Analysis of chloroplast transit peptide function using mutations in the carboxyl-terminal region

Protein import into chloroplasts requires a transit peptide, which interacts with the chloroplast transport apparatus and leads to translocation of the protein across the chloroplast envelope. While the amino acid sequences of many transit peptides are known, functional domains have been difficult to identify. Previous studies suggest that the carboxyl terminus of the transit peptide for ribulose bisphosphate carboxylase small subunit is important for both translocation across the chloroplast envelope and proper processing of the precursor protein. We dissected this region using in vitro mutagenesis, creating a set of mutants with small changes in primary structure predicted to cause alterations in secondary structure. The import behavior of the mutant proteins was assessed using isolated chloroplasts. Our results show that removal of a conserved arginine residue in this region results in impaired processing, but does not necessarily affect import rates. In contrast, substituting amino acids with low reverse turn or amphiphilic potential for other original residues affected import rate but not processing.     

Prediction of protein secondary structure from amino acid sequence

The conformational parametersP _k for each amino acid species (j=1–20) of sequential peptides in proteins are presented as the product ofP _i,k , wherei is the number of the sequential residues in thekth conformational state (k=-helix,-sheet,-turn, or unordered structure). Since the average parameter for ann-residue segment is related to the average probability of finding the segment in the kth state, it becomes a geometric mean of (P _k )_av=(P _i,k ) ^1/n with amino acid residuei increasing from 1 ton. We then used ln(P_k)_av to convert a multiplicative process to a summation, i.e., ln(P _k ) _av =(1/n)P _i,k (i=1 ton) for ease of operation. However, this is unlike the popular Chou-Fasman algorithm, which has the flaw of using the arithmetic mean for relative probabilities. The Chou-Fasman algorithm happens to be close to our calculations in many cases mainly because the difference between theirP _k and our InP _k is nearly constant for about one-half of the 20 amino acids. When stronger conformation formers and breakers exist, the difference become larger and the prediction at the N- and C-terminal-helix or-sheet could differ. If the average conformational parameters of the overlapping segments of any two states are too close for a unique solution, our calculations could lead to a different prediction.     

Phosphorylation loops in synthetic peptides of the human neurofilament protein middle-sized subunit

Peptides containing 13 and 39 amino acid residues and serine-side-chain-phosphorylated (P) analogues thereof, corresponding to human neurofilament protein middle-sized subunit (NF-M), have been synthesized in order to localize the phosphorylation site of this protein. The secondary structure of the nonphosphorylated peptides, determined by circular dichroism (CD) measurements, predicted secondary structural calculations and energy conformational calculations, was suggested to be a series of alternating type I (III) -turns and 3₁₀ or -helices. By contrast, the phosphorylated peptides exhibit a unique conformation, probably due to salt bridges between the phosphoserine and the lysine residues. This has provided the first clear evidence that phosphorylation induces conformational changes among these synthetic peptides and presumably, in NF proteins as well. These phosphorylation loops might be the major recognition sites of the neurofilament protein-directed kinases.     

Conformation of the metastasis-inhibiting laminin pentapeptide

The binding of cancer cells to the basement membrane glycoprotein laminin appears to be a critical step in the metastatic process. This binding can be inhibited competitively by a specific pentapeptide sequence (Tyr-Ile-Gly-Ser-Arg) of the laminin B1 chain, and this peptide can prevent metastasis formationin vivo. However, other similar pentapeptide sequences (e.g., Tyr-Ile-Gly-Ser-Glu) have been found to be much less active in metastasis inhibition, raising the possibility that such amino acid substitutions produce structural changes responsible for altering binding to the laminin receptor. In this study, conformational energy analysis has been used to determine the three-dimensional structures of these peptides. The results indicate that the substitution of Glu for the terminal Arg produces a significant conformational change in the peptide backbone at the middle Gly residue. These results have important implications for the design of drugs that may be useful in preventing metastasis formation and tumor spread.     

