Filamentous bacteriophage Pf1 structure determined at 7 Å resolution by refinement of models for the α-helical subunit

The molecular structure of filamentous bacteriophage Pf1 has been determined to 7 Å resolution by analysis of X-ray diffraction data from partially oriented fibers of virus particles. The continuous intensity distribution along layer-lines was measured by numerically separating contributions from overlapping layer-lines. The data were phased by an iterative refinement technique that used the known spatial extent and high α-helical content of the virus particle to refine a structural model. This refinement converges to a unique structural solution that is consistent with the X-ray data and with information derived from physical and chemical studies. The coat protein consists of two α-helical segments: one, almost parallel to the particle axis, is centered at a radius of about 15 Å; the other, at about 25 Å radius, is tilted by about 25 ° to the particle axis. This structure is consistent with every generalization about α-helical packing. The inner and outer segments interlock along most of their length with a crossing angle of 20.5 °. The inner α-helical segments also interact with symmetry-related copies of themselves, as do the outer segments. The double layer of tightly packed, intricately interlocked α-helices forms a stable, 20 Å thick protein coat around the viral DNA.     

Structures of segments of α-synuclein fused to maltose-binding protein suggest intermediate states during amyloid formation

Aggregates of the protein α-synuclein are the main component of Lewy bodies, the hallmark of Parkinson's disease. α-Synuclein aggregates are also found in many human neurodegenerative diseases known as synucleinopathies. In vivo, α-synuclein associates with membranes and adopts α-helical conformations. The details of how α-synuclein converts from the functional native state to amyloid aggregates remain unknown. In this study, we use maltose-binding protein (MBP) as a carrier to crystallize segments of α-synuclein. From crystal structures of fusions between MBP and four segments of α-synuclein, we have been able to trace a virtual model of the first 72 residues of α-synuclein. Instead of a mostly α-helical conformation observed in the lipid environment, our crystal structures show α-helices only at residues 1-13 and 20-34. The remaining segments are extended loops or coils. All of the predicted fiber-forming segments based on the 3D profile method are in extended conformations. We further show that the MBP fusion proteins with fiber-forming segments from α-synuclein can also form fiber-like nano-crystals or amyloid-like fibrils. Our structures suggest intermediate states during amyloid formation of α-synuclein.     

Restriction in the conformational flexibility of apoproteins in the presence of organic cosolvents: A consequence of the formation of “native-like conformation”

