Polypeptide Backbone Chains Research Articles

In vitro inhibitory effect of five natural sweeteners on α-glucosidase and α-amylase.

A promising and efficacious approach to manage diabetes is inhibiting α-glucosidase and α-amylase activity. Therefore, the inhibitory activities of five natural sweeteners (mogrosides (Mog), stevioside (Ste), glycyrrhizinic acid (GA), crude trilobatin (CT), and crude rubusoside (CR)) against α-glucosidase and α-amylase and their interactions were evaluated in vitro using enzyme kinetics, fluorescence spectroscopy, Fourier infrared spectroscopy, and molecular docking. The inhibitor sequence was CT > GA > Ste, as GA competitively inhibited α-glycosidase activity while CT and Ste exhibited mixed inhibitory effects. Compared to a positive control acarbose, the inhibitory activity of CT was higher. For α-amylase, the mixed inhibitors CT, CR, and Mog and the competitive inhibitor Ste effectively inhibited the enzyme, with the following order: CT > CR > Ste > Mog; nevertheless, the inhibitors were slightly inferior to acarbose. Three-dimensional fluorescence spectra depicted that GA, CT, and CR bound to the hydrophobic cavity of α-glucosidase or α-amylase and changed the polarity of the hydrophobic amino acid-based microenvironment and structure of the polypeptide chain backbone. Infrared spectroscopy revealed that GA, CT, and CR could disrupt the secondary structure of α-glucosidase or α-amylase, which decreased enzyme activity. GA, trilobatin and rubusoside bound to amino acid residues through hydrogen bonds and hydrophobic interactions, changing the conformation of enzyme molecules to decrease the enzymatic activity. Thus, CT, CR and GA exhibit promising inhibitory effects against α-glucosidase and α-amylase.

The refinement of the parameters of β-turns using neutron diffraction data

Beta-bends are a difficult to interpret type of polypeptide chain backbone structure of globular proteins. Beta-bends are usually classified according to the dihedral angles φ and ψ of amino acid residues i + 1 and i + 2. Ramachandran map analysis of amino acid residues i + 1 and i + 2 indicates the resulting conformational stresses in bending. The mechanism of stabilization of their energetically disadvantageous conformations is still unclear. This kind of conformation stresses can only be compensated by additional interactions, such as additional hydrogen bonds, whose geometry and energy compensates for the beta-bending stress. Neutronography is the only available direct method for determining the position of hydrogen atoms in the structures of chemical compounds, including proteins. In this work, beta-bends from 176 protein structures from PDB established by neutronography are studied. In these structures, 3733 beta-bends were found using the i → i + 3 hydrogen bonding criterion. Using clustering by the magnitude of conformational angles, eight types of bends were newly identified. The magnitudes of conformational angles for each type of bend were determined. The hypothesis of additional hydrogen bonding to stabilize the bend was not confirmed, suggesting that the bending stress is compensated by other factors.

Typical Umami Ligand-Induced Binding Interaction and Conformational Change of T1R1-VFT.

Umami taste receptor type 1 member 1/3 (T1R1/T1R3) heterodimer has multiple ligand-binding sites, most of which are located in T1R1-Venus flytrap domain (T1R1-VFT). However, the critical binding process of T1R1-VFT/umami ligands remains largely unknown. Herein, T1R1-VFT was prepared with a sufficient amount and functional activity, and its binding characteristics with typical umami molecules (monosodium l-glutamate, disodium succinate, beefy meaty peptide, and inosine-5'-monophosphate) were explored via multispectroscopic techniques and molecular dynamics simulation. The results showed that, driven mainly by hydrogen bond, van der Waals forces, and electrostatic interactions, T1R1-VFT bound to umami compound at 1:1 (stoichiometric interaction) and formed T1R1-VFT/ligand complex (static fluorescence quenching) with a weak binding affinity (Ka values: 252 ± 19 to 1169 ± 112 M-1). The binding process was spontaneous and exothermic (ΔG, -17.72 to -14.26 kJ mol-1; ΔH, -23.86 to -12.11 kJ mol-1) and induced conformational changes of T1R1-VFT, which was mainly reflected in slight unfolding of α-helix (Δα-helix < 0) and polypeptide chain backbone structure. Meanwhile, the binding of the four ligands stabilized the active conformation of the T1R1-VFT pocket. This work provides insight into the binding interaction between T1R1-VFT/umami ligands and improves understanding of how umami receptor recognizes specific ligand molecules.