Analysis of Chameleon Sequences by Energy Decomposition on a Pairwise Per-residue Basis

The conversion from α-helix to β-strand that has been widely observed in so-called chameleon sequences has received considerable attention since such a structural change may induce many amyloidogenic proteins to self-assemble into fibrils thus causing fatal diseases. Here we report a large scale-analysis of the energetics of secondary structural conversions in a collection of chameleon sequences retrieved from the Protein Data Bank. Major energetic contributions to the secondary structural conversion were analyzed by carrying out energy decomposition on a pairwise per-residue basis, i.e., (i,i), (i,i ± 1), (i,i ± 2), (i,i ± 3), (i,i ± 4) and > (i,i ± 4) intra-/inter-residual interactions. While the overall potential energy differences were subtle, individual residue-based interacting energy differences were observed to vary significantly depending on the specific type of secondary structural conversion. The average energy difference between α-helix and β-strand, <ΔE _α→β>, in the chameleon sequences varied significantly in (i,i), (i,i ± 1) and > (i,i ± 4) interactions. The major energetic factors in secondary structure conversions were electrostatic interactions and the polar term for solvation energy. In addition, residue-based average energy differences in α-helix → β-strand conversions were well-correlated to those in α-helix → random coil → β-strand conversions (R ² = 0.92). Assuming that three secondary structural elements can transform in either direction, this strong correlation indicates that the present energy decomposition method using database structures of chameleon sequences provides a reliable tool for the characterization of secondary structure fluctuations in amino acid sequences.     

Predicting conformational switches in proteins.

We describe a new computational technique to predict conformationally switching elements in proteins from their amino acid sequences. The method, called ASP (Ambivalent Structure Predictor), analyzes results from a secondary structure prediction algorithm to identify regions of conformational ambivalence. ASP identifies ambivalent regions in 16 test protein sequences for which function involves substantial backbone rearrangements. In the test set, all sites previously described as conformational switches are correctly predicted to be structurally ambivalent regions. No such regions are predicted in three negative control protein sequences. ASP may be useful as a guide for experimental studies on protein function and motion in the absence of detailed three-dimensional structural data.     

三联体密码子在mRNA二级结构中的分布

对编码成熟肽的ｍＲＮＡ二级结构的分析显示,每个密码子在ｍＲＮＡ二级结构中的位置有一定的倾向性,这种倾向性似乎与相应氨基酸的构象性质相一致。大多数编码疏水氨基酸的密码子位于ｍＲＮＡ二级结构中较稳定的茎区;反之,大多数编码亲水氨基酸的密码子位于柔性的环区。这个结果支持了最近得到的关于ｍＲＮＡ与蛋白质之间存在丰三维结构信息传递的结论。     

Origins of structural diversity within sequentially identical hexapeptides

Efforts to predict protein secondary structure have been hampered by the apparent structural plasticity of local amino acid sequences. Kabsch and Sander (1984, Proc. Natl. Acad. Sci. USA 81, 1075–1078) articulated this problem by demonstrating that identical pentapeptide sequences can adopt distinct structures in different proteins. With the increased size of the protein structure database and the availability of new methods to characterize structural environments, we revisit this observation of structural plasticity. Within a set of proteins with less than 50% sequence identity, 59 pairs of identical hexapeptide sequences were identified. These local structures were compared and their surrounding structural environments examined. Within a protein structural class (α/α, β/β, α/β, α + β), the structural similarity of sequentially identical hexapeptides usually is preserved. This study finds eight pairs of identical hexapeptide sequences that adopt β-strand structure in one protein and α-helical structure in the other. In none of the eight cases do the members of these sequence pairs come from proteins within the same folding class. These results have implications for class dependent secondary structure prediction algorithms.     

Molecular dynamics-based analyses of the structural instability and secondary structure of the fibrinogen gamma chain protein with the D356V mutation

Mutations in the fibrinogen gamma chain (FGG) gene have been associated with various disorders, such as dysfibrinogenemia, thrombophilia, and hypofibrinogenemia. A literature survey showed that a residue exchange in fibrinogen Milano I from γ Asp to Val at position 330 impairs fibrin polymerization. The D356V (D330V) mutation located in the C-terminus was predicted to be highly deleterious and to affect the function of the protein. The pathogenicity of the altered gene and changes in protein functions were predicted using in silico methods, such as SIFT, PolyPhen 2, I-Mutant 3.0, Align GV–GD, PhD–SNP, and SNPs&GO. The secondary structure of the mutant protein was unwound by the end of the 50-ns simulation period, and a structural change in the helix-turn transition of the alpha-helical (352–356) region residues was observed. Moreover, a change in the length of the helical region was visualized in the mutant trajectory file, indicating the local transient unfolding of the protein. The obtained computational results suggest that the substitution of the neutral amino acid valine for the acidic amino acid aspartic acid at position 356 results in an unwound conformation within 50 ns, which might contribute to defective polymerization. Our analysis also provides insights into the effect of the conformational change in the D356V (D330V) mutant on protein structure and function.     