The influence of n-propanol on the overall α-helical conformation of β-globin, apocytochrome C, and the functional domain of streptococcal M49 protein (pepM49) and its consequence on the proteolysis of the respective proteins has been investigated. A significant amount of α-helical conformation is induced into these proteins atpH 6.0 and 4°C in the presence of relatively low concentrations of n-propanol. The induction of α-helical conformation into the proteins increased as a function of the propanol concentration, the maximum induction occurring around 30% n-propanol. In the case of α-globin, the fluorescence of its tryptophyl residues also increased as a function of n-propanol concentration, the midpoint of this transition being around 20% n-propanol. Furthermore, concomitant with the induction of helical conformation into these proteins, the proteolysis of their polypeptide chain by V8 protease also gets restricted. The α-helical conformation induced into α- and β-globin by n-propanol decreased as the temperature is raised from 4 to 24°C. In contrast, the α-helical conformation of both α- and β-chain (i.e., globin with noncovalently bound heme) did not exhibit such a sensitivity to this change in temperature. However, distinct differences exist between the n-propanol induced “α-helical conformation” of globins and the “α-helical conformation” of α- and β-chains. A cross-correlation of the n-propanol induced increase in the fluorescence of β-globin with the corresponding increase in the α-helical conformation of the polypeptide chain suggested that the fluorescence increase represents a structural change of the protein that is secondary to the induction of the α-helical conformation into the protein (i.e., an integration of the helical conformation induced to the segments of the polypeptide chain to influence the microenvironment of the tryptophyl residues). Presumably, the fluorescence increase is a consequence of the packing of the helical segments of globin to generate a “native-like structure.” The induction of α-helical conformation into these proteins in the presence of n-propanol and the consequent generation of “native-like conformation” is not unique to n-propanol. Trifluoroethanol, another helix-inducing organic solvent, also behaves in the same fashion as n-propanol. However, in contrast to the proteins described above, n-propanol could neither induce an α-helical conformation into performic acid oxidized RNAse-A nor restrict its proteolysis by proteases. Thus, the high sensitivity of apoproteins and the protein domains to assume α-helical conformation in the presence of low concentration of n-propanol with a concomitant restriction of the proteolytic susceptibility of their polypeptide chain appears to be unique to those proteins that exhibit high α-helical propensities. Apparently, this phenomenon of helix induction and the restriction of proteolysis reflects the formation of rudimentary tertiary interaction of the native protein and is unique to apoproteins or structural domains of α-helical proteins. Consistent with this concept, the induction of α-helical conformation into shorter polypeptide fragments of 30 residues, (e.g., α_1-30, which exists in an α-helical conformation in hemoglobin) is very low. Besides, this peptide exhibited neither the high sensitivity to the low concentrations of n-propanol seen with the apoproteins/protein domains nor the resistance toward proteolysis. The results suggest that the organic cosolvent induced decrease in the conformational flexibility of the apoprotein, and the consequent restriction of their proteolytic cleavage provides an opportunity to develop new strategies for protease catalyzed segment condensation reactions.     

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.     

A new approach to secondary structure evaluation: Secondary structure prediction of porcine adenylate kinase and yeast guanylate kinase by CD spectroscopy of overlapping synthetic peptide segments

A new approach for evaluating the secondary structure of proteins by CD spectroscopy of overlapping peptide segments is applied to porcine adenylate kinase (AK1) and yeast guanylate kinase (GK3). One hundred seventy-six peptide segments of a length of 15 residues, overlapping by 13 residues and covering the complete sequences of AK1 and GK3, were synthesized in order to evaluate their secondary structure composition by CD spectroscopy. The peptides were prepared by solid phase multiple peptide synthesis method using the 9-fluorenylmethoxycarbonyl/tert-butyl strategy. The individual peptide secondary structures were studied with CD spectroscopy in a mixture of 30% trifluoroethanol in phosphate buffer (pH 7) and subsequently compared with x-ray data of AK1 and GK3. Peptide segments that cover α-helical regions of the AK1 or GK3 sequence mainly showed CD spectra with increasing and decreasing Cotton effects that were typical for appearing and disappearing α-helical structures. For segments with dominating β-sheet conformation, however, the application of this method is limited due to the stability and clustering of β-sheet segments in solution and due to the difficult interpretation of random-coiled superimposed β-sheet CD signals. Nevertheless, the results of this method especially for α-helical segments are very impressive. All α-helical and 71% of the β-sheet containing regions of the AK1 and GK3 could be identified. Moreover, it was shown that CD spectra of consecutive peptide content reveal the appearance and disappearance of α-helical secondary structure elements and help localizing them on the sequence string. © 1997 John Wiley & Sons, Inc. Biopoly 41: 213–231, 1997     

Synthetic peptide-targeted selection of phage display mimotopes highlights immunogenic features of α-helical vs non-helical epitopes of Taenia solium paramyosin: implications for parasite- and host-protective roles of the protein

