Solution NMR structures provide first structural coverage of the large protein domain family PF08369 and complementary structural coverage of dark operative protochlorophyllide oxidoreductase complexes

High-quality NMR structures of the C-terminal domain comprising residues 484–537 of the 537-residue protein Bacterial chlorophyll subunit B (BchB) from Chlorobium tepidum and residues 9–61 of 61-residue Asr4154 from Nostoc sp. (strain PCC 7120) exhibit a mixed α/β fold comprised of three α-helices and a small β-sheet packed against second α-helix. These two proteins share 29 % sequence similarity and their structures are globally quite similar. The structures of BchB(484–537) and Asr4154(9–61) are the first representative structures for the large protein family (Pfam) PF08369, a family of unknown function currently containing 610 members in bacteria and eukaryotes. Furthermore, BchB(484–537) complements the structural coverage of the dark-operating protochlorophyllide oxidoreductase.     

Development of the multiple sequence approximation within the AGADIR model of α-helix formation: Comparison with Zimm-Bragg and Lifson-Roig formalisms

In this work we present the development of the multiple sequence approximation (AGADIRms) and the standard one-sequence approximation (AGADIRIs) within the framework of AGADIR's α-helix formation model. The extensive comparison between these new formulations and the original one [AGADIR; v. Muñoz and L. Serrano (1994),Nat. Struct. Biol., Vol. 1, pp. 399–409] indicates that the standard one-sequence approximation is virtually identical to the multiple sequence approximation, while the previously used residue partition function approximation [Muñoz and Serrano (1994); (1995), J. Mol. Biol., Vol. 245, pp. 275–296] is less precise. The calculations of the average helical content performed with AGADIR are precise for peptides of less than 30 residues and progressively diverge from the multiple sequence formulation for longer peptides. The helicity distribution of heteropolypeptides with less than 50% average helical content is also well described, while those of quasi-homopolymers with high helical content tend to be flattened. These inaccuracies lead to an underestimation of 0.017 kcal/mol for the mean-residue enthalpic contribution in AGADIR, as compared to AGADIRms and AGADIRIs. The other energy contributions to α-helix stability are not affected by the original statistical approximation. We also discuss the particularities of the model for α-helix formation utilized in AGADIR and compare it with the classical Zimm-Bragg and Lifson-Roig theories. Moreover, we develop the mathematical relationships between the basic AGADIR energy contributions and helix nucleation and elongation, which permit the quantitative comparison between formalisms. Remarkably, the comparison between AGADIRms and the Lifson-Roig formalism shows that, despite the differences on treating helix/coil cooperativity, both theories give virtually identical results when an equivalent set of parameters is used. This indicates that the helix/coil transition is a solid theory independent of the particularities of the model for α-helix formation. © 1997 John Wiley & Sons, Inc. Biopoly 41: 495–509, 1997.     

The measurement of transmembrane helices by the deconvolution of CD spectra of membrane proteins: A review

The interpretation of the CD spectra of proteins to date requires additional secondary structural information of the proteins to be analyzed, such as x-ray or nmr data. Therefore, these methods are inappropriate for a CD data base whose secondary structures are unknown, as in the case of the membrane proteins. The Convex Constraint Analysis algorithm [A. Perczel, M. Hollósi, G. Tusnády, and G. D. Fasman (1991) Protein Engineering, Vol. 4, 669–679], on the other hand, operates only on a collection of spectral data to extract the common spectral components with their spectral weights. The linear combinations of these derived “pure” CD curves can reconstruct the original data set with great accuracy. For a membrane protein data set, the five-component spectra so obtained from the deconvolution consisted of two different types of α-helices (the α-helix in the soluble domain and the α_T-helix, for the transmembrane α-helix), a β-pleated sheet, a class C-like spectrum related to β-turns, and a spectrum correlated with the unordered conformation. The deconvoluted CD spectrum for the α_T-helix was characterized by a positive red-shifted band in the range 195–200 nm (+95,000 deg cm² dmol^?l), with the intensity of the negative band at 208 nm being slightly less negative than that of the 222 nm band (?50,000 and ?60,000 deg cm² dmol^?1, respectively) in comparison with the regular α-helix, with a positive band at 190 nm and two negative bands at 208 and 222 nm with magnitudes of + 70,000, ?30,000, and ?30,000 deg cm² dmol^?1, respectively. © 1994 John Wiley & Sons, Inc.     