Sequence-dependence of secondary structure formation: conformational studies of host-guest peptides in alpha-helix and beta-structure supporting media

A newly designed host–guest approach is introduced as a experimental tool to explore the relationship between the sequence of peptides and their secondary structure. From the CD spectra of the host–guest peptides studied, a tentative scale for the α-helix potential in 2,2,2-trifluorethanol of guest amino acids is delineated. The conformational preferences are also examined in β-structure supporting media (solid state, CH₂Cl₂, CH₃OH, H₂O) using ir-absorption and CD techniques. Scales for the β-forming tendency of guest amino acid residues in the different media are delineated. It is shown that the preferred conformation of the host–guest peptides is a function of the medium, the chain length, and the protecting groups. Given the fact that conformational effects are important in peptide synthesis, the tentative scales may serve as a guideline to predict secondary structures of side-chain-protected or -deprotected peptides in a given solvent, complementing the well-known empirical conformational prediction parameters.     

Conformational analysis of peptides corresponding to all the secondary structure elements of protein L B1 domain: secondary structure propensities are not conserved in proteins with the same fold.

The solution conformation of three peptides corresponding to the two beta-hairpins and the alpha-helix of the protein L B1 domain have been analyzed by circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMR). In aqueous solution, the three peptides show low populations of native and non-native locally folded structures, but no well-defined hairpin or helix structures are formed. In 30% aqueous trifluoroethanol (TFE), the peptide corresponding to the alpha-helix adopts a high populated helical conformation three residues longer than in the protein. The hairpin peptides aggregate in TFE, and no significant conformational change occurs in the NMR observable fraction of molecules. These results indicate that the helical peptide has a significant intrinsic tendency to adopt its native structure and that the hairpin sequences seem to be selected as non-helical. This suggests that these sequences favor the structure finally attained in the protein, but the contribution of the local interactions alone is not enough to drive the formation of a detectable population of native secondary structures. This pattern of secondary structure tendencies is different to those observed in two structurally related proteins: ubiquitin and the protein G B1 domain. The only common feature is a certain propensity of the helical segments to form the native structure. These results indicate that for a protein to fold, there is no need for large native-like secondary structure propensities, although a minimum tendency to avoid non-native structures and to favor native ones could be required.     

Manipulation of peptide conformations by fine-tuning of the environment and/or the primary sequence

The widely observed phenomenon that peptides are capable of adopting multiple conformations in different environments suggests that secondary structure formation in a peptide segment is a process involving not only the peptide itself hut also the surrounding solvent media. The influence of the primary sequence and the molecular environment on peptide conformations are now investigated using synthetic peptides of amino acid sequence H₂N-(Ser-Lys)₂-Ala-X-Gly-Ala-X-Gly-Trp-Ala-X-Gly-(Lys-Ser)₃-OH, where X = Ile or Val. These two peptides, namely 3I (X = Ile) and 3V (X = Val), are found to lack defined secondary structure in aqueous buffer. However, discrete conformational states, e.g., α-helices and β-sheets, are readily generated and interconverted for both peptides when the buffer is modulated with the addition of methanol, sodium dodecyl sulfate (SDS) micelles, or phospholipid vesicles. The role of the primary sequence in affecting peptide conformations is manifested in that peptides 3I and 3V, which differ respectively in their content of β-branched Ile or Val residues, differ in their secondary structures at monomeric concentrations in 2 mM SDS and in mixed lipid vesicles of phosphatidic acid and phosphatidylcholine. The overall results suggest that peptide segments can be conformationally flexible entities poised to react to minor modulations in cither the molecular environment or the primary sequence, a circumstance that may he relevant to protein functioning and folding. © 1995 John Wiley & Sons, Inc.     