Paramyosin of the pig-human parasite Taenia solium (TPmy) is a α-helical protein located on the worm surface that is suggested to fulfill an immunomodulatory role protecting the parasite against host immune system. Besides, in challenging experiments the protein shows a vaccine potential. These observations imply that TPmy harbors antigenic determinants for each of these contrasting actions. However the suggestion was not given a support from experimental data because respective epitopes have not been described thus far. To circumvent this difficulty, we use synthetic peptides with sequences of regions composed of α-helical or linear structure to induce rabbit antibody responses for phage-display mapping of epitope core amino-acid sets. Antibodies to α-helical regions were weak binders and M13 phage-displayed peptides selected by them from two different libraries exhibited no amino-acid similarities with the original protein site. In contrast, the antibodies produced in response to non-helical segment within α-helical structure were better binders and selectors of perfect structural mimics of the protein site. This first phage display epitope analysis of TPmy supports the notion that the rod-like α-helix, which encompasses over 90% of the total amino acids, may serve as an immunomodulatory shield that protects the parasite. Further, the seven non-helical segments of the TPmy molecule may represent the only anti-parasite discrete immunogenic epitopes whose representative mimotopes can be utilized in development of pure epitope vaccines.     

Interconnection between the globule architecture and disposition of conformationally stanch oligopeptides in proteins of the same structural family

It has been shown that selective interactions between helical segments of macromolecules can be realized in globular proteins in the segments characterized by the same periodicities of charge distribution, i.e., conformationally stanch oligopeptides (CSOPs). It has been found that in the macromolecules of α-helical proteins, CSOPs are disposed at distances characteristic of direct interactions. For representatives of many structural families of α-type proteins, family-specific disposition of CSOPs is observed. The similar disposition of conformationally stanch segments is not related to amino acid sequence homology but reflects peculiarities of native 3D architectures of protein globules.     

Crystal structure of a core domain of stomatin from Pyrococcus horikoshii Illustrates a novel trimeric and coiled-coil fold

Stomatin is a major integral membrane protein of human erythrocytes, the absence of which is associated with a form of hemolytic anemia known as hereditary stomatocytosis. However, the function of stomatin is not fully understood. An open reading frame, PH1511, from the hyperthermophilic archaeon Pyrococcus horikoshii encodes p-stomatin, a prokaryotic stomatin. Here, we report the first crystal structure of a stomatin ortholog, the core domain of the p-stomatin PH1511p (residues 56-234 of PH1511p, designated as PhSto^CD). PhSto^CD forms a novel homotrimeric structure. Three α/β domains form a triangle of about 50 Å on each side, and three α-helical segments of about 60 Å in length extend from the apexes of the triangle. The α/β domain of PhSto^CD is partly similar in structure to the band-7 domain of mouse flotillin-2. While the α/β domain is relatively rigid, the α-helical segment shows conformational flexibility, adapting to the neighboring environment. One α-helical segment forms an anti-parallel coiled coil with another α-helical segment from a symmetry-related molecule. The α-helical segment shows a heptad repeat pattern, and mainly hydrophobic residues form a coiled-coil interface. According to chemical cross-linking experiments, PhSto^CD would be able to assemble into an oligomeric form. The coiled-coil fold observed in the crystal probably contributes to self-association.     

The mitochondrial import protein Mim1 promotes biogenesis of multispanning outer membrane proteins

The mitochondrial outer membrane contains translocase complexes for the import of precursor proteins. The translocase of the outer membrane complex functions as a general preprotein entry gate, whereas the sorting and assembly machinery complex mediates membrane insertion of β-barrel proteins of the outer membrane. Several α-helical outer membrane proteins are known to carry multiple transmembrane segments; however, only limited information is available on the biogenesis of these proteins. We report that mitochondria lacking the mitochondrial import protein 1 (Mim1) are impaired in the biogenesis of multispanning outer membrane proteins, whereas overexpression of Mim1 stimulates their import. The Mim1 complex cooperates with the receptor Tom70 in binding of precursor proteins and promotes their insertion and assembly into the outer membrane. We conclude that the Mim1 complex plays a central role in the import of α-helical outer membrane proteins with multiple transmembrane segments.     