Secondary structure perturbations in salt-induced protein precipitates

The secondary structure implications of precipitation induced by a chaotropic salt, KSCN, and a structure stabilizing salt, Na₂SO₄, were studied for twelve different proteins. α-helix and β-sheet content of precipitate and native structures were estimated from the analysis of amide I band Raman spectra. A statistical analysis of the estimated perturbations in the secondary structure contents indicated that the most significant event is the formation of β-sheet structures with a concomitant loss of α-helix on precipitation with KSCN. The conformational changes for each protein were also analyzed with respect to elements of primary, secondary and tertiary structure existing in the native protein; primary structure was quantified by the fractions of hydrophobic and charged amino acids, secondary structure by x-ray estimates of α-helix and β-sheet contents of native proteins and tertiary structure by the dipole moment and solvent-accessible surface area. For the KSCN precipitates, factors affecting β-sheet content included the fraction of charged amino acids in the primary sequence and the surface area. Changes in α-helix content were influenced by the initial helical content and the dipole moment. The enhanced β-sheet contents of precipitates observed in this work parallel protein structural changes occurring in other aggregative phenomena.     

Insights into thermal resistance of proteins from the intrinsic stability of their α-helices

To investigate the role of α helices in protein thermostability, we compared energy characteristics of α helices from thermophilic and mesophilic proteins belonging to four protein families of known three-dimensional structure, for at least one member of each family. The changes in intrinsic free energy of α-helix formation were estimated using the statistical mechanical theory for describing helix/coil transitions in peptide helices [Munoz, V., Serrano, L. Nature Struc. Biol. 1:399–409, 1994; Munoz, V., Serrano, L. J. Mol. Biol. 245:275–296, 1995; Munoz, V., Serrano, L. J. Mol. Biol. 245:297–308, 1995]. Based on known sequences of mesophilic and thermophilic RecA proteins we found that (1) a high stability of α helices is necessary but is not a sufficient condition for thermostability of RecA proteins, (2) the total helix stability, rather than that of individual helices, is the factor determining protein thermostability, and (3) two facets of intrahelical interactions, the intrinsic helical propensities of amino acids and the side chain–side chain interactions, are the main contributors to protein thermostability. Similar analysis applied to families of L-lactate dehydrogenases, seryl-tRNA synthetases, and aspartate amino transferases led us to conclude that an enhanced total stability of α helices is a general feature of many thermophilic proteins. The magnitude of the observed decrease in intrinsic free energy on α-helix formation of several thermoresistant proteins was found to be sufficient to explain the experimentally determined increase of their thermostability. Free energies of intrahelical interactions of different RecA proteins calculated at three temperatures that are thought to be close to its normal environmental conditions were found to be approximately equal. This indicates that certain flexibility of RecA protein structure is an essential factor for protein function. All RecA proteins analyzed fell into three temperature-dependent classes of similar α-helix stability (ΔG_int = 45.0 ± 2.0 kcal/mol). These classes were consistent with the natural origin of the proteins. Based on the sequences of protein α helices with optimized arrangement of stabilizing interactions, a natural reserve of RecA protein thermoresistance was estimated to be sufficient for conformational stability of the protein at nearly 200°C. Proteins 29:309–320, 1997. © 1997 Wiley-Liss, Inc.     