An internal AUU codon initiates a smaller peptide encoded by bacteriophage T4 baseplate gene 26

Summary Bacteriophage T4 baseplate gene 26 codes for two in-frame overlapping peptides with identical C-terminal regions. By site-directed mutagenesis we have now determined that an internal AUU, codon 114 of gene 26, is used as the initiation codon for the synthesis of a smaller peptide (gp26^*). Thus gene 26^* gives rise to a peptide of 95 amino acid residues with an M_r of 10873, while the complete gene 26 encodes a peptide of 208 amino acid residues of M_r 23 880. Expression of gene 26^* is shown to depend on the RNA secondary structure in the translational initiation region of this gene.     

Structural comparison in solution of a native and retro peptide derived from the third helix of Staphylococcus aureus protein A,domain B: retro peptides,a useful tool for the discrimination of helix stabilization factors dependent on the peptide chain orientation

A peptide fragment corresponding to the third helix of Staphylococcus Aureus protein A, domain B, was chosen to study the effect of the main‒chain direction upon secondary structure formation and stability, applying the retro‒enantio concept. For this purpose, two peptides consisting of the native (Ln) and reversed (Lr) sequences were synthesized and their conformational preferences analysed by CD and NMR spectroscopy. A combination of CD and NMR data, such as molar ellipcitity, NOE connectivities, Hα and NH chemical shifts, ³J_αN coupling constants and amide temperature coefficients indicated the presence of nascent helices for both Ln and Lr in water, stabilized upon addition of the fluorinated solvents TFE and HFIP. Helix formation and stabilization appeared to be very similar in both normal and retro peptides, despite the unfavourable charge–macrodipole interactions and bad N-capping in the retro peptide. Thus, these helix stabilization factors are not a secondary structure as determined for this specific peptide. In general, the synthesis and confirmational analysis of peptide pairs with opposite main‒chain directions, normal and retro peptides, could be useful in the determination of secondary structure stabilization factors dependent on the direction. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

Improved prediction for N-termini of alpha-helices using empirical information

The prediction of the secondary structure of proteins from their amino acid sequences remains a key component of many approaches to the protein folding problem. The most abundant form of regular secondary structure in proteins is the alpha-helix, in which specific residue preferences exist at the N-terminal locations. Propensities derived from these observed amino acid frequencies in the Protein Data Bank (PDB) database correlate well with experimental free energies measured for residues at different N-terminal positions in alanine-based peptides. We report a novel method to exploit this data to improve protein secondary structure prediction through identification of the correct N-terminal sequences in alpha-helices, based on existing popular methods for secondary structure prediction. With this algorithm, the number of correctly predicted alpha-helix start positions was improved from 30% to 38%, while the overall prediction accuracy (Q3) remained the same, using cross-validated testing. Although the algorithm was developed and tested on multiple sequence alignment-based secondary structure predictions, it was also able to improve the predictions of start locations by methods that use single sequences to make their predictions. Furthermore, the residue frequencies at N-terminal positions of the improved predictions better reflect those seen at the N-terminal positions of alpha-helices in proteins. This has implications for areas such as comparative modeling, where a more accurate prediction of the N-terminal regions of alpha-helices should benefit attempts to model adjacent loop regions. The algorithm is available as a Web tool, located at http://rocky.bms.umist.ac.uk/elephant.     

Synthetic peptide and template-assembled synthetic protein models of the hen egg white lysozyme 87–97 helix: Importance of a protein-like framework for conformational stability in a short peptide sequence

In the native structure of hen egg white lysozyme (HEL), the amino acid sequence 87–97 (HEL 87–97) forms an amphiphilic helix, with hydrophilic residues in the sequence directed toward the solvent. A synthetic version of the HEL 87–97 sequence (with the cysteine corresponding to position 94 of HEL replaced by alanine) displays conformational features in solution typical of an unordered structure as judged by CD. However, various modifications in the sequence result in increased helix-forming potential of the HEL 87–97 analogues. Further stabilization of the helical conformation in the most helical analogue of the HEL 87–97 sequence is obtained when 4 copies of this peptide sequence are coupled on a peptide carrier molecule following the template-assembled synthetic protein (TASP) approach M. Mutter and S. Vuilleumier (1989) Angew. Chem. Int. Ed. Engl., Vol. 28, pp. 535–554 “A Chemical Approach to Protein Design–Template-Assembled Synthetic Proteins (TASP).” This suggests that long-range interactions of the peptide with its environment contribute to conformational stability in short peptide sequences. TASP molecules may prove useful for the study of the factors that determine secondary structure formation in short peptides by providing a protein-like framework. © 1993 John Wiley & Sons, