Toxin III of the scorpionAndroctonus australis Hector: Proton nuclear magnetic resonance assignments and secondary structure

Summary ¹H NMR has been applied to a3.5 mM, pH 5.4, solution of toxin III (64 amino acids) from venom of the scorpionAndroctonus australis Hector. The resonance assignment strategy began by applying a generalized main-chain directed method for rapid identification and resonance assignments of secondary structures. The remaining resonances were assigned by the sequential method. Major structural features include a helix of 2 1/2 turns (residues 20–28) which is linked by two disulfide bridges to the central strand of a triple-stranded antiparallel -sheet. Turns were identified at residues 15–17, 47–49 and also at residues 51–53. Numerous NOEs have been observed between hydrophobic residues which suggest the presence of a hydrophobic core; these include Leu³⁷, Leu²³, Val⁴⁷, Tyr¹⁴, Trp⁴⁵ and Tyr⁵. The Trp⁴⁵ and Tyr⁵ rings lie orthogonal to one another. No crystal structure has been solved for this AaH III toxin. Comparisons are made with other members of the scorpion toxin family.Thenomenclature used is similar to that described by Wütrich, 1986.     

A Motif from Lys216 to Lys222 in Human BUB3 Protein Is a Nuclear Localization Signal and Critical for BUB3 Function in Mitotic Checkpoint

Human BUB3 is a key mitotic checkpoint factor that recognizes centromeric components and recruits other mitotic checkpoint molecules to the unattached kinetochore. The key amino acid residues responsible for its localization are not yet defined. In this study, we identified a motif from Lys²¹⁶ to Lys²²² in BUB3 as its nuclear localization signal. A BUB3 mutant with deletion of this motif (Del216–222) was found to localize to both the cytoplasm and the nucleus, distinct from the exclusively nuclear distribution of wild-type BUB3. Further analysis revealed that residues Glu²¹³, Lys²¹⁶, Lys²¹⁷, Lys²¹⁸, Tyr²¹⁹, and Phe²²¹, but not Lys²²², contribute to nuclear localization. Interestingly, the nuclear localization signal was also critical for the kinetochore localization of BUB3. The deletion mutant Del216–222 and a subtle mutant with four residue changes in this region (E213Q/K216E/K217E/K218E (QE)) did not localize to the kinetochore efficiently or mediate mitotic checkpoint arrest. Protein interaction data suggested that the QE mutant was able to interact with BUB1, MAD2, and BubR1 but that its association with the centromeric components CENP-A and KNL1 was impaired. A motif from Leu⁶¹ to Leu⁶⁵ in CENP-A was found to be involved in the association of BUB3 and CENP-A in cells; however, further assays suggested that CENP-A does not physically interact with BUB3 and does not affect BUB3 localization. Our findings help to dissect the mechanisms of BUB3 in mitotic checkpoint signaling.     

The lipid-associated 3D structure of SPA, a broad-spectrum neuropeptide antagonist with anticancer properties

[D-Arg¹, D-Trp^5,7,9, Leu¹¹] substance P (SPA) belongs to a family of peptides including antagonist G and SpD that act as broad-spectrum neuropeptide antagonists at several peripheral receptors. The lipid-induced structure of these peptides may be important for the receptor interactions of these analogs. Thus we describe the tertiary structure of SPA in the presence of sodium dodecylsulfate micelles at pH 5.0, and 25°C as determined from two-dimensional ¹H-NMR data recorded at 500 MHz. The resulting three-dimensional structure can be generally described as two type IV nonstandard turns around Arg¹*, Pro², Lys³, and Pro⁴ and Gln⁶, Trp⁷*, Phe⁸, and Trp⁹* residues, respectively, inserted into the interfacial region of the micelles (the asterisks denote D-form amino acid). These turns juxtapose the N- and C-termini of SPA and may form the basis of this peptide's unique ability to inhibit peptide receptor interactions at multiple receptor types.     

Molecular and Conformational Basis of a Specific and High-Affinity Interaction between AlbA and Albicidin Phytotoxin

The albA gene of Klebsiella oxytoca encodes a protein of 221 amino acids that binds the albicidin phytotoxin with a high affinity (dissociation constant = 6.4 × 10⁻⁸ M). For this study, circular dichroism (CD) spectrometry and an alanine scanning mutagenesis approach were used in combination to investigate the molecular and conformational mechanisms of this high-affinity protein-ligand interaction. CD analysis revealed that AlbA contains a high-affinity binding site, and binding of the albicidin ligand to AlbA in a low-ionic-strength environment induced significant conformational changes. The ligand-dependent conformational changes of AlbA were specific and rapid and reached a stable plateau within seconds after the addition of the antibiotic. However, such conformational changes were not detected when AlbA and albicidin were mixed in the high-ionic-strength buffer that is required for maximal binding activity. Based on the conceptual model of protein-ligand interaction, we propose that a threshold ion strength allows AlbA to complete its conformational rearrangement and resume its original stable structure for accommodation of the bound albicidin. Mutagenesis analysis showed that the replacement of Lys¹⁰⁶, Trp¹¹⁰, Tyr¹¹³, Leu¹¹⁴, Tyr¹²⁶, Pro¹³⁴, and Trp¹⁶² with alanine did not change the overall conformational structure of AlbA but decreased the albicidin binding activity about 30 to 60%. We conclude that these residues, together with the previously identified essential residue His¹²⁵, constitute a high-affinity binding pocket for the ligand albicidin. The results also suggest that hydrophobic and electrostatic potentials of these key amino acid residues may play important roles in the AlbA-albicidin interaction.     

Analysis of the DNA binding site of Escherichia coli RecA protein

To investigate the DNA binding site of RecA protein, we constructed 15 recA mutants having alterations in the regions homologous to the other ssDNA binding proteins. The in vivo analyses showed that the mutational change at Arg²⁴³, Lys²⁴⁸, Tyr²⁶⁴, or simultaneously at Lys⁶ and Lys¹⁹, or Lys⁶ and Lys²³ caused severe defects in the recA functions, while other mutational changes did not. Purified RecA-K6A-K23A (Lys⁶ and Lys²³ changed to Ala and Ala, respectively) protein was indistinguishable from the wild-type RecA protein in its binding to DNA. However, the RecA-R243A (Arg²⁴³ changed to Ala) and RecA-Y264A (Tyr²⁶⁴ changed to Ala) proteins were defective in binding to both ss- and ds-DNA. In self-oligomerization property, RecA-R243A was proficient but RecA-Y264A was deficient, suggesting that the RecA-R243A protein had a defect in DNA binding site and the RecA-Y264A protein was defective in its interaction with the adjacent RecA molecule. The region of residues 243–257 including the Arg²⁴³ is highly homologous to the DNA binding motif in the ssDNA binding proteins, while the eukaryotic RecA homologues have a similar structure at the amino-terminal side proximal to the nucleotide binding core. The region of residues 243–257 would be a part of the DNA binding site. The other parts of this site would be the Tyr¹⁰³ and the region of residues 178–183, which were cross-linked to ssDNA. These three regions lie in a line in the crystal structure.     

Structural Insights into the Substrate Specificity of a 6-Phospho-β-glucosidase BglA-2 from Streptococcus pneumoniae TIGR4

The 6-phospho-β-glucosidase BglA-2 (EC 3.2.1.86) from glycoside hydrolase family 1 (GH-1) catalyzes the hydrolysis of β-1,4-linked cellobiose 6-phosphate (cellobiose-6′P) to yield glucose and glucose 6-phosphate. Both reaction products are further metabolized by the energy-generating glycolytic pathway. Here, we present the first crystal structures of the apo and complex forms of BglA-2 with thiocellobiose-6′P (a non-metabolizable analog of cellobiose-6′P) at 2.0 and 2.4 Å resolution, respectively. Similar to other GH-1 enzymes, the overall structure of BglA-2 from Streptococcus pneumoniae adopts a typical (β/α)₈ TIM-barrel, with the active site located at the center of the convex surface of the β-barrel. Structural analyses, in combination with enzymatic data obtained from site-directed mutant proteins, suggest that three aromatic residues, Tyr¹²⁶, Tyr³⁰³, and Trp³³⁸, at subsite +1 of BglA-2 determine substrate specificity with respect to 1,4-linked 6-phospho-β-glucosides. Moreover, three additional residues, Ser⁴²⁴, Lys⁴³⁰, and Tyr⁴³² of BglA-2, were found to play important roles in the hydrolytic selectivity toward phosphorylated rather than non-phosphorylated compounds. Comparative structural analysis suggests that a tryptophan versus a methionine/alanine residue at subsite −1 may contribute to the catalytic and substrate selectivity with respect to structurally similar 6-phospho-β-galactosidases and 6-phospho-β-glucosidases assigned to the GH-1 family.     

Biophysical Investigations of Complement Receptor 2 (CD21 and CR2)-Ligand Interactions Reveal Amino Acid Contacts Unique to Each Receptor-Ligand Pair

Human complement receptor type 2 (CR2 and CD21) is a cell membrane receptor, with 15 or 16 extracellular short consensus repeats (SCRs), that promotes B lymphocyte responses and bridges innate and acquired immunity. The most distally located SCRs, SCR1–2, mediate the interaction of CR2 with its four known ligands (C3d, EBV gp350, IFNα, and CD23). To ascertain specific interacting residues on CR2, we utilized NMR studies wherein gp350 and IFNα were titrated into ¹⁵N-labeled SCR1–2, and chemical shift changes indicative of specific inter-molecular interactions were identified. With backbone assignments made, the chemical shift changes were mapped onto the crystal structure of SCR1–2. With regard to gp350, the binding region of CR2 is primarily focused on SCR1 and the inter-SCR linker, specifically residues Asn¹¹, Arg¹³, Ala²², Arg²⁸, Ser³², Arg³⁶, Lys⁴¹, Lys⁵⁷, Tyr⁶⁴, Lys⁶⁷, Tyr⁶⁸, Arg⁸³, Gly⁸⁴, and Arg⁸⁹. With regard to IFNα, the binding is similar to the CR2-C3d interaction with specific residues being Arg¹³, Tyr¹⁶, Arg²⁸, Ser⁴², Lys⁴⁸, Lys⁵⁰, Tyr⁶⁸, Arg⁸³, Gly⁸⁴, and Arg⁸⁹. We also report thermodynamic properties of each ligand-receptor pair determined using isothermal titration calorimetry. The CR2-C3d interaction was characterized as a two-mode binding interaction with K_d values of 0.13 and 160 μm, whereas the CR2-gp350 and CR2-IFNα interactions were characterized as single site binding events with affinities of 0.014 and 0.035 μm, respectively. The compilation of chemical binding maps suggests specific residues on CR2 that are uniquely important in each of these three binding interactions.     

Cunning Simplicity of a Stoichiometry Driven Protein Folding Thesis

Abstract

Mastoparan B (MP-B) is an antimicrobial cationic tetradecapeptide amide isolated from the venom of the hornet Vespa basalis. NMR spectroscopy was used to study the membrane associated structures of MP-B in various model membrane systems such as 120 mM DPC micelles, 200 mM SDS micelles, and 3%(w/v) DMPC/DHPC (1:2) bicelles. In all systems, MP-B has an amphiphilic α-helical structure from Lys² to Leu¹⁴. NOESY experiments performed on MP-B in nondeuterated SDS micelles show that protons in the indole ring of Trp⁹ are in close contact with methylene protons of SDS micelles. T₁ relaxation data and NOE data revealed that the bound form of MP-B may be dominant in SDS micelles. The interactions between MP-B and zwitterionic DPC micelles were much weaker than those between MP-B and anionic SDS micelles. By substitution of Trp⁹ with Ala⁹, the pore-forming activity of MP-B was decreased dramatically. All of these results imply that strong electrostatic interactions between the positively charged Lys residues in MP-B and the anionic phospholipid head groups must be the primary factor for MP-B binding to the cell membrane. Then, insertion of the indole ring of Trp⁹ into the membrane, as well as the amphiphilic α-helical structures of MP-B may allow MP-B to span the lipid bilayer through the C-terminal portion. These structural features are crucial for the potent antibiotic activities of MP-B.     

Reassessment of the Unique Mode of Binding between Angiotensin II Type 1 Receptor and Their Blockers

While the molecular structures of angiotensin II (Ang II) type 1 (AT₁) receptor blockers (ARBs) are very similar, they are also slightly different. Although each ARB has been shown to exhibit a unique mode of binding to AT₁ receptor, different positions of the AT₁ receptor have been analyzed and computational modeling has been performed using different crystal structures for the receptor as a template and different kinds of software. Therefore, we systematically analyzed the critical positions of the AT₁ receptor, Tyr¹¹³, Tyr¹⁸⁴, Lys¹⁹⁹, His²⁵⁶ and Gln²⁵⁷ using a mutagenesis study, and subsequently performed computational modeling of the binding of ARBs to AT₁ receptor using CXCR4 receptor as a new template and a single version of software. The interactions between Tyr¹¹³ in the AT₁ receptor and the hydroxyl group of olmesartan, between Lys¹⁹⁹ and carboxyl or tetrazole groups, and between His²⁵⁶ or Gln²⁵⁷ and the tetrazole group were studied. The common structure, a tetrazole group, of most ARBs similarly bind to Lys¹⁹⁹, His²⁵⁶ and Gln²⁵⁷ of AT₁ receptor. Lys¹⁹⁹ in the AT₁ receptor binds to the carboxyl group of EXP3174, candesartan and azilsartan, whereas oxygen in the amidecarbonyl group of valsartan may bind to Lys¹⁹⁹. The benzimidazole portion of telmisartan may bind to a lipophilic pocket that includes Tyr¹¹³. On the other hand, the n-butyl group of irbesartan may bind to Tyr¹¹³. In conclusion, we confirmed that the slightly different structures of ARBs may be critical for binding to AT₁ receptor and for the formation of unique modes of binding.     

Ligand-binding effects on the kringle 4 domain from human plasminogen: a study by laser photo-CIDNP1H-NMR spectroscopy

Photo-chemically induced dynamic nuclear polarization (photo-CIDNP) one-dimensional and two-dimensional (2D)¹H-NMR techniques have been applied to the study of the kringle 4 domain of human plasminogen both ligand-free and complexed to the antifibrinolytic drugs ɛ-aminocaproic acid and p-benzylaminesulfonic acid (BASA). A number of aromatic side-chains (His³, Trp⁷², Tyr⁴¹, Tyr⁵⁰ and Tyr⁷⁴) appear to be exposed and accessible to 3-N-car☐ymethyl-lumiflavin, the photopolarizing flavin dye, both in the presence and in the absence of ligands. A lesser exposure is observed for the Trp²⁵ and Trp⁶² indole groups in the presence of BASA. The spin-spin (J-coupling) and dipolar (Overhauser) connectivities in the 2D experiments afford absolute assignment of aromatic resonances for the above residues, as well as of those stemming from the Trp⁷² ring in the presence of BASA. Moreover, a number of H^β resonances can be identified and sorted according to specific types of amino acid residues.     

Site-directed mutagenesis of α-<Emphasis Type="SmallCaps">l</Emphasis>-rhamnosidase from <Emphasis Type="Italic">Alternaria</Emphasis> sp. L1 to enhance synthesis yield of reverse hydrolysis based on rational design

The α-l-rhamnosidase catalyzes the hydrolytic release of rhamnose from polysaccharides and glycosides and is widely used due to its applications in a variety of industrial processes. Our previous work reported that a wild-type α-l-rhamnosidase (RhaL1) from Alternaria sp. L1 could synthesize rhamnose-containing chemicals (RCCs) though reverse hydrolysis reaction with inexpensive rhamnose as glycosyl donor. To enhance the yield of reverse hydrolysis reaction and to determine the amino acid residues essential for the catalytic activity of RhaL1, site-directed mutagenesis of 11 residues was performed in this study. Through rationally designed mutations, the critical amino acid residues which may form direct or solvent-mediated hydrogen bonds with donor rhamnose (Asp²⁵², Asp²⁵⁷, Asp²⁶⁴, Glu⁵³⁰, Arg⁵⁴⁸, His⁵⁵³, and Trp⁵⁵⁵) and may form the hydrophobic pocket in stabilizing donor (Trp²⁶¹, Tyr³⁰², Tyr³¹⁶, and Trp³⁶⁹) in active-site of RhaL1 were analyzed, and three positive mutants (W261Y, Y302F, and Y316F) with improved product yield stood out. From the three positive variants, mutant W261Y accelerated the reverse hydrolysis with a prominent increase (43.7 %) in relative yield compared to the wild-type enzyme. Based on the 3D structural modeling, we supposed that the improved yield of mutant W261Y is due to the adjustment of the spatial position of the putative catalytic acid residue Asp²⁵⁷. Mutant W261Y also exhibited a shift in the pH-activity profile in hydrolysis reaction, indicating that introducing of a polar residue in the active site cavity may affect the catalysis behavior of the enzyme.     