Structure-activity relationships of a snake cathelicidin-related peptide, BF-15

Cathelicidin-BF15 (BF-15) is a 15-mer peptide derived from Cathelicidin-BF (BF-30), which is found in the venom of the snake Bungarus fasciatus and exhibits broad antimicrobial activity. Since BF-15 retains most part of the antimicrobial activity of BF-30 but has significantly reduced haemolytic activity and a much shorter sequence length (and less cost), it is a particularly attractive template around which to design novel antimicrobial peptides. However, the structure–activity relationship of it is still unknown. We designed and synthesized a series of C-terminal amidated analogs of BF-15 based on its amphipathic α-helix structure. And we characterized their antimicrobial potency and haemolytic activity. We identified the amidated BF-15 (analog B1) with potent antimicrobial activity against several antibiotic-resistant bacteria (MICs between 1 and 64 μg/mL, 2–16-folds higher than BF-30) and much lower haemolytic activity. The subsequent circular dichroism study results showed a typical α-helix pattern of analog B1 and the content of the α-helix structure of it increased significantly comparing with BF-30, which indicates the peptide sequence of BF-15 may provide a major contribution to the α-helix content of the whole BF-30 sequence. The peptide induced chaotic membrane morphology and cell debris as determined by electron microscopy. This suggests that the antimicrobial activity of B1 is based on cytoplasmic membrane permeability. Taken together, our results suggested that peptide B1 should be considered as an excellent candidate for developing therapeutic drugs.     

NMR study of silk I structure of Bombyx mori silk fibroin with 15N- and 13C-NMR chemical shift contour plots

The metastable state silk I structures of Bombyx mori silk fibroin in the solid state were studied on the basis of ¹⁵N- and ¹³C-nmr chemical shifts of Ala, Ser, and Gly residues. The ¹⁵N cross-polarization magic angle spinning (CP/MAS) nmr spectra of the precipitated fraction after chymotrypsin hydrolysis of B. mori silk fibroin with the silk I and silk II forms were measured to determine the ¹⁵N chemical shifts of Gly, Ala, and Ser residues. For comparison, ¹⁵N CP/MAS nmr chemical shifts of Ala were measured for [¹⁵N] Ala Philosamia cynthia ricini silk fibroin with antiparallel β-sheet and α-helix forms. The ¹³C CP/MAS nmr chemical shifts of Ala, Ser, and Gly residues of B. mori silk fibroin with the silk I and silk II forms, as well as ¹³C CP/MAS nmr chemical shifts of Ala residue of P. c. ricini silk fibroin with β-sheet and α-helix forms, are used for the examination of the silk I structure. Both silk I and α-helix peaks are shifted to a lower field than silk II (β-sheet) for the Cα carbons of the Ala residues, while both Cβ carbon peaks are shifted to higher field. However, the silk I peak of the ¹⁵N nucleus of the Ala residue is shifted to lower field than the silk II peak, but the α-helix peak is shifted to high field. Thus, the difference in the structure between the silk I and α-helix is reflected in a different manner between the ¹³C and ¹⁵N chemical shifts. The Cα and Cβ chemical shift contour plots for Ala and Ser residues, and the Cα plot for the Gly residue, were prepared from the Protein Data Bank data obtained for 12 proteins and used for discussing the silk I structure quantitatively from the conformation-dependent chemical shifts. The plots reported by Le and Oldfield for ¹⁵N chemical shifts were also used for the purpose. All these chemical shift data support Fossey's model (Ala: ϕ = −80°, φ = 150°, Gly: ϕ = −150°, φ = 80°) and do not support Lotz and Keith's model (Ala: ϕ = −104.6°, φ = 112.2°, Gly: ϕ = 79.8°, φ = 49.7° or Ala: ϕ = −124.5°, φ = 88.2°, Gly: ϕ = −49.8°, φ = −76.1°) as the silk I structure. © 1997 John Wiley & Sons, Inc.     

ω-Helices in Proteins

A modification of the α-helix, termed the ω-helix, has four residues in one turn of a helix. We searched the ω-helix in proteins by the HELFIT program which determines the helical parameters—pitch, residues per turn, radius, and handedness—and p = rmsd/(N ? 1)^1/2 estimating helical regularity, where “rmsd” is the root mean square deviation from the best fit helix and “N” is helix length. A total of 1,496 regular α-helices 6–9 residues long with p ≤ 0.10 Å were identified from 866 protein chains. The statistical analysis provides a strong evidence that the frequency distribution of helices versus n indicates the bimodality of typical α-helix and ω-helix. Sixty-two right handed ω-helices identified (7.2% of proteins) show non-planarity of the peptide groups. There is amino acid preference of Asp and Cys. These observations and analyses insist that the ω-helices occur really in proteins.     

