrane domains 3 (TM3), 5 and 6, and the target G protein C-terminal region. However, the initial interactions formed between GPCRs and their specific G proteins remain unclear. Alanine scanning mutagenesis of the murine olfactory receptor S6 (mOR-S6) indicated that the N-terminal acidic residue of helix 8 of mOR-S6 is responsible for initial transient and specific interactions with chimeric Gα15_olf, resulting in a response that is 2.2-fold more rapid and 1.7-fold more robust than the interaction with Gα15. Our mutagenesis analysis indicates that the hydrophobic core buried between helix 8 and TM1–2 of mOR-S6 is important for the activation of both Gα15_olf and Gα15. This review focuses on the functional role of the C-terminal amphipathic helix 8 based on several recent GPCR studies.