Quantification of the Influence of Extracellular Laccase and Intracellular Reactions on the Isomer-Specific Biotransformation of the Xenoestrogen Technical Nonylphenol by the Aquatic Hyphomycete Clavariopsis aquatica

The aquatic hyphomycete Clavariopsis aquatica was used to quantify the effects of extracellular laccase and intracellular reactions on the isomer-specific biotransformation of technical nonylphenol (t-NP). In laccase-producing cultures, maximal removal rates of t-NP and the isomer 4-(1-ethyl-1,4-dimethylpentyl)phenol (NP112) were about 1.6- and 2.4-fold higher, respectively, than in laccase-lacking cultures. The selective suppression of either laccase or intracellular reactions resulted in essentially comparable maximal removal rates for both compounds. Evidence for an unspecific oxidation of t-NP isomers was consistently obtained from laccase-expressing fungal cultures when intracellular biotransformation was suppressed and from reaction mixtures containing isolated laccase. This observation contrasts with the selective degradation of t-NP isomers by bacteria and should prevent the enrichment of highly estrogenic isomers in remaining t-NP. In contrast with laccase reactions, intracellular fungal biotransformation caused a significant shift in the isomeric composition of remaining t-NP. As a result, certain t-NP constituents related to more estrogenic isomers were less efficiently degraded than others. In contrast to bacterial degradation via ipso-hydroxylation, the substitution pattern of the quaternary α-carbon of t-NP isomers does not seem to be very important for intracellular transformation in C. aquatica. As-yet-unknown intracellular enzymes are obviously induced by nonylphenols. Mass spectral data of the metabolites resulting from the intracellular oxidation of t-NP, NP112, and 4-(1-ethyl-1,3-dimethylpentyl)phenol indicate nonyl chain hydroxylation, further oxidation into keto or aldehyde compounds, and the subsequent formation of carboxylic acid derivatives. Further metabolites suggest nonyl chain desaturation and methylation of carboxylic acids. The phenolic moieties of the nonylphenols remained unchanged.Nonylphenol ethoxylates (NPEOs) represent a major group of industrial nonionic surfactants. Technical nonylphenol (t-NP), used for the production of NPEOs, is synthesized by Friedel-Crafts alkylation of phenol with a mixture of differently branched nonenes. It therefore comprises a great variety of mainly para-substituted isomers, with variously branched nonyl chains. About 50 to 80 t-NP isomers were estimated to occur in environmentally relevant matrices (19). The incomplete bioconversion of NPEOs in wastewater treatment plants results in the formation of the less biodegradable t-NP and is considered a major source of this contaminant in the aquatic environment (57). The recalcitrance of t-NP to biodegradation is partly due to the presence in more than 85% of the t-NP isomers of a quaternary α-carbon in the branched nonyl chain. Such structural characteristics are considered to limit biological nonyl chain oxidation (11, 53, 55). Nonylphenols are known to disrupt normal endocrine functions in vertebrates (57). Certain isomers contained in t-NP have been reported to possess a considerably higher estrogenic activity than the t-NP mixture (15). Due to increasing concerns with respect to their largely unknown environmental fate and potentially adverse environmental and human health effects, nonylphenols have been listed as priority hazardous substances in the EU water framework directive.In light of the concerns above, microbial reactions with the potential to reduce nonylphenol concentrations in the environment but also offering new possibilities for applications such as effluent treatment have received increasing attention (11). Among environmental microorganisms, both aquatic and terrestrial fungi, as well as bacteria, have been shown to degrade t-NP (11). Fungal attack on nonylphenols differs from bacterial nonylphenol degradation. In the case of intracellular nonylphenol biotransformation reactions catalyzed by fungi, only metabolites modified in the alkyl chain have been described (23, 52). Metabolites indicative of oxidation of the phenolic ring have not been described to date. Bacterial degradation pathways have only been documented in the genera Sphingomonas and Sphingobium. Bacterial mineralization of the aromatic moiety of t-NP isomers to CO₂ and H₂O is initiated via ring hydroxylation at the ipso (C-4) position of the phenolic ring, and nonanols are produced from the nonyl chains (10, 11, 15, 16). Bacteria have been shown to utilize branched-chain nonylphenols as growth substrates (11, 12, 17, 43). In contrast, only one report describes the growth of a fungus, the yeast Candida aquaetextoris, on nonylphenol (the isomer 4-n-NP containing a linear nonyl chain) (52). With respect to fungal attack on t-NP, cometabolism seems to be the dominating process (11). Recent literature data indicate that certain t-NP isomers with an estrogenic potency higher than those of the original t-NP mixture can be enriched in remaining t-NP. This results from the selective removal of individual isomers upon bacterial ipso-substitution degradation mechanisms (15). However, the effects of fungal biotransformation reactions on the isomeric profile of t-NP have not yet been quantified.Laccases are extracellular multicopper oxidases. These have most frequently been described in white-rot basidiomycetes, which unspecifically oxidize via one-electron abstraction certain lignin constituents, as well many xenobiotic compounds. Thereby, organic radicals are generated as the primary oxidation products (3). Among the several groups of fungi found in aquatic environments, aquatic hyphomycetes (AQH) are a phylogenetically diverse group of mitosporic fungi specifically adapted to their exclusively aquatic lifestyle. AQH have been shown to metabolize several organic environmental pollutants, including t-NP (23), polycyclic musk fragrances (31), pesticide metabolites (2), and synthetic dyes (22). Therefore, with respect to the fungal attack on organic pollutants found in aquatic ecosystems, AQH are of special importance. Laccase production by strictly aquatic fungi such as AQH has already been demonstrated and discussed in the context of lignocellulose decay in aquatic ecosystems (1). A role of this enzyme in the AQH-catalyzed breakdown of aquatic environmental pollutants has been recently suggested. Here, laccase isolated from the AQH Clavariopsis aquatica was shown to act on nonylphenol (23) and polycyclic musk fragrances (31). Laccase has also been implicated in nonylphenol degradation by white-rot fungi (44, 45). Isolated extracellular laccases from several aquatic and terrestrial fungi were shown to catalyze the formation of oligomeric coupling products from nonylphenols via organic radical intermediates (6, 11). However, the effects of laccase reactions on the isomeric patterns of t-NP have not been assessed to date.The aim of the present study was to quantify the influence of extracellular laccase catalysis and intracellular biotransformation on nonylphenol removal rates and on the isomeric composition of t-NP. For this, C. aquatica was used as a model organism. The derived data were compared to effects of bacteria on nonylphenol isomers reported by other authors (15), and environmental and biotechnological implications of fungal t-NP biotransformation were deduced. At the same time we addressed metabolite formation from t-NP and the two major t-NP isomers 4-(1-ethyl-1,3-dimethylpentyl)phenol (NP111) and 4-(1-ethyl-1,4-dimethylpentyl)phenol (NP112) (Fig. ​(Fig.1).1). This was done to substantiate the apparent differences between fungi and bacteria in the intracellular oxidation of t-NP (11, 15).Open in a separate windowFIG. 1.Chemical structures of the nonylphenol isomers NP111 and NP112.     

Cpn20: Siamese twins of the chaperonin world

The chloroplast cpn20 protein is a functional homolog of the cpn10 co-chaperonin, but its gene consists of two cpn10-like units joined head-to-tail by a short chain of amino acids. This double protein is unique to plastids and was shown to exist in plants as well plastid-containing parasites. In vitro assays showed that this cpn20 co-chaperonin is a functional homolog of cpn10. In terms of structure, existing data indicate that the oligomer is tetrameric, yet it interacts with a heptameric cpn60 partner. Thus, the functional oligomeric structure remains a mystery. In this review, we summarize what is known about this distinctive chaperonin and use a bioinformatics approach to examine the expression of cpn20 in Arabidopsis thaliana relative to other chaperonin genes in this species. In addition, we examine the primary structure of the two homologous domains for similarities and differences, in comparison with cpn10 from other species. Lastly, we hypothesize as to the oligomeric structure and raison d’être of this unusual co-chaperonin homolog. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Histological examinations of facial glands in <Emphasis Type="Italic">Saccopteryx</Emphasis><Emphasis Type="Italic">bilineata</Emphasis> (Chiroptera,Emballonuridae), and their potential use in territorial marking

Scent marking is widespread among individuals of Mammalia species, especially in resource defence social systems. Apart from urine and faeces that are used for claiming resource ownership, specialised scent glands are the main source of secretions in scent marking individuals. Most previous studies have described secretory epithelia macroscopically, since many glands are conspicuous. But macroscopically inconspicuous scent glands or morphological structures might then be overlooked. In Saccopteryx bilineata (greater sac-winged bat), behavioural observations suggest that both sexes have, apart from the conspicuous gular glands of males, specialised facial glands to display territorial marking. We investigated the facial glands of two males and one female S. bilineata histologically and found, first, that both sexes possess a bilateral symmetrically intermandibular gland, which is composed of a bed of modified apocrine sudoriferous cells. Second, we found lip glands consisting of modified apocrine sudoriferous cell units with pigmented ducts around the upper and the lower lip. Both gland types are probably involved during territorial marking.     