Transmembrane orientation of α-helices in the thylakoid membrane and in the light-harvesting complex. A polarized infrared spectroscopy study

The structure and orientation of the major protein constituent of photosynthetic membranes in green plants, the chlorophyll $\text{a}\text{b}$ light-harvesting complex (LHC) have been investigated by ultraviolet circular dichroism (CD) and polarized infrared spectroscopies. The isolated purified LHC has been reconstituted into phosphatidylcholine vesicles and has been compared to the pea thylakoid membrane. The native orientation of the pigments in the LHC reconstituted in vesicles was characterized by monitoring the low-temperature polarized absorption and fluorescence spectra of reconstituted membranes. Conformational analysis of thylakoid and LHC indicate that a large proportion of the thylakoid protein is in the α-helical structure (56 ± 4%), while the LHC is for 44 ± 7% α-helical. By measuring the infrared dichroism of the amide absorption bands of air-dried oriented multilayers of thylakoids and LHC reconstituted in vesicles, we have estimated the degree of orientation of the α-helical chains with respect to the membrane normal. Infrared dichroism data demonstrate that transmembrane α-helices are present in both thylakoid and LHC with the α-helix axes tilted at less than 30° in LHC and 40° in thylakoid with respect to the membrane normal. In thylakoids, an orientation of the polar C=O ester groups of the lipids parallel to the membrane plane is detected. Our results are consistent with the existence of 3–5 transmembrane α-helical segments in the LHC molecules.     

Synthesis and characterization of poly(LysAla3)

The synthesis and characterization of poly(LysAla₃) are described. The polytetrapeptide is a model for short sequences found in proelastin, and is presumably involved in desmosine or isodesmosine cross-link formation in the native protein. Poly(LysAla₃) is found to possess a mixture of conformations in aqueous solution dependent on molecular weight and pH. Low-molecular-weight (ca. 3000) material appears to be a mixture of random and extended helix at neutral pH. However, as the molecular weight is increased an increasing amount of α-helix is observed rising to >50% for mol wt = 21,000. The α-helical chain segments are thermally stable, melting to a mixture of extended and random forms at T_m = 25°C. High pH (10.5) promotes further α-helix formation but at pH >11.0 the polypeptide becomes insoluble. The inference is that short chain segments of the peptide in elastin are unlikely to be α-helical in the equilibrium state but may fluctuate through such a conformation.     

The transmembrane segment of Tom20 is recognized by Mim1 for docking to the mitochondrial TOM complex

Mitochondria cannot be made de novo. Mitochondrial biogenesis requires that up to 1000 proteins are imported into mitochondria, and the protein import pathway relies on hetero-oligomeric translocase complexes in both the inner and outer mitochondrial membranes. The translocase in the outer membrane, the TOM complex, is composed of a core complex formed from the β-barrel channel Tom40 and additional subunits each with single, α-helical transmembrane segments. How α-helical transmembrane segments might be assembled onto a transmembrane β-barrel in the context of a membrane environment is a question of fundamental importance. The master receptor subunit of the TOM complex, Tom20, recognizes the targeting sequence on incoming mitochondrial precursor proteins, binds these protein ligands, and then transfers them to the core complex for translocation across the outer membrane. Here we show that the transmembrane segment of Tom20 contains critical residues essential for docking the Tom20 receptor into its correct environment within the TOM complex. This crucial docking reaction is catalyzed by the unique assembly factor Mim1/Tom13. Mutations in the transmembrane segment that destabilize Tom20, or deletion of Mim1, prevent Tom20 from functioning as a receptor for protein import into mitochondria.     

Macrocyclic α helical peptide therapeutic modality: A perspective of learnings and challenges

Macrocyclic α-helical peptides have emerged as a compelling new therapeutic modality to tackle targets confined to the intracellular compartment. Within the scope of hydrocarbon-stapling there has been significant progress to date, including the first stapled α-helical peptide to enter into clinical trials. The principal design concept of stapled α-helical peptides is to mimic a cognate (protein) ligand relative to binding its target via an α-helical interface. However, it was the proclivity of such stapled α-helical peptides to exhibit cell permeability and proteolytic stability that underscored their promise as unique macrocyclic peptide drugs for intracellular targets. This perspective highlights key learnings as well as challenges in basic research with respect to structure-based design, innovative chemistry, cell permeability and proteolytic stability that are essential to fulfill the promise of stapled α-helical peptide drug development.     

A structural model for ovine rhodopsin

An experimentally testable structure for ovine rhodopsin has been modelled from a combination of several secondary-structure prediction methods. The proposed structure agrees well with available experimental data. The model envisages seven transmembrane segments that are largely, but not entirely, α-helical. The prediction of roughly adjacent regions of more irregular structure within these segments (which could introduce significant changes in helix pitch or rotation) may provide a good working model for considering the structural mobility of the protein.     

Discordant and chameleon sequences: their distribution and implications for amyloidogenicity

Identification of ambiguous encoding in protein secondary structure is paramount to develop an understanding of key protein segments underlying amyloid diseases. We investigate two types of structurally ambivalent peptides, which were hypothesized in the literature as indicators of amyloidogenic proteins: discordant α-helices and chameleon sequences. Chameleon sequences are peptides discovered experimentally in different secondary-structure types. Discordant α-helices are α-helical stretches with strong β-strand propensity or prediction. To assess the distribution of these features in known protein structures, and their potential role in amyloidogenesis, we analyzed the occurrence of discordant α-helices and chameleon sequences in nonredundant sets of protein domains (n = 4263) and amyloidogenic proteins extracted from the literature (n = 77). Discordant α-helices were identified if discordance was observed between known secondary structures and secondary-structure predictions from the GOR-IV and PSIPRED algorithms. Chameleon sequences were extracted by searching for identical sequence words in α-helices and β-strands. We defined frustrated chameleons and very frustrated chameleons based on varying degrees of total β propensity ≥α propensity. To our knowledge, this is the first study to discern statistical relationships between discordance, chameleons, and amyloidogenicity. We observed varying enrichment levels for some categories of discordant and chameleon sequences in amyloidogenic sequences. Chameleon sequences are also significantly enriched in proteins that have discordant helices, indicating a clear link between both phenomena. We identified the first set of discordant-chameleonic protein segments we predict may be involved in amyloidosis. We present a detailed analysis of discordant and chameleons segments in the family of one of the amyloidogenic proteins, the Prion Protein.     

α-Helical assembly of biologically active peptides and designed helix bundle protein

The formation of α-helical assembly by complexing biologically active peptides with de novo designed protein is described. The de novo designed protein described here is a cystinelinked 4-helix bundle protein constructed with 80 amino acid residues and forms a hydrophobic core region surrounded by 4 helices in an aqueous solution. The biologically active peptides, such as melittin and human growth hormone releasing factor, contain the sequences that are able to form amphiphilic helices. These peptides alone do not form the α-helix structure in a diluted solution with low ion strength. But on mixing with the designed helix bundle protein, the peptides are strongly bound to the protein with the induction of α-helical structure in the biologically active peptides. The content of induced α-helix is in accord with that estimated from the amphiphilic sequence. The results mean that a novel architecture composed of α-helices is formed. Fluorescent and temperature-scanning measurement revealed that the α-helical assembly is constructed with hydrophobic interaction. Also, it is shown by means of fluorescence depolarization that the assembly has a compact globular form corresponding to 1 : 1 complex. © 1994 John Wiley & Sons, Inc.     

The influence of nearest-neighboring amino acid residues on aspects of secondary structure of proteins. Attempts to locate -helices and -sheets

The influence of nearest-neighbor pairs of amino acids (n ? 1) and (n + 1) on the conformation of amino acid (n) in proteins has been studied. From experimental data on eleven proteins of known three dimensional structures, our definition of an α-helical domain in the Φ,Ψ plot has been reexamined and found to be satisfactory. On the same principle, a regular β-sheet domain has been delineated. We then revised our 20 × 20 table of frequencies of occurrences of various conformations tabulating three values: α-helical, β-sheet, and neither. These frequencies were then used to locate the helixbreaking positions in cytochrome b₅, papain, thermolysin, and calcium-binding protein. In conjuction with the helical wheel method, they were useful for predicting the locations of most α-helical segments. Similarly the β-sheet breaking positions in papain were located and most of the β-sheets found by X-ray diffraction were close to or between these positions. Data on β-sheets are extremely sparse so that extensive tests were not possible. The application of this method to abnormal hemoglobins suggested possible distortions of helices and in several instances correlated with abnormal properties of the hemoglobins and association with disease. The variable region of human immunoglobin heavy chains was found to have a very low α-helical content though β-sheet structures might exist.     

The First N-terminal Amino Acids of α-Synuclein Are Essential for α-Helical Structure Formation In Vitro and Membrane Binding in Yeast

α-Synuclein (α-syn), a protein implicated in Parkinson's disease, is structurally diverse. In addition to its random-coil state, α-syn can adopt an α-helical structure upon lipid membrane binding or a β-sheet structure upon aggregation. We used yeast biology and in vitro biochemistry to detect how sequence changes alter the structural propensity of α-syn. The N-terminus of the protein, which adopts an α-helical conformation upon lipid binding, is essential for membrane binding in yeast, and variants that are more prone to forming an α-helical structure in vitro are generally more toxic to yeast. β-Sheet structure and inclusion formation, on the other hand, appear to be protective, possibly by sequestering the protein from the membrane. Surprisingly, sequential deletion of residues 2 through 11 caused a dramatic drop in α-helical propensity, vesicle binding in vitro, and membrane binding and toxicity in yeast, part of which could be mimicked by mutating aspartic acid at position 2 to alanine. Variants with distinct structural preferences, identified here by a reductionist approach, provide valuable tools for elucidating the nature of toxic forms of α-syn in neurons.     

Ser/Thr Motifs in Transmembrane Proteins: Conservation Patterns and Effects on Local Protein Structure and Dynamics

We combined systematic bioinformatics analyses and molecular dynamics simulations to assess the conservation patterns of Ser and Thr motifs in membrane proteins, and the effect of such motifs on the structure and dynamics of α-helical transmembrane (TM) segments. We find that Ser/Thr motifs are often present in β-barrel TM proteins. At least one Ser/Thr motif is present in almost half of the sequences of α-helical proteins analyzed here. The extensive bioinformatics analyses and inspection of protein structures led to the identification of molecular transporters with noticeable numbers of Ser/Thr motifs within the TM region. Given the energetic penalty for burying multiple Ser/Thr groups in the membrane hydrophobic core, the observation of transporters with multiple membrane-embedded Ser/Thr is intriguing and raises the question of how the presence of multiple Ser/Thr affects protein local structure and dynamics. Molecular dynamics simulations of four different Ser-containing model TM peptides indicate that backbone hydrogen bonding of membrane-buried Ser/Thr hydroxyl groups can significantly change the local structure and dynamics of the helix. Ser groups located close to the membrane interface can hydrogen bond to solvent water instead of protein backbone, leading to an enhanced local solvation of the peptide.     

The mode of α-synuclein binding to membranes depends on lipid composition and lipid to protein ratio

Interactions of the presynaptic protein α-synuclein with membranes are involved in its physiological action as well as in the pathological misfolding and aggregation related to Parkinsons's disease. We studied the conformation and orientation of α-synuclein bound to model vesicular membranes using multiparametric response polarity-sensitive fluorescent probes together with CD and EPR measurements. At low lipid to α-synuclein ratio the protein binds membranes through its N-terminal domain. When lipids are in excess, the α-helical content and the role of the C-terminus in binding increase. Highly rigid membranes also induce a greater α-helical content and a lower polarity of the protein microenvironment.