Three-dimensional 1H NMR structure of the nucleocapsid protein NCp10 of Moloney murine leukemia virus

Summary The nucleocapsid protein of Moloney murine leukemia virus (NCp10) is a 56-amino acid protein which contains one zinc finger of the CysX₂CysX₄HisX₄Cys form, a highly conserved motif present in most retroviruses and retroelements. At pH5, NCp10 binds one zinc atom and the complexation induces a folding of the CysX₂CysX₄HisX₄Cys box, similar to that observed for the zinc-binding domains of HIV-1 NC protein. The three-dimensional structure of NCp10 has been determined in aqueous solution by 600 MHz ¹H NMR spectroscopy. The proton resonances could be almost completely assigned by means of phase-sensitive double-quantum-filtered COSY, TOCSY and NOESY techniques. NOESY spectra yielded 597 relevant structural constraints, which were used as input for distance geometry calculations with DIANA. Further refinement was performed by minimization with the program AMBER, which was modified by introducing a zinc force field. The solution structure is characterized by a well-defined central zinc finger (rmsd of 0.747±0.209 Å for backbone atoms and 1.709±0.187 Å when all atoms are considered), surrounded by flexible N- and C-terminal domains. The Tyr²⁸, Trp³⁵, Lys³⁷, Lys⁴¹ and Lys⁴² residues, which are essential for activity, lie on the same face of the zinc finger, forming a bulge structure probably involved in viral RNA binding. The significance of these structural characteristics for the various biological functions of the protein is discussed, taking into account the results obtained with various mutants.     

Crystal Structure of Histamine Dehydrogenase from Nocardioides simplex

Histamine dehydrogenase (HADH) isolated from Nocardioides simplex catalyzes the oxidative deamination of histamine to imidazole acetaldehyde. HADH is highly specific for histamine, and we are interested in understanding the recognition mode of histamine in its active site. We describe the first crystal structure of a recombinant form of HADH (HADH) to 2.7-Å resolution. HADH is a homodimer, where each 76-kDa subunit contains an iron-sulfur cluster ([4Fe-4S]²⁺) and a 6-S-cysteinyl flavin mononucleotide (6-S-Cys-FMN) as redox cofactors. The overall structure of HADH is very similar to that of trimethylamine dehydrogenase (TMADH) from Methylotrophus methylophilus (bacterium W3A1). However, some distinct differences between the structure of HADH and TMADH have been found. Tyr⁶⁰, Trp²⁶⁴, and Trp³⁵⁵ provide the framework for the “aromatic bowl” that serves as a trimethylamine-binding site in TMADH is comprised of Gln⁶⁵, Trp²⁶⁷, and Asp³⁵⁸, respectively, in HADH. The surface Tyr⁴⁴² that is essential in transferring electrons to electron-transfer flavoprotein (ETF) in TMADH is not conserved in HADH. We use this structure to propose the binding mode for histamine in the active site of HADH through molecular modeling and to compare the interactions to those observed for other histamine-binding proteins whose structures are known.     

The amino acid sequence of human APOA-I, an apolipoprotein isolated from high density lipoproteins.

The complete amino acid sequence of human A-I has been determined by manual and automated Edman degradation of intact and peptide fragments of A-I. A-I is a single chain protein of 243 residues with the following amino acid composition: Asp₁₆, Asn₅, Thr₁₀, Ser₁₅, Glu₂₇, Gln₁₉, Pro₁₀, Gly₁₀, Ala₁₉, Val₁₃, Met₃, Leu₃₇, Tyr₇, Phe₆, Trp₄, Lys₂₁, His₅, and Arg₁₆. The amino acid sequence contains no linear segments of hydrophobic or hydrophilic residues. A detailed correlation of the amino acid sequence, conformation, and self association of A-I will add further insight into the molecular mechanisms involved in protein-protein and protein-lipid interactions.     

Structure-function studies on gastrointestinal hormones: I. Synthesis of secretin analogs and their biological and immunological properties

Syntheses by conventional procedures of the three analogs corresponding to the porcine secretin sequence crossed at position 6 by the N-terminal hexapeptide sequences of VIP, GIP, and glucagon are described, viz., Ala⁴,Val⁵-, Tyr¹,Ala²,Glu³-, and Gln³-secretin (VIP-SN, GIP-SN, and GLU-SN). The analog Phe¹,Phe²,Trp³,Lys⁴-secretin (SOMA-SN), designed on the basis of the surprising homology of the sequence portions 10–13 of somatostatin and 5–8 of secretin, was also prepared. Finally, the synthesis of N^α-3-(4-hydroxyphenyl)propionyl-β-alanyl-secretin (DATA-SN), a tracer suitable for secretin radioimmunoassay and as an N-terminus modified secretin analog, is reported. The analogs are compared, in terms of their biological and immunological properties in different assay systems, with pure synthetic secretin.     

Analytical studies on crotamine hydrochloride.

The neurotoxin crotamine, which is a basic low molecular weight protein, was isolated, in the form of its hydrochloride, from a South Brazilian rattlesnake (Crotalus durissus crotaminicus, Crotalus durissus terrificus) venom.Disc electrophoresis, carried out in 7% acrylamide by the method of Reisfeld, at pH 4.5, showed a single band for the purified toxin.The toxin showed the following amino acid composition: Asx₂, Ser₃, Glx₂, Pro₃, Gly₅, Cys₅, Met₁, Ileu₁, Leu₁, Tyr₁, Phe₂, Trp₂, Lys₉, His₂, and Arg₂, which corresponds to a minimum molecular weight of 4760. This assignment of minimum molecular weight is supported by the recovery of 1.0 mole of N-terminal $\text{Tyr}\text{5800}\text{g}$ of crotamine by the Sanger dinitrophenol (DNP) method, 1.0 mole of $\text{Tyr}\text{4880}\text{g}$ by the Udenfriend method, and by uv analysis, which gave a value of 4820. The odd number of half-cystine residues ($\text{5}\text{4760}\text{g}$) cannot be explained on the basis of available analytical data.Tyrosine was the only amino-terminal residue detectable by the Sanger DNP method. Glycine was identified as the only carboxyl-terminal residue by the hydrazinolysis method of Akabori and by release upon treatment with yeast carboxypeptidase.The H⁺ electrometric and Cl^? complexometric titrations of crotamine hydrochloride showed that about 13 moles of HCl are bound per 4760 g of the free base.     

Neutral Proteinases I and II of Aspergillus sojae: Some Physicochemical Properties and Amino Acid Composition

Some physicochemical properties of neutral proteinases I and II, zinc-containing metalloenzymes, from Aspergillus sojae were investigated.

Neutral proteinase I: The enzyme protein had a sedimentation coefficient of 3.90S, an intrinsic viscosity of 0.0315 dl/g, and a partial specific volume, calculated from the amino acid and carbonhydrate composition, of 0.715 cm³/g. The molecular weight was 42,200 from the Yphantis’ procedure, and was 42,500 from the calculation according to the Scheraga-Mandel-kern’s formula. The integral numbers of amino acid residues per molecule calculated on the basis of 42,200 as molecular weight were as follows; Lys₁₆, His₆, Arg₁₃, Trp₈, Asp₅₆, Thr₂₅, Ser₂₃, Glu₃₁, Pro₁₈, Gly₄₀, Ala₃₃, l/2Cys₄, Val₁₁, Met₆, Ile₁₅, Leu₂₅, Tyr₂₀, Р?е₁₀, (amide-ammonia)₂₉, in addition to mannose₆, galactose₁, hexosamine₃.

Neutral proteinase II: The enzyme protein had a sedimentation coefficient of 2.32S, an intrinsic viscosity of 0.0270 dl/g, and a calculated partial specific volume of 0.714 cm³/g. The molecular weight was 16,800 from the Yphantis’ procedure, and was 18,000 from the sedimentation and intrinsic viscosity. The following amino acid compositions was calculated on the basis of 16,800 as molecular weight; Lys₈, His₃, Arg₃, Asp₁₉, Thr₁₇, Ser₁₁, GIu₂₃, Pro₅, Gly₉, Ala₂₄, l/2Cys₄, Val₅, Ile₃, Leu₁₃, Tyr₁₀, Phe₃, (amide-ammonia)₁₅. In the enzyme preparation, neither methionine nor tryptophan was detected and carbohydrate was also absent.