Generation and identification of variants with improved purification yield of Bowman-Birk protease inhibitors carrying protein binding loops

Replacing the chymotrypsin inhibitory loop of soybean Bowman-Birk inhibitor (sBBI) with a VEGF binding peptide (BBI-AV) significantly reduces the overall purification yield when BBI-AV is produced as a fusion protein in a Bacillus subtilis expression system. The low purification yield is primarily due to a higher fraction of molecules with incorrect disulfide bond configurations after production and also after disulfide bond shuffling induced by 2-mercaptoethanol. To improve production yields, site-saturation libraries were generated at 39 out of the 66 amino acid residues of BBI-AV. Initial screens were designed to select for variants with higher trypsin inhibitory activities than the parent after treatment with a reducing agent. Secondary screens were developed to select for variants with the highest purification yields, and to also eliminate any false positives. From the screens, it was found that positively charged substitutions in the exposed hydrophobic patch region (sites 27, 29, 40, 50 & 52) are especially productive. In fact, one substitution, F50R, improves the purification yield to nearly the same level as wild-type sBBI. Productive amino acid substitutions were combined to select for the variant with the best overall yield after purification. Several variants were obtained with higher purification yields than even sBBI. The octuple variants, A13I-S25R-M27A-L29P-S31A-A40H-F50K-V52T and A13I-S25K-M27A-L29R-S31E-A40K-F50Q-V52Q, are particularly productive having greater than a five fold increase in final purification yield over the parent.     

Design of an Insulin Analog with Enhanced Receptor Binding Selectivity: RATIONALE, STRUCTURE, AND THERAPEUTIC IMPLICATIONS*

Insulin binds with high affinity to the insulin receptor (IR) and with low affinity to the type 1 insulin-like growth factor (IGF) receptor (IGFR). Such cross-binding, which reflects homologies within the insulin-IGF signaling system, is of clinical interest in relation to the association between hyperinsulinemia and colorectal cancer. Here, we employ nonstandard mutagenesis to design an insulin analog with enhanced affinity for the IR but reduced affinity for the IGFR. Unnatural amino acids were introduced by chemical synthesis at the N- and C-capping positions of a recognition α-helix (residues A1 and A8). These sites adjoin the hormone-receptor interface as indicated by photocross-linking studies. Specificity is enhanced more than 3-fold on the following: (i) substitution of Gly^A1 by d-Ala or d-Leu, and (ii) substitution of Thr^A8 by diaminobutyric acid (Dab). The crystal structure of [d-Ala^A1,Dab^A8]insulin, as determined within a T₆ zinc hexamer to a resolution of 1.35 Å, is essentially identical to that of human insulin. The nonstandard side chains project into solvent at the edge of a conserved receptor-binding surface shared by insulin and IGF-I. Our results demonstrate that modifications at this edge discriminate between IR and IGFR. Because hyperinsulinemia is typically characterized by a 3-fold increase in integrated postprandial insulin concentrations, we envisage that such insulin analogs may facilitate studies of the initiation and progression of cancer in animal models. Future development of clinical analogs lacking significant IGFR cross-binding may enhance the safety of insulin replacement therapy in patients with type 2 diabetes mellitus at increased risk of colorectal cancer.     

A cis-Proline in ��-Hemoglobin Stabilizing Protein Directs the Structural Reorganization of ��-Hemoglobin

α-Hemoglobin (αHb) stabilizing protein (AHSP) is expressed in erythropoietic tissues as an accessory factor in hemoglobin synthesis. AHSP forms a specific complex with αHb and suppresses the heme-catalyzed evolution of reactive oxygen species by converting αHb to a conformation in which the heme is coordinated at both axial positions by histidine side chains (bis-histidyl coordination). Currently, the detailed mechanism by which AHSP induces structural changes in αHb has not been determined. Here, we present x-ray crystallography, NMR spectroscopy, and mutagenesis data that identify, for the first time, the importance of an evolutionarily conserved proline, Pro³⁰, in loop 1 of AHSP. Mutation of Pro³⁰ to a variety of residue types results in reduced ability to convert αHb. In complex with αHb, AHSP Pro³⁰ adopts a cis-peptidyl conformation and makes contact with the N terminus of helix G in αHb. Mutations that stabilize the cis-peptidyl conformation of free AHSP, also enhance the αHb conversion activity. These findings suggest that AHSP loop 1 can transmit structural changes to the heme pocket of αHb, and, more generally, highlight the importance of cis-peptidyl prolyl residues in defining the conformation of regulatory protein loops.Mammalian adult hemoglobin (HbA)⁵ is a tetramer of two αHb and two βHb subunits, which is produced to extremely high concentrations (∼340 mg/ml) in red blood cells. Numerous mechanisms exist to balance and coordinate HbA synthesis in normal erythropoiesis, and problems with the production of either HbA subunit give rise to thalassemia, a common cause of anemia worldwide. Previously, we identified α-hemoglobin stabilizing protein (AHSP) as an accessory factor in normal HbA production (1). AHSP forms a dimeric complex with αHb (see Fig. 1A) (2) but does not interact with βHb or HbA. AHSP also binds heme-free (apo) αHb (3) and may serve functions in both the folding of nascent αHb (4) and the detoxification of excess αHb that remains following HbA assembly (2, 5). Mice carrying an Ahsp gene knock-out display mild anemia, ineffective erythropoiesis, and enhanced sensitivity to oxidative stress (1, 6), features also observed in β-thalassemia patients due to the cytotoxic effects of free αHb.Open in a separate windowFIGURE 1.Summary of αHb·AHSP interactions. A, the αHb·AHSP complex(PDB code 1Z8U) (2). The interface is formed from helices 1 and 2 and the intervening loop 1 (green) of AHSP, together with helices G-H and the B-C corner of αHb (cyan). B, detailed views of the heme binding site of αHb as it appears in oxy-HbA (PDB code 1GZX) (69) and the final bis-histidyl αHb·AHSP complex (PDB code 1Z8U) with two histidine ligands to the iron. Typical visible absorption spectra in the region 450–700 nm are shown.Free αHb promotes the formation of harmful reactive oxygen species as a result of reduction/oxidation reactions involving the heme iron (7, 8). Reactive oxygen species can damage heme, αHb, and other cellular structures, resulting in hemoglobin precipitates and death of erythroid precursor cells (9–12). The presence of AHSP may explain how cells tolerate the slight excess of αHb that is observed in normal erythropoiesis, which is postulated to inhibit the formation of non-functional βHb tetramers, thus providing a robust mechanism for achieving the correct subunit stoichiometry during HbA assembly (13).Structural and biochemical studies have begun to elucidate the molecular mechanism by which AHSP detoxifies αHb. AHSP binds to oxygenated αHb to generate an initial complex that retains the oxy-heme, as evidenced by a characteristic visible absorption spectrum (see Fig. 1B, middle) and resonance Raman spectrum (5). This initial oxy-αHb·AHSP complex then converts to a low spin Fe³⁺ complex (2), in which the heme iron is bound at both axial positions by the side chains of His⁵⁸ and His⁸⁷ from αHb (see Fig. 1B, right). The formation of this complex inhibits αHb peroxidase activity and heme loss (2). Bis-histidyl heme coordination is becoming increasingly recognized as a feature of numerous vertebrate and non-vertebrate globins (14) and has been shown previously to confer a relative stabilization of the Fe³⁺ over the Fe²⁺ oxidation state (15–17). Although bis-histidyl heme coordination has previously been detected in solutions of met-Hb, formed through spontaneous autoxidation of Hb (18–21), the bishis-αHb·AHSP complex provides the first evidence that the bis-histidyl heme may play a positive functional role in Hb biochemistry by inhibiting the production of harmful reactive oxygen species.Despite its potential importance, the mechanism by which AHSP influences heme coordination in its binding partner is still unknown. As shown in Fig. 1A, AHSP binds αHb at a surface away from the heme pocket, and thus structural changes must somehow be transmitted through the αHb protein. It is intriguing that the free AHSP protein switches between two alternative conformations linked to cis/trans isomerization of the Asp²⁹-Pro³⁰ peptide bond in loop 1 (22) and that, in complex with αHb, this loop is located at the αHb·AHSP interface (see Fig. 1A). Peptide bonds preceding proline residues are unique in that the cis or trans bonding conformations have relatively similar stabilities (23), allowing an interconversion between these conformations that can be important for protein function (24, 25). Previous x-ray crystal structures of αHb·AHSP complexes have been obtained only with a P30A mutant of AHSP, in which isomerization is abolished and the Asp²⁹-Ala³⁰ peptide bond adopts a trans conformation, leaving the potential structural and functional significance of the evolutionarily conserved Pro³⁰ undisclosed. Here, we demonstrate a functional role for AHSP Pro³⁰ in conversion of oxy-αHb to the bis-histidyl form and identify a specific structural role for a cis Asp²⁹-Pro³⁰ peptide bond in this process. From a mechanistic understanding of how AHSP promotes formation of bis-histidyl αHb, we may eventually be able to engineer AHSP function as a tool in new treatments for Hb diseases such as β-thalassemia.     

64Cu-AMD3100—A novel imaging agent for targeting chemokine receptor CXCR4

CXCR4 is a chemokine receptor which has been shown to be exploited by various tumors for increased survival, invasion, and homing to target organs. We developed a one step radiosynthesis for labeling the CXCR4-specific antagonist AMD3100 with Cu-64 to produce ⁶⁴Cu-AMD3100 with a specific activity of 11.28 Ci/μmol (417 GBq/μmol) at the end of radiosynthesis. Incorporation of Cu(II) ion into AMD3100 did not change its ability to inhibit cellular migration in response to the (only) CXCR4 ligand, SDF-1/CXCL12. ⁶⁴Cu-AMD3100 binding affinity to CXCR4 was found to be 62.7 μM. Biodistribution of ⁶⁴Cu-AMD3100 showed accumulation in CXCR4-expressing organs and tissues, a renal clearance pathway, and an anomalous specific accumulation in the liver. We conclude that ⁶⁴Cu-AMD3100 exhibits promise as a potential PET imaging agent for visualization of CXCR4-positive tumors and metastases and might be used to guide and monitor anti-CXCR4 tumor therapy.     

Tropoelastin

Tropoelastin is a 60-72 kDa alternatively spliced extracellular matrix protein and a key component of elastic fibres. It is found in all vertebrates except for cyclostomes. Secreted tropoelastin is tethered to the cell surface, where it aggregates into organised spheres for cross-linking and incorporation into growing elastic fibres. Tropoelastin is characterised by alternating hydrophobic and hydrophilic domains and is highly flexible. The conserved C-terminus is an area of the molecule of particular biological importance in that it is required for both incorporation into elastin and for cellular interactions. Mature cross-linked tropoelastin gives elastin, which confers resilience and elasticity on a diverse range of tissues. Elastin gene disruptions in disease states and knockout mice emphasise the importance of proper tropoelastin production and assembly, particularly in vascular tissue. Tropoelastin constructs hold promise as biomaterials as they mimic many of elastin's physical and biological properties with the capacity to replace damaged elastin-rich tissue.     

The Escherichia coli Cell Division Protein and Model Tat Substrate SufI (FtsP) Localizes to the Septal Ring and Has a Multicopper Oxidase-Like Structure

The Escherichia coli protein SufI (FtsP) has recently been proposed to be a component of the cell division apparatus. The SufI protein is also in widespread experimental use as a model substrate in studies of the Tat (twin arginine translocation) protein transport system. We have used SufI-GFP (green fluorescent protein) fusions to show that SufI localizes to the septal ring in the dividing cell. We have also determined the structure of SufI by X-ray crystallography to a resolution of 1.9 Å. SufI is structurally related to the multicopper oxidase superfamily but lacks metal cofactors. The structure of SufI suggests it serves a scaffolding rather than an enzymatic role in the septal ring and reveals regions of the protein likely to be involved in the protein-protein interactions required to assemble SufI at the septal ring.     

Enhancing the Activity of a Protein by Stereospecific Unfolding: CONFORMATIONAL LIFE CYCLE OF INSULIN AND ITS EVOLUTIONARY ORIGINS*S??

A central tenet of molecular biology holds that the function of a protein is mediated by its structure. An inactive ground-state conformation may nonetheless be enjoined by the interplay of competing biological constraints. A model is provided by insulin, well characterized at atomic resolution by x-ray crystallography. Here, we demonstrate that the activity of the hormone is enhanced by stereospecific unfolding of a conserved structural element. A bifunctional β-strand mediates both self-assembly (within β-cell storage vesicles) and receptor binding (in the bloodstream). This strand is anchored by an invariant side chain (Phe^B24); its substitution by Ala leads to an unstable but native-like analog of low activity. Substitution by d-Ala is equally destabilizing, and yet the protein diastereomer exhibits enhanced activity with segmental unfolding of the β-strand. Corresponding photoactivable derivatives (containing l- or d-para-azido-Phe) cross-link to the insulin receptor with higher d-specific efficiency. Aberrant exposure of hydrophobic surfaces in the analogs is associated with accelerated fibrillation, a form of aggregation-coupled misfolding associated with cellular toxicity. Conservation of Phe^B24, enforced by its dual role in native self-assembly and induced fit, thus highlights the implicit role of misfolding as an evolutionary constraint. Whereas classical crystal structures of insulin depict its storage form, signaling requires engagement of a detachable arm at an extended receptor interface. Because this active conformation resembles an amyloidogenic intermediate, we envisage that induced fit and self-assembly represent complementary molecular adaptations to potential proteotoxicity. The cryptic threat of misfolding poses a universal constraint in the evolution of polypeptide sequences.How insulin binds to the insulin receptor (IR)² is not well understood despite decades of investigation. The hormone is a globular protein containing two chains, A (21 residues) and B (30 residues) (Fig. 1A). In pancreatic β-cells, insulin is stored as Zn²⁺-stabilized hexamers (Fig. 1B), which form microcrystal-line arrays within specialized secretory granules (1). The hexamers dissociate upon secretion into the portal circulation, enabling the hormone to function as a zinc-free monomer. The monomer is proposed to undergo a change in conformation upon receptor binding (2). In this study, we investigated a site of conformational change in the B-chain (Phe^B24) (arrow in Fig. 1A). In classical crystal structures, this invariant aromatic side chain (tawny in Fig. 1B) anchors an antiparallel β-sheet at the dimer interface (blue in Fig. 1C). Total chemical synthesis is exploited to enable comparison of corresponding d- and l-amino acid substitutions at this site, an approach designated “chiral mutagenesis” (3-5). In the accompanying article, the consequences of this conformational change are investigated by photomapping of the receptor-binding surface (6). Together, these studies redefine the interrelation of structure and activity in a protein central to the hormonal control of metabolism.Open in a separate windowFIGURE 1.Sequence and structure of insulin. A, sequences of the B-chain (upper) and A-chain (lower) with disulfide bridges as indicated. The arrow indicates invariant Phe^B24. The B24-B28 β-strand is highlighted in blue. B, crystal structure of the T₆ zinc insulin hexamer (Protein Data Bank code 4INS): ribbon model (left) and space-filling model (right). The B24-B28 β-strand is shown in blue, and the side chain of Phe^B24 is highlighted in tawny. The B-chain is otherwise dark gray; the A-chain, light gray; and zinc ions, magenta. Also shown at the left are the side chains of His^B10 at the axial zinc-binding sites. C, cylinder model of the insulin dimer showing the B24-B26 antiparallel β-sheet (blue) anchored by the B24 side chain (tawny circle). The A- and B-chains are shown in light and dark gray, respectively. The protomer at the left is shown in the R-state, in which the central α-helix of the B-chain is elongated (B3-B19 in the frayed R^f protomer of T₃R^f₃ hexamers and B1-B19 in the R protomer of R₆ hexamers). The three types of zinc insulin hexamers share similar B24-B26 antiparallel β-sheets as conserved dimerization elements.The structure of an insulin monomer in solution resembles a crystallographic protomer (Fig. 2A) (7-9). The A-chain contains an N-terminal α-helix, non-canonical turn, and second helix; the B-chain contains an N-terminal segment, central α-helix, and C-terminal β-strand. The β-strand is maintained in an isolated monomer wherein the side chain of Phe^B24 (tawny in Fig. 2A), packing against the central α-helix of the B-chain, provides a “plug” to seal a crevice in the hydrophobic core (Fig. 2B). Anomalies encountered in previous studies of insulin analogs suggest that Phe^B24 functions as a conformational switch (4, 7, 10-14). Whereas l-amino acid substitutions at B24 generally impair activity (even by such similar residues as l-Tyr) (15), a seeming paradox is posed by the enhanced activities of nonstandard analogs containing d-amino acids (10-12).

TABLE 1

Previous studies of insulin analogs