Protein and Peptide Based Drug Delivery- A Review

Biologics include vaccines, recombinant proteins, genes, synthetic tissues and viruses, which have emerged from molecular and cellular biology, with respect to unmet clinical needs and expanding indications. Hence it is important at this stage to outline the essential events that have led to the current level of interest in protein and peptide drug delivery systems. Polymers consisting of amino acids that are covalently linked by peptide bonds are known as Proteins and inturn peptides are small proteins composed of few dozen amino acids. Due to large molecular sizes of the proteins ranging from thousands to several millions atomic masses sizes; absorption through the epithelial barriers in the gastrointestinal tract is low. Moreover, proteins are rapidly degraded by digestive enzymes. As the bioavailability by oral route is poor they can be administered by other routes mainly by parenteral (IV, SC, IM) injections. The biological activity of proteins is strongly dependent on their molecular structure i.e 10 structure; the amino acid sequence, which ultimately dictates the non-covalent interactions and the 20i.e alpha helices and beta helices; the periodic spatial arrangement of the polypeptide chain backbone and 30; the 3-dimensional conformation of the whole molecule, including the positions of all amino acid side chains. Some proteins may consist of multiple peptide chains grouped together by non-covalent intermolecular interactions. The arrangement of these subunits relative to each other constitutes the 40structure. An alteration at any level of molecular structure leads to a change or loss of biological activity. The present review focuses on the development of various routes of drug administration, formulation as well as stability aspects of protein and peptide drug delivery system.

The investigation of protein flexibility of various soybean cultivars in relation to physicochemical and conformational properties

The conformation and physicochemical attributes of proteins play important roles in determining their flexibility. This study investigates the structural flexibility of twelve different soybean proteins. Results suggest that amino acids not only affects protein flexibility but its spatial structure as well. In addition, β-conglycinin protein contained more flexible subunits than glycinin. The HN-87, HN-48, HH-44 and HH-53 proteins were more flexible with lower α-helix content. Other key factors that influence higher structural flexibility include higher surface hydrophobicity and fewer disulfide bonds. Furthermore, more flexible proteins ensured the unfolding of the polypeptide chain backbone and endowed them with excellent emulsifying characteristics. In all, flexible proteins showed stronger surface activity than rigid ones. These findings thus provide clear evidence on the structural flexibility-functional properties interactions of different soybean proteins.

The relationship between the sign of the polypeptide backbone angle omega and the type of the side chain radical of amino-acid residues

The dihedral angle ω, which reflects the nonplanarity of the peptide group, was found to be essential for describing the conformation of the polypeptide chain backbone in the context of particular side chain radicals of amino-acid residues. Conformational clusters corresponding to conformationally stable peptides identified previously were observed.

Descriptive statistics of disallowed regions and various protein secondary structures in the context of studying twisted β-hairpins

A detailed analysis of polypeptide-chain backbone conformations was carried out for polypeptide-chain segments adjacent to β-turn regions, including the sites of disallowed conformations. A cross comparison of conformations was performed for disallowed regions of the Ramachandran plot and main types of β-turns and adjacent secondary structures. Based on the results, disallowed region 2 (II, II') in the Ramachandran plot was shown to coincide mainly with β-hairpins and, more exactly, twisted β-hairpins. The frequency of residues with angles ϕi, ψi that fall in region 2 (II, II') in the latter is 140 times higher than in common β-hairpins.

Foldamer Stability Coupled to Aggregation Propensity of Elongated Trp‐Cage Miniproteins

