Leucyl Residues Research Articles

Polarity and Charge as Determinants for Translocase Requirement for Membrane Protein Insertion

Membrane protein biogenesis in bacteria is a well-studied but poorly understood area in the field of membrane proteins. In bacteria, the Sec translocon and the YidC insertase promote the membrane insertion process. Both proteins are essential, and universally conserved, and together insert about 95% of the proteins in E.coli. Membrane insertion of SecYEG-dependent proteins likely occurs at the lateral gate of SecY, the channel-forming subunit of the bacterial Sec translocon. On the other hand, YidC catalyzes insertion by possessing a hydrophilic groove, which can accommodate the hydrophilic region of the substrate that inserts across the membrane. Recently, Soman et al (2014) have reported that increasing the charge of the translocated region of procoat can route substrates from the YidC pathway into the Sec pathway. Here, we have further tested this polarity/charge hypothesis by showing that the major coat protein of bacteriophage M13 can become increasingly YidC/Sec dependent by making the periplasmic loop highly polar in the absence of charged residues. We also show that adding hydrophobic amino acids to highly polar or charged loop can decrease the Sec-dependence of the otherwise strictly Sec-dependent substrates. Additionally we demonstrate that increasing the driving force of insertion by adding four leucyl residues to the transmembrane segment leads to translocation of a highly charged region that was not inserted by native TM segment. Finally, we show that the length of procoat loop is a determinant for Sec dependent insertion. The combined results support the polarity/charge hypothesis and is consistent with the notion that membrane insertion occurs at the interface of SecYEG and YidC.

L-Leucylglycylglycine

In the title compound, C10H19N3O4, the N- and C-termini are protonated and ionized, respectively, and the mol­ecule forms a zwitterion. The main chain is in a folded form. In the crystal, the N-terminal –NH3 + group hydrogen bonds to three C-terminal –COO groups and one carbonyl O atom, forming a three-dimensional network. In addition, an N—H⋯O hydrogen bond between the amide groups of the middle glycine residue and a C—H⋯O inter­action continue along the a-axis direction. The side chains of the leucyl residues form a hydro­phobic region along the a axis.

L-Leucylglycine 0.67-hydrate and [(4S)-2,2-dimethyl-4-(2-methylpropyl)-5-oxoimidazolidin-3-ium-1-yl]acetate

Crystals of L-leucylglycine (L-Leu-Gly) 0.67-hydrate, C(8)H(16)N(2)O(3)·0.67H(2)O, (I), were obtained from an aqueous solution. There are three symmetrically independent dipeptide zwitterionic molecules in (I) and they are parallel to one another. The hydrogen-bond network composed of carboxylate and amino groups and water molecules extends parallel to the ab plane. Hydrophilic regions composed of main chains and hydrophobic regions composed of the isobutyl groups of the leucyl residues are aligned alternately along the c axis. An imidazolidinone derivative was obtained from L-Leu-Gly and acetone, viz. [(4S)-2,2-dimethyl-4-(2-methylpropyl)-5-oxoimidazolidin-3-ium-1-yl]acetate, C(11)H(20)N(2)O(3), (II), and was crystallized from a methanol-acetone solution of L-Leu-Gly. The unit-cell parameters coincide with those reported previously for L-Leu-Gly dihydrate revealing that the previously reported values should be assigned to the structure of (II). One of the imidazolidine N atoms is protonated and the ring is nearly planar, except for the protonated N atom. Protonated N atoms and deprotonated carboxy groups of neighbouring molecules form hydrogen-bonded chains. The ring carbonyl group is not involved in hydrogen bonding.

Identification and Isolation of an Azoreductase fromEnterococcus faecium

Azo dyes are commonly used in many commercial industries. Some of the azo dyes can produce carcinogenic compounds after being metabolized by azoreductase. Several human intestinal microbiota possess azoreductase activity which plays an important role in the toxicity and mutagenicity of these azo dye compounds. The acpD gene product (AzoEf1) responsible for the azoreductase activity of Enterococcus faecium, an intestinal bacterium, was heterologously expressed, purified and characterized. The protein sequence shares 67% identity with the azoreductase from Enterococcus faecalis, AzoA. Although AzoEf1 possesses many commonalities with AzoA, there are differences in coenzyme preference, residues associated with FMN binding, substrate specificity, and specific activity. AzoEf1 utilized both NADH and NADPH for the reduction of azo dyes, and it contains a leucyl residue at position 104 and threonyl residue at position 19 which differ from AzoA at the active site. Its specific activity was 5095 M/min/mg and its catalytic efficiency for Methyl red reduction was lower than AzoA.