Calorimetric measurement of enthalpy change in the isothermal helix--coil transition of poly-L-lysine in aqueous solution

The heat ΔH° for converting an uncharged lysine residue from a coil to an α-helical state in poly-L -lysine in 0.1N KCl has been determined calorimetrically to be ?1200 cal/mole at both 15°C and 25°C. Essentially the same value has been obtained for the conversion of an uncharged residue from a coil to a β-pleated sheet state. Titration data provided information about the state of charge of the polymer in the calorimetric experiments, and optical rotatory dispersion data about its conformation. In order to compute ΔH°, the observed Calorimetric heat was corrected for the heat of breaking the sample cell, the heal of dilution of HCl, the heat of neutralization of OH^? ion, and the heat of ionization of the ε-amino group in the random coil. The latter was obtained from similar Calorimetric measurements on poly-D ,L -lysine, which was shown to be a good model for the random coil form of poly-L -lysine. The measured transition heat was ～0.7 cal., which is only 7% of the total heat liberated when a 40 ml solution of 0.25% w/v poly-L -lysine is brought, from pH 11 to pH 7; nevertheless it could be determined with a precision of ±8%. The conformation of poly-L -lysine at pH 11 appears to be completely helical at 15°C, but a mixture of 90% α-helix, 5% β form, and 5% coil at 25°C. Since ΔH° ～ 0 for the α ? β conversion, the polymer behaves like one of 95% α-helix and 5% coil in the calorimeter at 25°C. At neutral pH, poly-L -lysine is an extended coil, like poly-D ,L -lysine.     

Vibrational analysis of peptides,polypeptides, and proteins. XVIII. Conformational sensitivity of the α-helix spectrum: αI- and αII-Poly(L-alanine)

The α_II-helix (? = ?70.47°, ψ = ?35.75°) is a structure having the same n and h as the (standard) α_I-helix (? = ?57.37°, ψ = ?47.49°). Its conformational angles are commonly found in proteins. Using an improved α-helix force field, we have compared the vibrational frequencies of these two structures. Despite the small conformational differences, there are significant predicted differences in frequencies, particularly in the amide A, amide I, and amide II bands, and in the conformation-sensitive region below 900 cm^?1. This analysis indicates that α_II-helices are likely to be present in bacteriorhodopsin [Krimm, S. & Dwivedi, A. M. (1982) Science 216 , 407–408].     

Secondary structure of 11 S globulin in aqueous solution investigated by FT-IR derivative spectroscopy

Secondary structure of 11 S globulin, a major storage protein of soybean seeds, has been investigated in aqueous solution by FT-IR spectroscopy. Conformational changes in the native protein upon thermal and chemical denaturation have been monitored by observing changes in the frequency position and peak intensity of the various bands. The frequency of the Amide I band of the native protein shifts by 4 cm⁻¹ from 1643 cm⁻¹ to 1647 cm⁻¹ when denatured, while the corresponding intensity of the Amide I band compared to the native protein, decreases by 30 and 67%, respectively, for the urea and thermally denatured proteins, indicating gross conformational changes in the secondary structure. Trifluoroethanol, an α-helix promoter shifts the Amide I band from 1643 cm⁻¹ to 1651 cm⁻¹, typical of α-helix, with a corresponding increase in intensity by 14% relative to the native protein. Derivative spectroscopy, allowing resolution of overlapping bands, shows that the native protein mainly consists of ß-sheet, ß-turns and disordered structure with very little α-helix. On denaturation, ß-sheet disappeared almost completely with urea, while this is less so with thermal denaturation.     

Efficacy of α‐Lactalbumin and Milk Protein on Weight Loss and Body Composition During Energy Restriction

Our objective was to examine whether elevated α‐lactalbumin (αlac) protein intake compared to elevated supra sustained milk protein (SSP) and sustained milk protein (SP) intake results into a difference in body weight and body composition over a 6‐month energy‐restriction intervention. Body weight, body composition, resting energy expenditure (REE), satiety and blood‐ and urine‐parameters of 87 subjects (BMI 31 ± 5 kg/m² and fat percentage 40 ± 8%) were assessed before and after daily energy intakes of 100, 33, and 67% for 1, 1, and 2 months respectively (periods 1, 2, and 3), with protein intake from meal replacements and 2 months of 67% with ad libitum protein intake additional to the meal replacements (period 4). The diets resulted in 0.8 ± 0.3 g/kg body mass (BM) for SP and significant higher protein intake (24‐h nitrogen) of 1.2 ± 0.3 and 1.0 ± 0.3 g/kgBM for SSP and αlac (P < 0.05). Body weight and fat percentage was decreased in all groups after 6 months (SP ?7 ± 5 kg and ?5 ± 3%; SSP ?6 ± 3 kg and ?5 ± 3%; αlac ?6 ± 4 kg and ?4 ± 4%, P < 0.001; there was no significant group by time difference). Furthermore, sparing of fat‐free mass (FFM) and preservation of REE in function of FFM during weight loss was not significantly different between the αlac‐group and the SSP‐ and SP‐groups. In conclusion, the efficacy of αlac in reduction of body weight and fat mass (FM), and preservation of FFM does not differ from the efficacy of similar daily intakes of milk protein during 6 months of energy restriction.     

Structure–activity relationships of a snake cathelicidin-related peptide,BF-15

Cathelicidin-BF15 (BF-15) is a 15-mer peptide derived from Cathelicidin-BF (BF-30), which is found in the venom of the snake Bungarus fasciatus and exhibits broad antimicrobial activity. Since BF-15 retains most part of the antimicrobial activity of BF-30 but has significantly reduced haemolytic activity and a much shorter sequence length (and less cost), it is a particularly attractive template around which to design novel antimicrobial peptides. However, the structure–activity relationship of it is still unknown. We designed and synthesized a series of C-terminal amidated analogs of BF-15 based on its amphipathic α-helix structure. And we characterized their antimicrobial potency and haemolytic activity. We identified the amidated BF-15 (analog B1) with potent antimicrobial activity against several antibiotic-resistant bacteria (MICs between 1 and 64 μg/mL, 2–16-folds higher than BF-30) and much lower haemolytic activity. The subsequent circular dichroism study results showed a typical α-helix pattern of analog B1 and the content of the α-helix structure of it increased significantly comparing with BF-30, which indicates the peptide sequence of BF-15 may provide a major contribution to the α-helix content of the whole BF-30 sequence. The peptide induced chaotic membrane morphology and cell debris as determined by electron microscopy. This suggests that the antimicrobial activity of B1 is based on cytoplasmic membrane permeability. Taken together, our results suggested that peptide B1 should be considered as an excellent candidate for developing therapeutic drugs.     

Structure of the three-chain unit of the bovine epidermal keratin filament

The characteristic α-type X-ray diffraction pattern displayed by bovine epidermal keratin filaments can be ascribed to the presence of segments of triple-chain coiled coil α-helix in the repeating three-chain unit of the filaments.Limited proteolysis of filaments polymerized in vitro or a citrate-soluble protein derived from them with crystalline trypsin releases two types of α-helix-enriched particles which provide information on the structure of the three-chain unit. The smaller, particle 2, of molecular weight 42,500, α-helix content of 92% and dimensions of 180 Å × 20 Å, consists of three chains aligned side-by-side that presumably form a coiled coil. The high yields of particle 2 allow the conclusion that all of the α-helix of the epidermal keratin filament is present in the form of these discrete three-chain α-helical segments. The larger, particle 1, recovered during the earlier stages of digestion has a molecular weight of 100,000 to 110,000, α-helix content of 75%, average dimensions of 400 Å × 20 Å and also consists of three chains aligned side-by-side. It contains two α-helical segments corresponding to particle 2 which are located at the amino -terminal and carboxyl-terminal ends and are separated by a region of non-helix. Particle 1 contains all of the α-helix and therefore is the major portion of the three-chain unit of the keratin filament. The products resulting from reaction of intact filament subunits with N-bromosuccinimide suggest that particle 1 is formed during digestion by removal of regions of non-helix from each end of this unit.The structure of the three-chain unit of the bovine epidermal keratin filament may thus be viewed as three polypeptide subunits aligned side-by-side with two discrete coiled coil α-helical segments interspersed with regions of non-helix.     

Structure and dynamics of the M13 coat signal sequence in membranes by multidimensional high-resolution and solid-state NMR spectroscopy

The polypeptide corresponding to the signal sequence of the M13 coat protein and the five N-terminal residues of the mature protein was prepared by solid-phase peptide synthesis with a ¹⁵N isotopic label at the alanine-12 position. Multidimensional solution NMR spectroscopy and molecular modeling calculations indicate that this polypeptide assumes helical conformations between residues 5 and 20, in the presence of sodium dodecylsulfate micelles. This is in good agreement with circular dichroism spectroscopic measurement, which shows an α-helix content of approximately 42%. The α-helix comprises an uninterrupted hydrophobic stretch of ≤12 amino acids, which is generally believed to be too short for a stable transmembrane alignment in a biological bilayer. The monoexponential proton-deuterium exchange kinetics of this hydrophobic helical region is characterized by half-lives of 15–75 minutes (pH 4.2, 323 K). When the polypeptide is reconstituted into phospholipid bilayers, the broad anisotropy of the proton-decoupled ¹⁵N solid-state NMR spectroscopy indicates that the hydrophobic helix is immobilized close to the lipid bilayer surface at the time scale of ¹⁵N solid-state NMR spectroscopy (10⁻⁴ seconds). By contrast, short correlation times, immediate hydrogen-deuterium exchange as well as nuclear Overhauser effect crosspeak analysis suggest that the N and C termini of this polypeptide exhibit a mobile random coil structure. The implications of these structural findings for possible mechanisms of membrane insertion and translocation as well as for membrane protein structure prediction algorithms are discussed. © 1997 Wiley-Liss Inc.     

Thermal and charge-induced coil to -helix transition of poly-L-glutamic acid and random L-glutamic acid-L-alanine copolymers

Thermal and charge induced random coil to α-helix transitions of poly-L -glutamic acid (PGA) were measured by optical rotatory dispersion in various solvents. The data of PGA in 0.1M Nacl were analyzed by the Zimm-Rice theory. Enthalpy and entropy changes for the coil-to-helix transition in the unionized state were obtained: ΔH° = ?1020 ± 100 cal/residue mole; ΔH° = ?3.0 ± 0.4 e.u./residue mole. The initiation parameter, σ, of the Zimm-Rice theory was given by a value of 5 ± 1 × 10^-3. Random copolymers of L -glutamic acid and L -alanine containing 10, 30, and 40 molar percents of alanyl residue were synthesized. Stabilities of α-helix of the copolymers were compared to that of PGA. In water and water-ethanol solutions, stabilities of the polymers were almost equal after the simple correction about the ionized charge density of the polymers. In 0.1 M NaCl solution these copolymers showed some deviations from the transition curve of PGA, which would suggest the hydrophobic contribution of the alanyl residues.     

Solution structure of P01, a natural scorpion peptide structurally analogous to scorpion toxins specific for apamin-sensitive potassium channel

The venom of the North African scorpion Androctonus mauretanicus mauretanicus possesses numerous highly active neurotoxins that specifically bind to various ion channels. One of these, P05, has been found to bind specifically to calcium-activated potassium channels and also to compete with apamin, a toxin extracted from bee venom. Besides the highly potent ones, several of these peptides (including that of P01) have been purified and been found to possess only a very weak, although significant, activity in competition with apamin. The amino acid sequence of P01 shows that it is shorter than P05 by two residues. This deletion occurs within an α-helix stretch (residues 5–12). This α-helix has been shown to be involved in the interaction of P05 with its receptor via two arginine residues. These two arginines are absent in the P01 sequence. Furthermore, a proline residue in position 7 of the P01 sequence may act as an α-helix breaker. We have determined the solution structure of P01 by conventional two-dimensional ¹H nuclear magnetic resonance and show that 1) the proline residue does not disturb the α-helix running from residues 5 to 12; 2) the two arginines are topologically replaced by two acidic residues, which explains the drop in activity; 3) the residual binding activity may be due to the histidine residue in position 9; and 4) the overall secondary structure is conserved, i.e., an α-helix running from residues 5 to 12, two antiparallel stretches of β-sheet (residues 15–20 and 23–27) connected by a type I′ β-turn, and three disulfide bridges connecting the α-helix to the β-sheet.     