AbstractHere we present folding‐associated aggregation propensity of three Trp‐cage foldamers: E0 (20 aa), E5 (25 aa) and E10 (30 aa), models of different sizes but comparable molecular properties. Electronic circular dichroism (ECD), vibrational circular dichroism (VCD) and FT‐IR spectroscopic measurements were used to monitor their concentration‐dependent, heat‐induced (5 °C → 65 °C) “α→β” fold transition. The ECD curves of E0 display an ensemble of highly dynamic structures. ECD of both E5 and E10 foldamers show the expected Trp‐cage fold, dominated by their α‐helical properties. No sign of β‐structures was revealed by ECD at any conditions (5 °C &lt; T &lt; 65 °C, 5 &lt; pH &lt; 7, c ≈ 30 μM) for any of these miniproteins. However, at higher concentration (c ≈ 1–30 mM) both VCD and FT‐IR spectral features of E5 as well as E10 resemble that of a β‐strand (ca. 1615 cm–1), accompanied with “free β‐edges”, or native β‐sheets (ca. 1635 cm–1). E5 at lower concentrations (c ≈ 1–3 mM), and E10 at higher concentration (c ≈ 30 mM) display the α→native‐β→β‐sheet folding transitions, monitored by the characteristic C=O vibrational normal mode frequency shift as follows: ca. 1650 cm–1 → ca. 1635 cm–1 → ca. 1615 cm–1, respectively. The latter folding path is irreversible. The shortest polypeptide E0 has an “unordered” fold, while E10 presents the most tightly packed Trp‐cage 3D‐structure. We have found that both high dynamicity and/or tight molecular core packing are different in nature, but common in efficacy in preventing the polypeptide backbone chain against self‐aggregation. However, E5 is intermediate in size and stability, and thus among these three polypeptides it is the quickest to aggregate. The present molecular triad, E0, E5 and E10, serves as a good example of larger globular proteins for which aggregation and amyloid fiber‐like nanoparticle formations are often associated with Alzheimer's, Creutzfeldt–Jakob, or prion diseases.

Poly(l-lysine) as a polychelatogen to remove toxic metals using ultrafiltration and bactericide properties of poly(l-lysine)–Cu2+ complexes

Poly(l-lysine) is a water-soluble, synthetic polypeptide containing functional amine groups that help remove di- and trivalent metal ions from aqueous solutions. This polymer’s removal properties were studied under different experimental conditions: (1) competitive and non-competitive conditions; (2) different pHs; and (3) filtration factors. Under the conditions of the Liquid-Phase Polymer-Based Retention (LPR) technique, the cooper (II) ion interaction was found to be selective and efficient when compared to other divalent metal ions studied. However, interestingly, this selectivity disappeared when trivalent metal ions were present. The polymer–metal ion interactions are based on the amino groups of the side-chains as well as the polypeptide backbone chain. The removal of metal ions was strongly dependent on the pH. By structural characterization with FT-IR and EPR spectroscopy, participation of the amide and mainly amine groups was found to take place for the coordination. For the Cu2+, coordination through four amine nitrogen donor atoms in the primary coordination sphere was detected. Antibacterial activity tests were conducted with the poly(l-lysine)–Cu2+ complex and showed a higher activity in comparison with the precursors Cu2+ and poly(l-lysine) at the same concentrations for E. coli (6538P), a Gram-negative bacterium, and S. aureus (ATCC), a Gram-positive bacterium.

Left helix of polyproline II type and genesis of β-structures in spidroins 1 and 2 and their recombinant analogs

The distribution of secondary structures along the polypeptide chains of spider proteins spidroins 1 and 2 and their recombinant analogs has been studied by statistical methods. It was found that these proteins in the monomolecular form contain only trace amounts of beta-structures. At the same time, the regions of the sequence including Ala and Gly are predicted as helical-containing (with alpha-helices and left-helices of polyproline II type). An analysis of literature data and our data obtained in this study shows that the main conformation of the polypeptide chain solutions of spidroins 1 and 2 and their recombinant analogs in water solutions is the left-helix of polyproline II type with some contaminations of alpha-helices and a very small share of beta-structures. The transition to the state with extended conformations, which are peculiar to mature filaments of spider webs, requires the dehydration of the polypeptide chain backbone. Thus, the genesis of beta-structure in spider web proteins is determined by the conditions of conformation transitions between the main regular conformations of the polypeptide chain backbone.

Active site geometry of glucose‐1‐phosphate uridylyltransferase

Glucose-1-phosphate uridylyltransferase, or UGPase, catalyzes the production of UDP-glucose from glucose-1-phosphate and UTP. Because of the biological role of UDP-glucose in glycogen synthesis and in the formation of glycolipids, glycoproteins, and proteoglycans, the enzyme is widespread in nature. Recently this laboratory reported the three-dimensional structure of UGPase from Escherichia coli. While the initial X-ray analysis revealed the overall fold of the enzyme, details concerning its active site geometry were limited because crystals of the protein complexed with either substrates or products could never be obtained. In an effort to more fully investigate the active site geometry of the enzyme, UGPase from Corynebacterium glutamicum was subsequently cloned and purified. Here we report the X-ray structure of UGPase crystallized in the presence of both magnesium and UDP-glucose. Residues involved in anchoring the ligand to the active site include the polypeptide chain backbone atoms of Ala 20, Gly 21, Gly 117, Gly 180, and Ala 214, and the side chains of Glu 36, Gln 112, Asp 143, Glu 201, and Lys 202. Two magnesium ions are observed coordinated to the UDP-glucose. An alpha- and a beta-phosphoryl oxygen, three waters, and the side chain of Asp 142 ligate the first magnesium, whereas the second ion is coordinated by an alpha-phosphoryl oxygen and five waters. The position of the first magnesium is conserved in both the glucose-1-phosphate thymidylyltransferases and the cytidylyltransferases. The structure presented here provides further support for the role of the conserved magnesium ion in the catalytic mechanisms of the sugar-1-phosphate nucleotidylyltransferases.

Secondary structure determines protein topology

Using a test set of 13 small, compact proteins, we demonstrate that a remarkably simple protocol can capture native topology from secondary structure information alone, in the absence of long-range interactions. It has been a long-standing open question whether such information is sufficient to determine a protein's fold. Indeed, even the far simpler problem of reconstructing the three-dimensional structure of a protein from its exact backbone torsion angles has remained a difficult challenge owing to the small, but cumulative, deviations from ideality in backbone planarity, which, if ignored, cause large errors in structure. As a familiar example, a small change in an elbow angle causes a large displacement at the end of your arm; the longer the arm, the larger the displacement. Here, correct secondary structure assignments (alpha-helix, beta-strand, beta-turn, polyproline II, coil) were used to constrain polypeptide backbone chains devoid of side chains, and the most stable folded conformations were determined, using Monte Carlo simulation. Just three terms were used to assess stability: molecular compaction, steric exclusion, and hydrogen bonding. For nine of the 13 proteins, this protocol restricts the main chain to a surprisingly small number of energetically favorable topologies, with the native one prominent among them.

High Resolution Structures of an Oxidized and Reduced Flavoprotein: THE WATER SWITCH IN A SOLUBLE FORM OF (S)-MANDELATE DEHYDROGENASE

The crystal structures of a soluble mutant of the flavoenzyme mandelate dehydrogenase (MDH) from Pseudomonas putida and of the substrate-reduced enzyme have been analyzed at 1.35-A resolution. The mutant (MDH-GOX2) is a fully active chimeric enzyme in which residues 177-215 of the membrane-bound MDH are replaced by residues 176-195 of glycolate oxidase from spinach. Both structures permit full tracing of the polypeptide backbone chain from residues 4-356, including a 4-residue segment that was disordered in an earlier study of the oxidized protein at 2.15 A resolution. The structures of MDH-GOX2 in the oxidized and reduced states are virtually identical with only a slight increase in the bending angle of the flavin ring upon reduction. The only other structural changes within the protein interior are a 10 degrees rotation of an active site tyrosine side chain, the loss of an active site water, and a significant movement of six other water molecules in the active site by 0.45 to 0.78 A. Consistent with solution studies, there is no apparent binding of either the substrate, mandelate, or the oxidation product, benzoylformate, to the reduced enzyme. The observed structural changes upon enzyme reduction have been interpreted as a rearrangement of the hydrogen bonding pattern within the active site that results from binding of a proton to the N-5 position of the anionic hydroquinone form of the reduced flavin prosthetic group. Implications for the low oxidase activity of the reduced enzyme are also discussed.

Combinations of turns in proteins.

We observed that beta- and gamma-turns in protein structure may be associated as peptides representing combinations of turns that span between nine and 26 amino acid residues along the polypeptide backbone chain and often correspond to loops in the protein structure. Around 475 peptides resulted from the analysis of a non-redundant data set corresponding to 248 protein crystal structures selected from the Protein Data Bank. Nearly 40% protein chains are associated with two or more peptides and the peptides with nine and 10 amino acid residues are more frequent. A maximum of four distinct peptides varying in number of amino acid residues were observed in at least 10 proteins along the same protein chain. Nearly 80% peptides comprise type IV beta-turns that are associated with irregular dihedral angle values suggesting this may be important for the conformational diversity associated with the loops in proteins. In general, predominant interactions that possibly stabilize these peptides involve main-chain and side-chain interactions with solvent, in addition to hydrogen bond, salt-bridge and non-bonded interactions. Majority of the peptides were observed in hydrolase, oxidoreductase, transferase, serine proteinase/inhibitor complex, electron transport/electron transfer and lyase proteins.