A New Optical Parametric Amplifier Based on Lithium Thioindate Used for Sum Frequency Generation Vibrational Spectroscopic Studies of the Amide I Mode of an Interfacial Model Peptide

We describe a new optical parametric amplifier (OPA) that employs lithium thioindate, LiInS2 (LIS), to create tunable infrared light between 1500 cm(-1) and 2000 cm(-1). The OPA based on LIS described within provides intense infrared light with a good beam profile relative to similar OPAs built on silver gallium sulfide, AgGaS2 (AGS), or silver gallium selenide, AgGaSe2 (AGSe). We have used the new LIS OPA to perform surface-specific sum frequency generation (SFG) vibrational spectroscopy of the amide I vibrational mode of a model peptide at the hydrophobic deuterated polystyrene (d8-PS)-phosphate buffered saline interface. This model polypeptide (which is known to be an alpha-helix in the bulk solution under the high ionic strength conditions employed here) contains hydrophobic leucyl (L) residues and hydrophilic lysyl (K) residues, with sequence Ac-LKKLLKLLKKLLKL-NH2. The amide I mode at the d8-PS-buffer interface was found to be centered around 1655 cm(-1). This can be interpreted as the peptide having maintained its alpha-helical structure when adsorbed on the hydrophobic surface, although other interpretations are discussed.

The ability of lower peptides to form co-crystals: inclusion compounds of Leu-Leu-Leu tripeptide with pyridine and picolines

Co-crystals of L-leucyl-L-leucyl-L-leucine tripeptide (LLL) with pyridine (Py), α-picoline (2-MePy), β-picoline (3-MePy) and γ-picoline (4-MePy) have been prepared and studied. Single-crystal XRD analyses reveal the following formulas: LLL*(Py) (1), LLL*(2-MePy)*0.12(H2O) (2), LLL*(3-MePy) (3), and LLL*(4-MePy)*0.14(H2O) (4). The compounds may be described as inclusion compounds of LLL (host), pyridine or picolines (guest) and water (secondary guest). The host LLL molecules are arranged in anti-parallel β-sheet layers and have conformations consistent with this type of peptide organization. The layers are packed by van der Waals forces with an interlayer distance of 11.5–11.8 A. The isobutyl side groups of leucyl residues protrude into the interlayer space and form large channels on one side of the layer. The guest molecules reside in the channels and form H-bonds to the host NH3+ groups. The crystal structures have two host molecules in the asymmetric unit, which in most cases, exhibit rarely observed whole-molecule disorder. Three of the compounds invariably form as twinned crystals. TGA measurements of bulk products reveal the following compositions: LLL*1.04(Py) (1), LLL*0.99(2-MePy) (2), LLL*0.96(3-MePy) (3), and LLL*0.92(4-MePy) (4), in good agreement with the XRD results. The character of thermal dissociation implies the preservation of the host layered structure as guest molecules diffuse out of the channels. Compound 2 dissociates with the formation of an intermediate with the stoichiometry of LLL*0.5(2-MePy).

Carbon-13 NMR method for the detection of correlated hydrogen exchange at adjacent backbone peptide amides and its application to hydrogen exchange in five antiparallel beta strands within the hydrophobic core of Streptomyces subtilisin inhibitor (SSI).