ΔμH+ dependency of in vitro protein translocation into Escherichia coli inner-membrane vesicles varies with the signal-sequence core-region composition

Signal sequences frequently contain α-helix-destabilizing amino acids in the hydrophobic core. Nuclear magnetic resonance studies on the conformation of signal sequences in membrane mimetic environments revealed that these residues cause a break in the α-helix. In the precursor of the Escherichia coli outer membrane protein PhoE (pre-PhoE), a glycine residue at position -10 (Gly^?10) is thought to be responsible for the break in the α-helix. We investigated the role of this glycine residue in the translocation process by employing site-directed mutagenesis. SDS-PAGE analysis showed drastic variations in the electrophoretic mobilities of the mutant precursor proteins, suggesting an important role of the glycine residue in determining the conformation of the signal sequence. In vivo, no drastic differences in the translocation kinetics were observed as compared with wild-type PhoE, except when a charged residue (Arg) was substituted for Gly^?10. However, the in vitro translocation of all mutant proteins into inverted inner-membrane vesicles was affected. Two classes of precursors could be distinguished. Translocation of one class of mutant proteins (Ala, Cys and Leu for Gly^?10) was almost independent of the presence of a ΔμH⁺, whereas translocation of the other class of precursors (wild type or Ser) was strongly decreased in the absence of the ΔμH⁺. Apparently, the ΔμH⁺ dependency of in vitro protein translocation varies with the signal-sequence core-region composition. Furthermore, a proline residue at position -10 resulted in a signal sequence that did not prevent the folding of the precursor in an in vitro trimerization assay.     

Conformation and aggregation of bovine myelin proteins

CD and PMR spectra were obtained on three major protein fractions of bovine CNS myelin: the basic A-1 protein, the Folch-Lees proteolipid apoprotein (APL), and the Wolfgram proteolipid protein (WPP). Most PMR peaks of the A-1 broadened on going from D₂O to salt solutions or to 100% 2-Chloroethanol (2-CE). CD spectra showed no α-helix in water or salt solutions, but showed 42% in 2-CE. The APL showed no PMR in D₂O, but did show aromatic amino acid peaks in 1.5% SDS. CD spectra showed 37% α-helix in both cases. The PMR of the WPP in 1.5% SDS showed aromatic amino acids, and the CD showed <20% α-helix. All three proteins showed sharp PMR spectra in trifluoroacetic acid with α-CH chemical shifts characteristic of random coils. It was concluded that the A-1 and the APL aggregate.     

Analysis and prediction of the packing of α-helices against a β-sheet in the tertiary structure of globular proteins

The packing of α-helices and β-sheets in six $\text{α}\text{β}$ proteins (e.g. flavodoxin) has been analysed. The results provide the basis for a computer algorithm to predict the tertiary structure of an $\text{α}\text{β}$ protein from its amino acid sequence and actual assignment of secondary structure.The packing of an individual α-helix against a β-sheet generally involves two adjacent ± 4 rows of non-polar residues on the α-helix at the positions i, i + 4, i + 8, i + 1, i + 5, i + 9. The pattern of interacting β-sheet residues results from the twisted nature of the sheet surface and the attendant rotation of the side-chains. At a more detailed level, four of the α-helical residues (i + 1, i + 4, i + 5 and i + 8) form a diamond that surrounds one particular β-sheet residue, generally isoleucine, leucine or valine. In general, the α-helix sits 10 Å above the sheet and lies parallel to the strand direction.The prediction follows a combinational approach. First, a list of possible β-sheet structures (10⁶ to 10¹⁴) is constructed by the generation of all β-sheet topologies and β-strand alignments. This list is reduced by constraints on topology and the location of non-polar residues to mediate the sheet/helix packing, and then rank-ordered on the extent of hydrogen bonding. This algorithm was uniformly applied to 16 $\text{α}\text{β}$ domains in 13 proteins. For every structure, one member of the reduced list was close to the crystal structure; the root-mean-square deviation between equivalenced C^α atoms averaged 5.6 Å for 100 residues. For the $\text{α}\text{β}$ proteins with pure parallel β-sheets, the total number of structures comparable to or better than the native in terms of hydrogen bonds was between 1 and 148. For proteins with mixed β-sheets, the worst case is glyceraldehyde-3-phosphate dehydrogenase, where as many as 3800 structures would have to be sampled. The evolutionary significance of these results as well as the potential use of a combinatorial approach to the protein folding problem are discussed.