Crystal structure of the oxidized cytochrome c2 from Blastochloris viridis

The crystal structure of the oxidized cytochrome c 2 from Blastochloris (formerly Rhodopseudomonas) viridis was determined at 1.9 Å resolution. Structural comparison with the reduced form revealed significant structural changes according to the oxidation state of the heme iron. Slight perturbation of the polypeptide chain backbone was observed, and the secondary structure and the hydrogen patterns between main-chain atoms were retained. The oxidation state-dependent conformational shifts were localized in the vicinity of the methionine ligand side and the propionate group of the heme. The conserved segment of the polypeptide chain in cytochrome c and cytochrome c 2 exhibited some degree of mobility, interacting with the heme iron atom by the hydrogen bond network. These results indicate that the movement of the internal water molecule conserved in various c-type cytochromes drives the adjustments of side-chain atoms of nearby residue, and the segmental temperature factor changes along the polypeptide chain.

Recognition of the lipoyl domain is the ultimate determinant of substrate channelling in the pyruvate dehydrogenase multienzyme complex

Reductive acetylation of the lipoyl domain (E2plip) of the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli is catalysed specifically by its partner pyruvate decarboxylase (E1p), and no productive interaction occurs with the analogous 2-oxoglutarate decarboxylase (E1o) of the 2-oxoglutarate dehydrogenase complex. Residues in the lipoyl-lysine β-turn region of the unlipoylated E2plip domain (E2plip apo) undergo significant changes in both chemical shift and transverse relaxation time ( T 2) in the presence of E1p but not E1o. Residue Gly11, in a prominent surface loop between β-strands 1 and 2 in the E2plip domain, was also observed to undergo a significant change in chemical shift. Addition of pyruvate to the mixture of E2plip apo and E1p caused larger changes in chemical shift and the appearance of multiple cross-peaks for certain residues, suggesting that the domain was experiencing more than one type of interaction. Residues in both β-strands 4 and 5, together with those in the prominent surface loop and the following β-strand 2, appeared to be interacting with E1p, as did a small patch of residues centred around Glu31. The values of T 2 across the polypeptide chain backbone were also lower than in the presence of E1p alone, suggesting that E2plip apo binds more tightly after the addition of pyruvate. The lipoylated domain (E2plip holo) also exhibited significant changes in chemical shift and decreases in the overall T 2 relaxation times in the presence of E1p, the residues principally affected being restricted to the half of the domain that contains the lipoyl-lysine (Lys41) residue. In addition, small chemical shift changes and a general drop in T 2 times in the presence of E1o were observed, indicating that E2plip holo can interact, weakly but non-productively, with E1o. It is evident that recognition of the protein domain is the ultimate determinant of whether reductive acetylation of the lipoyl group occurs, and that this is ensured by a mosaic of interactions with the Elp.

Functional assembly of fragments from bisected smooth muscle myosin light chain kinase.

The C-terminal regulatory segment of smooth muscle myosin light chain kinase folds back on its catalytic core to inhibit kinase activity. This regulatory segment consists of autoinhibitory residues linking the catalytic core to the calmodulin-binding sequence and perhaps additional C-terminal residues including an immunoglobulin-like motif. However, mutational and biochemical analyses showed no specific involvement of residues C-terminal to the calmodulin-binding sequence. To obtain additional insights on the proposed mechanisms for autoinhibition and Ca(2+)/calmodulin activation of the kinase, the polypeptide backbone chain of myosin light chain kinase was cleaved by genetic means to produce N- and C-terminal protein fragments. The N-terminal fragment containing the catalytic core was catalytically inactive when expressed alone. Co-expression of the N-terminal fragment with the C-terminal fragment containing the regulatory segment restored kinase activity. Deletion of the autoinhibitory linker residues without or with the calmodulin-binding sequence prevented restoration of kinase activity. In the presence or absence of Ca(2+)/calmodulin, regulatory segment binding occurred through the linker region connecting the catalytic core to the calmodulin-binding sequence. Collectively, these results indicate that residues C-terminal to the calmodulin-binding sequence (including the immunoglobulin-like motif) are not functional components of the regulatory segment. Furthermore, the principal autoinhibitory motif is contained in the sequence linking the catalytic core of myosin light chain kinase to the calmodulin-binding sequence.