A novel method for monitoring proton-deuteron (H/D) exchange at backbone amides is based on the observation of H/D isotope effects on the (13)C NMR signals from peptide carbonyls. The line shape of the carbonyl (13)C(i) signal is influenced by differential H/D occupancy at the two adjacent amides: the H(N)(i)(+1) (beta site) and the H(N)(i) (gamma site). At a carbon frequency of 75.4 MHz, the H --> D isotope shifts on the (13)C signal are about 5-7 Hz for exchange at the beta site and 2 Hz or less for exchange at the gamma site. Because the effects at the two sites are additive, the time dependence of the line shape of a particular carbonyl resonance can report not only the exchange rates at the individual sites but also the level of dual exchange. Therefore, the data can be analyzed to determine the rate (k(c)) and degree of correlated exchange (X(betagamma)) at the two sites. We have applied this approach to the investigation of the pH dependence of hydrogen exchange at several adjacent residues in Streptomyces subtilisin inhibitor (SSI). Two selectively labeled SSI proteins were produced: one with selective (13)C' labeling at all valyl residues and one with selective (13)C' labeling at all leucyl residues. This permitted the direct observation by one-dimensional (13)C NMR of selected carbonyl signals from residues with slowly exchanging amides at the i and i + 1 positions. The residues investigated were located in an alpha helix and in a five-stranded antiparallel beta sheet. Samples of the two labeled proteins were prepared at various pH values, and (13)C NMR spectra were collected at 50 degrees C prior to and at various times after transferring the sample from H(2)O to (2)H(2)O. Most of the slowly exchanging amides studied were intramolecular hydrogen-bond donors. In agreement with prior studies, the results indicated that the exchange rates of the amide hydrogens in proteins are governed not only by hydrogen bonding but also by other factors. For example, the amide hydrogen of Thr34 exchanges rapidly even though it is an intramolecular hydrogen-bond donor. Over nearly the whole pH range studied, the apparent rates of uncorrelated exchange (k(beta) and k(gamma)) were proportional to [OH(-)] and the apparent rates of correlated exchange at two adjacent sites (k(c)) were roughly proportional to [OH(-)](2). This enabled us to extract the pH-independent exchange rates (k(beta) degrees , k(gamma) degrees , and k(c) degrees ). In all cases in which correlated exchange could be measured, the observed sigmoidal pH dependence of X(betagamma) could be replicated roughly from the derived pH-independent rates.

Synthesis and dopamine receptor modulating activity of 3-substituted gamma-lactam peptidomimetics of L-prolyl-L-leucyl-glycinamide.

gamma-Lactam peptidomimetic 2 of Pro-Leu-Gly-NH(2) (PLG) was substituted at the 3-position with isobutyl, butyl, and benzyl moieties to give the PLG peptidomimetics 3-5, respectively. These compounds were synthesized to test the hypothesis that attaching a hydrophobic moiety to the lactam ring to mimic the isobutyl side chain of the leucyl residue of PLG would increase the dopamine receptor modulating activity of such peptidomimetics. These peptidomimetics were tested for their ability to enhance the binding of [(3)H]-N-propylnorapomorphine to dopamine receptors isolated from bovine striatal membranes. The rank order of effectiveness of the 3-substituent was benzyl > n-butyl > isobutyl > H.

A Calix[6]arene Dimer Linked Through Amino Acid Hydrogen Bond Interactions

A calix[6]arene containing three leucine residues at the lower rim dimerizes in apolar solvents with the formation of up to eighteen hydrogen bonds, giving rise to a self-assembled molecular capsule. The dimerization in chloroform was conformed by NMR spectroscopy and gel permeation chromatography, as well as by molecular mechanics and dynamics calculations. The highly flexible structure is unable to encapsulate guests of complementary size, probably because six methoxy groups and one of the leucyl residues permanently occupy the cavity.

Influence of N-terminal residue stereochemistry on the prolyl amide geometry and the conformation of 5-tert-butylproline type VI beta-turn mimics.

The effects of N-terminal amino acid stereochemistry on prolyl amide geometry and peptide turn conformation were investigated by coupling both L- and D-amino acids to (2S, 5R)-5-tert-butylproline and L-proline to generate, respectively, N-(acetyl)dipeptide N'-methylamides 1 and 2. Prolyl amide cis- and trans-isomers were, respectively, favored for peptides 1 and 2 as observed by proton NMR spectroscopy in water, DMSO and chloroform. The influence of solvent composition on amide proton chemical shift indicated an intramolecular hydrogen bond between the N'-methylamide proton and the acetamide carbonyl for the major conformer of dipeptides (S)-1, that became less favorable in (R)-1 and 2. The coupling constant (3J(NH,alpha)) values for the cis-isomer of (R)-1 indicated a phi2 dihedral angle value characteristic of a type VIb beta-turn conformation in solution. X-ray crystallographic analysis of N-acetyl-D-leucyl-5-tert-butylproline N'-methylamide (R)-lb showed the prolyl residue in a type VIb beta-turn geometry possessing an amide cis-isomer and psi3-dihedral angle having a value of 157 degrees, which precluded an intramolecular hydrogen bond. Intermolecular hydrogen bonding between the leucyl residues of two turn structures within the unit cell positioned the N-terminal residue in a geometry where their phi2 and psi2 dihedral angle values were not characteristic of an ideal type VIb turn. The circular dichroism spectra of tert-butylprolyl peptides (S)- and (R)-1b were found not to be influenced by changes in solvent composition from water to acetonitrile. The type B spectrum exhibited by (S)-1b has been previously assigned to a type VIa beta-turn conformation [Halab L, Lubell WD. J. Org. Chem. 1999; 64: 3312-3321]. The type C spectrum exhibited by the (R)-lb has previously been associated with type II' beta-turn and alpha-helical conformations in solution and appears now to be also characteristic for a type VIb geometry.

Attenuation of 19-9 Antigen Secretion in Human Colorectal Carcinoma Cell Cultures by Transfection with cDNA Encoding Novel ADP-Ribosylation Factor-like Proteins

We have used cDNAs coding for novel ADP-ribosylation factor-like molecules (ARL184 and ARL184Δ) to alter 19-9 antigen glycoprotein secretion in cultured human colorectal carcinoma cells SW1116 by transfection and cloning. This ARL contains a lipophilic N-terminal with an isoleucyl and 3 leucyl residues, 4 functioning consensus sequence GTP binding sites, and 184 total aminoacyl residues. An ARL cDNA was also constructed deleting the codon for the N-terminal glycyl moiety. The resulting cell clones were shown by Northern blots to overexpress ARL mRNA. Electron microscopy–immunocytochemistry also indicated the overexpression of ARL granules subcellularly. Secretion of the tumor-associated 19-9 antigen into apical medium was decreased 3- to 5-fold and the secretion of TCA/PTA precipitable3H-labeled glycoprotein was decreased by 34% in clone SW1116(ARL184)Δ. Western blot analyses of cell homogenates and media were in agreement with the secretion assays and showed a diminution of 170–200 kDa, 19-9, antigenicity in transfected cells and their media. Apical secretion of 19-9 antigen was diminished 14-fold in cells, SW1116 (ARL184)α, transfected with the complete ARL cDNA sequence, suggesting that the glycyl moiety may be required for maximal abatement. However, incorporation of label from [3H]myristate into 22-kDa bands of NP-40 extracts and ARL-antigenic molecules of parent cells was 3-fold greater than that in samples from the two transfectants; thus the transfected cells may not myristylate the overexpressed ARL efficiently. Notwithstanding the N-terminal glycyl moiety undergoing some other modification, we conclude that overexpression of this ARL is sufficient to generate a 19-9-deficient phenotype. These ARLs may eventually disrupt terminal oligosaccharide glycosylation, resulting in an apparent diminished exocytosis of 19-9 glycoprotein carriers by transfected and cloned cells.

Separate Agonist and Peptide Antagonist Binding Sites of the Oxytocin Receptor Defined by Their Transfer into the V2 Vasopressin Receptor

The neurohypophyseal nonapeptide oxytocin (OT) is the main hormone responsible for the initiation of labor; uterus contraction can be enhanced by application of oxytocin or suppressed by oxytocin antagonists. By transfer of domains from the G protein-coupled OT receptor into the related V2 vasopressin receptor, chimeric "gain in function" V2/OT receptors were produced that were able to bind either OT receptor agonists or a competitive peptide antagonist with high affinity. The binding site for the OT antagonist d(CH2)5[Tyr(Me)2,Thr4,Orn8,Tyr9]vasotocin was found to be formed by transmembrane helices 1, 2, and 7 with a major contribution to binding affinity by the upper part of helix 7. These transmembrane receptor regions could be excluded from participating in OT binding. For agonist binding and selectivity the first three extracellular receptor domains were most important. The interaction of the N-terminal domain and of the first extracellular loop of the OT receptor with the linear C-terminal tripeptidic part of oxytocin was demonstrated. Furthermore, the second extracellular loop of the OT receptor could be identified to interact with the cyclic hormone part. These three domains contribute to OT binding by synergistic interaction with oxytocin but not with the competitive antagonist. Our results provide evidence for the existence of separate domains and different conformations of a peptide hormone receptor involved in binding and selectivity for agonists and peptide antagonists.

Conformation and activity of +H2-Pro-Leu-Gly-NH2 and its analogues modified at the leucyl residue.

In order to investigate the relation between the conformation and the ability to modulate dopamine receptors, conformational free-energy calculations using an empirical potential (ECEPP/3) and hydration shell model were carried out on the tripeptide +H2-Pro-Leu-Gly-NH2 (PLG) and its analogues in which the Leu residue is replaced with Phe, Ahx (L-2-aminohexanoic acid), Ile, Abu and Ala residues in the unhydrated and hydrated states. PLG and two tripeptides possessing Phe and Ahx residues show dopamine receptor modulating activity, while the other tripeptides do not. Irrespective of activities, PLG and its analogues are found to have the high probabilities to form beta-bends of types II and I in the unhydrated state. However, in the hydrated state, the beta-bend probabilities of the PLG analogues decrease significantly compared with those in the unhydrated state. These results indicate that the beta-bend structure is a necessary factor for the PLG analogues to be active, but not a sufficient factor, and that the interactions of water molecules with the backbone may force the tripeptides to be more distorted or extended. The size and the orientation of the hydrophobic moiety of the second residue seem to be of consequence in eliciting the activity of the tripeptides.

Observation of a sterically unfavorable side‐chain conformation in a leucyl residue: Crystal and molecular structure of L‐leucyl–L‐leucine · DMSO solvate

The crystal structure of a dipeptide L-leucyl-L-leucine (C12H24N2O3) has been determined. The crystals are monoclinic, space group P2(1), with a = 5.434(4) A, b = 15.712(7) A, c = 11.275(2) A, beta = 100.41(1) degrees, and Z = 2. The crystals contain one molecule of dimethyl sulfoxide (DMSO) as solvent of crystallization for each dipeptide molecule. The structure has been solved by direct methods and refined to a final R index of 0.059 for 920 reflections (sin theta/lambda < or = 0.60 A-1) with I > or = 2 sigma (I). The trans peptide unit shows substantial degree of non-planarity (delta omega = 14 degrees). The peptide backbone adopts an extended conformation with torsion angles of psi 1 = 138(1) degrees, omega 1 = 166(1) degrees, phi 2 = -149.3(7) degrees, psi 21 = 164.2(7) degrees, and psi 22 = -15(1) degrees. For the first leucyl residue, the side-chain conformation is specified by the torsion angles 1 chi 1 = 176.7(7) degrees, 1 chi 21 = 62(1) degrees, 1 chi 22 = -177.4(8) degrees; the second leucyl residue adopts a sterically unfavorable conformation with 2 chi 1 = 61(1) degrees, 2 chi 21 = 97(1) degrees, and 2 chi 22 = -151(1) degrees. The packing involves head-to-tail interaction of peptide molecules and segregation of polar and nonpolar regions. The DMSO molecule is strongly hydrogen bonded to the terminal NH3+ group.

The NMR Structure of the Pulmonary Surfactant-Associated Polypeptide SP-C in an Apolar Solvent Contains a Valyl-Rich .alpha.-Helix

The nuclear magnetic resonance (NMR) structure of the pulmonary surfactant-associated lipoplypeptide C (SP-C) was determined in a mixed solvent of C2H3Cl/C2H3OH/ 1 M HCl 32:64:5 (v/v). Sequence-specific 1H NMR assignments and the collection of conformational constraints were achieved with two-dimensional 1H NMR, and the structure was calculated with the distance geometry program DIANA. The root mean square deviations for the well-defined polypeptide segment of residues 9-34 calculated for the 20 best energy-minimized DIANA conformers relative to their mean are 0.5 and 1.3 A for the polypeptide backbone atoms N, C alpha, and C', and for all heavy atoms, respectively. The 35-residue polypeptide chain of SP-C forms an alpha-helix between positions 9 and 34, which includes two segments of seven and four consecutive valyls that are separated by a single leucyl residue. The N-terminal hexapeptide segment, which includes two palmitoylcysteinyls, is flexibly disordered. The length of the alpha-helix is about 37 A, and the helical segment of residues 13-28, which contains exclusively aliphatic residues with branched side chains, is 23-A long and about 10 A in diameter. The alpha-helix is outstandingly regular, with virtually identical chi 1 angles for all valyl residues. The observation of a helical structure of SP-C was rather unexpected, considering that Val is generally underrepresented in alpha-helices, and it provides intriguing novel insights into the structural basis of SP-C functions as well as into general structural aspects of protein-lipid interactions in biological membranes.

Model peptides to study the effects of P 2 and P 3 substitutions in statine-containing HIV proteinase inhibitors

Through a series of synthetic model peptides, we have examined the structural requirements of the P 2 and P 3 residues in statine-based HIV protease (PR) inhibitors. Results agree with the general observations that, the more bulky the P 3 aromatic hydrophobic side chain, the more potent is the inhibitor. At P 2, an isopropyl side chain is critical in maintaining potency. Three-dimensional modeling demonstrates that the steric bulk of a leucyl residue or the unfavorable energy transfer, from water to enzyme, for a basic amino acid residue at P 2 markedly compromises activity. A naphthylalaninyl-valyl P 3-P 2 substituted analogue inhibits PR with an IC 50 value of 6 nM, and was also effective as an antiviral agent.

Molecular structures of L-Leu-L-Tyr, Gly-D,L-Met.p-toluenesulfonate and L-His-L-Leu

L-Leu-L-Tyr, (I), C15H22N2O4, M(r) = 294.35, crystallizes from MeOH/5% dimethyl sulfoxide in the orthorhombic space group P2(1)2(1)2(1). a = 5.644 (1), b = 12.094 (3), c = 22.548 (4) A, V = 1539.0 (5) A3, Z = 4, Dx = 1.270 g cm-3, Cu K alpha, lambda = 1.54184 A, mu = 7.228 cm-1, F(000) = 632, T = 173 K, final R (on F) = 0.033 for 1347 observations with I > or = 2 sigma (I). (I) crystallizes as a zwitterion with the N-terminus protonated and the C-terminus ionized. The peptide backbone adopts a distorted trans antiparallel beta pleated-sheet conformation, with principal torsion angles psi 1 = 163.7 (2), omega 1 = 158.7 (2), phi 2 = -110.9 (3) and psi 2 = 141.4 (2)degrees. The leucyl residue is in the g-(tg-) conformation while the tyrosyl residue adopts the g- conformation, with the phenol ring twisted from the low-energy perpendicular position. Gly-D,L-Met.p-toluenesulfonate, (II), C7H15N2O3S+.C7H7O3S-, M(r) = 378.47, crystallizes from MeOH/EtOAc in the orthorhombic space group Pbca. a = 33.642 (4), b = 15.951 (1), c = 6.785 (1) A, V = 3641.0 (4) A3, Z = 8, Dx = 1.381 g cm-3, Cu K alpha, lambda = 1.54184 A, mu = 28.865 cm-1, F(000) = 1600, T = 223 K, final R (on F) = 0.055 for 1669 observations with I > or = 3 sigma (I). Gly-D,L-Met exists as a cation with the N- and C-termini protonated, the p-toluenesulfonate being the counterion.(ABSTRACT TRUNCATED AT 250 WORDS)

Electronic and stereochemical characterizations of the photoinduced intermediates of nitrosyl complexes of metal (S = 5/2)-substituted hemoproteins trapped at low temperature.