In both neutral proteinases I and II, no free SH group was detected by the PCMB-titration in the presence of 8 M urea.     

Functional Differences of Invariant and Highly Conserved Residues in the Extracellular Domain of the Glycoprotein Hormone Receptors

Multiple interactions exist between human follicle-stimulating hormone (FSH) and the N-terminal hormone-binding fragment of the human FSH receptor (FSHR) extracellular domain (ECD). Binding of the other human glycoprotein hormones to their cognate human receptors (luteinizing hormone receptor (LHR) and thyroid-stimulating hormone receptor (TSHR)) was expected to be similar. This study focuses on amino acid residues in β-strands 2 (Lys⁷⁴), 4 (Tyr¹²⁴, Asn¹²⁹, and Thr¹³⁰), and 5 (Asp¹⁵⁰ and Asp¹⁵³) of the FSHR ECD identified in the human FSH·FSHR ECD crystal structure as contact sites with the common glycoprotein hormone α-subunit, and on noncontact residues in β-strands 2 (Ser⁷⁸) and 8 (Asp²²⁴ and Ser²²⁶) as controls. These nine residues are either invariant or highly conserved in LHR and TSHR. Mutagenesis and functional characterization of these residues in all three human receptors allowed an assessment of their contribution to binding and receptor activation. Surprisingly, the six reported α-subunit contact residues of the FSHR ECD could be replaced without significant loss of FSH binding, while cAMP signaling potency was diminished significantly with several replacements. Comparative studies of the homologous residues in LHR and TSHR revealed both similarities and differences. The results for FSH/FSHR were analyzed on the basis of the crystal structure of the FSH·FSHR ECD complex, and comparative modeling was used to generate structures for domains, proteins, and complexes for which no structures were available. Although structural information of hormone-receptor interaction allowed the identification of hormone-receptor contact sites, functional analysis of each contact site was necessary to assess its contribution to hormone binding and receptor activation.     

Synthesis and assay of structural intermediates of crustacean pigment-dispersing hormones (α and β-PDH)

Summary

The two characterized crustacean pigment-dispersing hormones (α-PDH; β-PDH) are octadecapeptides which differ in primary structure at six positions. Assays for melanophore pigment-dispersing activity showed β-PDH to be 21-fold more potent than α-PDH. In an effort to explain the difference in potencies between the two PDHs, we synthesized and purified six analogs of α-PDH (Leu^4?, Leu^11?, Lys^13?, Asn^16?, Asp^17?, and Glu³, Leu^4? α-PDH) in which the amino acid residues of α-PDH were substituted with those of β-PDH. Four analogs (Leu^11?, Lys^13?, Asn^16?, and Asp^17? α-PDH) possessed melanophore-dispersing activity equivalent to α-PDH. Leu^4? α-PDH and Glu³, Leu^4? α-PDH were 2.4? and 4-fold more potent than α-PDH, respectively. Glu^3-α-PDH was 3.3-fold more potent than α-PDH (Jorenby et al., 1987). These results suggest that the 21-fold increase in activity of β-PDH over α-PDH is due to an interactive effect of two or more substitutions rather than from the product of the effects brought about by individual substitutions.     

Cytokinin-Active Ribonucleosides in Phaseolus RNA: II. DISTRIBUTION IN tRNA SPECIES FROM ETIOLATED P. VULGARIS L. SEEDLINGS

The distribution of cytokinin-active ribonucleosides in tRNA species from etiolated Phaseolus vulgaris L. seedlings has been examined. Phaseolus tRNA was fractionated by benzoylated diethylaminoethyl-cellulose and RPC-5 chromatography, and the distribution of cytokinin activity was compared with the distribution of tRNA species expected to correspond to codons beginning with U. Phaseolus tRNA^Cys, tRNA^Trp, tRNA^Tyr, a major peak of tRNA^Phe, and a large fraction of tRNA^Leu were devoid of cytokinin activity in the tobacco bioassay. Cytokinin activity was associated with all fractions containing tRNA^Ser species and with minor tRNA^Leu species. In addition, several anomalous peaks of cytokinin activity that could not be directly attributed to U group tRNA species were detected.