Structural analysis of the H166G site-directed mutant of galactose-1-phosphate uridylyltransferase complexed with either UDP-glucose or UDP-galactose: detailed description of the nucleotide sugar binding site.

Galactose-1-phosphate uridylyltransferase plays a key role in galactose metabolism by catalyzing the transfer of a uridine 5'-phosphoryl group from UDP-glucose to galactose 1-phosphate. The enzyme from Escherichia coli is composed of two identical subunits. The structures of the enzyme/UDP-glucose and UDP-galactose complexes, in which the catalytic nucleophile His 166 has been replaced with a glycine residue, have been determined and refined to 1.8 A resolution by single crystal X-ray diffraction analysis. Crystals employed in the investigation belonged to the space group P2(1) with unit cell dimensions of a = 68 A, b = 58 A, c = 189 A, and beta = 100 degrees and two dimers in the asymmetric unit. The models for these enzyme/substrate complexes have demonstrated that the active site of the uridylyltransferase is formed by amino acid residues contributed from both subunits in the dimer. Those amino acid residues critically involved in sugar binding include Asn 153 and Gly 159 from the first subunit and Lys 311, Phe 312, Val 314, Tyr 316, Glu 317, and Gln 323 from the second subunit. The uridylyltransferase is able to accommodate both UDP-galactose and UDP-glucose substrates by simple movements of the side chains of Glu 317 and Gln 323 and by a change in the backbone dihedral angles of Val 314. The removal of the imidazole group at position 166 results in little structural perturbation of the polypeptide chain backbone when compared to the previously determined structure for the wild-type enzyme. Instead, the cavity created by the mutation is partially compensated for by the presence of a potassium ion and its accompanying coordination sphere. As such, the mutant protein structures presented here represent valid models for understanding substrate recognition and binding in the native galactose-1-phosphate uridylyltransferase.

Transmission electron microscopy studies on pig gastric mucin and its interactions with chitosan

Transmission electron microscopy has been employed to visualize the molecular structure and organization of a highly purified preparation of pig gastric mucin (molar mass M~9 × 10 6g/mol, as determined by low speed sedimentation equilibrium), prepared for microscopy by two completely independent methods. Samples were prepared for imaging by air drying on mica (in the presence of 50% glycerol) as well as critical point drying in acetone/CO 2. The data appear consistent with the accepted linear model for mucins, and are consistent with regions of variable degrees of glycosylation along the polypeptide backbone chain, with the overall conformation that of a loose or random coil. The behaviour of this mucin in a dilute solution mixture with the potential mucoadhesive polymer chitosan (M~1 60,000 g/mol) was then explored, and a clear interaction was demonstrated, consistent with dilute solution measurements using sedimentation velocity analysis in the analytical ultracentrifuge.

Three-dimensional solution structure of Callinectes sapidus metallothionein-1 determined by homonuclear and heteronuclear magnetic resonance spectroscopy.

Metallothionein is a cysteine-rich metal-binding protein whose biosynthesis is closely regulated by the level of exposure of an organism to zinc, copper, cadmium, and other metal salts. The metallothionein from Callinectes sapidus is known to bind six divalent metal ions in two separate metal-binding clusters. Heteronuclear 1H-113Cd and homonuclear 1H-1H NMR correlation experiments have been used to establish that the two clusters reside in two distinct protein domains. The three-dimensional solution structure of the metallothionein has been determined using the distance and angle constraints derived from these two-dimensional NMR data sets and a distance geometry/simulated annealing protocol. There are no interdomain short distance (< or = 4.5 A) constraints observed in this protein, enabling the calculation of structures for the N-terminal, beta domain and the C-terminal, alpha domain separately. A total of 18 structures were obtained for each domain. The structures are based on a total of 364 experimental NMR restraints consisting of 277 approximate interproton distance restraints, 12 chi 1 and 51 phi angular restraints, and 24 metal-to-cysteine connectivities obtained from 1H-113Cd correlation experiments. The only element of regular secondary structure in either of the two domains is a short segment of helix in the C-terminal alpha domain between Lys42 and Thr48. The folding of the polypeptide backbone chain in each domain, however, gives rise to several type I beta turns. There are no type II beta turns.