Low temperature photolysis of nitric oxide from the nitrosyl complexes of ferric myoglobin (NO-Fe(III)Mb) and manganese(II)-porphyrin-substituted myoglobin (NO-Mn(II)Mb) was examined by electron paramagnetic resonance (EPR) spectroscopy in order to elucidate the electronic and structural natures of the photoinduced intermediates of these hemoprotein-ligand complexes trapped at low temperature. The photoproduct of NO-Fe(III)Mb at 5 K exhibited entirely new X-band EPR absorptions in the magnetic field strength from 0 to 0.4 tesla. The widespread absorption together with distinct, sharp zero-field absorption was consistently observed in the photoproduct of the isoelectronic NO-Mn(II)Mb. These novel ERP signals indicate a spin-coupled pair with an effective spin of S = 2 between the high spin metal center (S = 5/2) and the photodissociated NO (S = 1/2) trapped adjacent to the metal center. On the other hand, the photolyzed form of nitrosyl complexes of Fe(III)- and Mn(II)-Glycera hemoglobins, in which the distal histidine of Mb is replaced by a leucyl residue, exhibited somewhat broader EPR absorptions similar to those of the corresponding native Fe(III)- or unliganded Mn(II)-Glycera hemoglobins, respectively, indicating that the photodissociated NO molecule moved farther away from the metal center in the heme pocket. These observations show the importance of the interaction of the distal residue with the ligand in determining the nature of the photolyzed states.

Role of recurrent hydrophobic residues in catalysis of helix formation by T cell-presented peptides in the presence of lipid vesicles.

We tested the hypothesis that the recurrence of hydrophobic amino acids in a polypeptide at positions falling in an axial, hydrophobic strip if the sequence were coiled as an alpha helix, can lead to helical nucleation on a hydrophobic surface. The hydrophobic surface could anchor such residues, whereas the peptide sequence grows in a helical configuration that is stabilized by hydrogen bonds among carbonyl and amido NH groups along the peptidyl backbone of the helix, and by other intercycle interactions among amino acid side chains. Such bound, helical structures might protect peptides from proteases and/or facilitate transport to a MHC-containing compartment and thus be reflected in the selection of T cell-presented segments. Helical structure in a series of HPLC-purified peptides was estimated from circular dichroism measurements in: 1) 0.01 M phosphate buffer, pH 7.0, 2) that buffer with 45% trifluoroethanol (TFE), and 3) that buffer with di-O-hexadecyl phosphatidylcholine vesicles. By decreasing the dielectric constant of the buffer, TFE enhances intrapeptide interactions generally, whereas the lipid vesicles only provide a surface for hydrophobic interactions. The peptides varied in their strip-of-helix hydrophobicity indices (SOHHI; the mean Kyte-Doolittle hydrophobicities of residues in an axial strip of an alpha helix) and in proline content. Structural order for peptides with helical circular dichroism spectra was estimated as percentage helicity from circular dichroism theta 222 nm values and peptide concentration. A prototypic alpha helical peptide with three cycles plus two amino acids and an axial hydrophobic strip of four leucyl residues (SOHHI = 3.8) was disordered in phosphate buffer, 58% helical in that buffer with 48% TFE, and 36% helical in that buffer with vesicles. Percentage helicity in the presence of vesicles of the subset of peptides without proline followed their SOHHI values. Peptides with multiple prolyl residues had circular dichroism spectra with strong signals, but since they did not have altered spectra in the presence of vesicles relative to phosphate buffer alone, the hydrophobic surface of the vesicle did not appear to stabilize those structures.

Carboxylterminal deletion mutants of ribulosebisphosphate carboxylase from Rhodospirillum rubrum

The carboxylterminal octapeptide of ribulosebisphosphate carboxylase from Rhodospirillum rubrum, which lacks small subunits, shows homology to a highly conserved region near the amino terminus of the small subunits of hexadecameric ribulosebisphosphate carboxylases, which are composed of large and small subunits. Truncations of the R. rubrum enzyme, which partially or completely deleted the region of homology, demonstrated that the region is not an important determinant of the catalytic efficiency of the enzyme. A further truncation, which replaced the carboxylterminal 19 amino acid residues with a single terminal leucyl residue, yielded a Rubisco whose substrate-saturated catalytic rate resembled that of the wild-type enzyme but which had weaker affinities for ribulose-P 2 and CO 2.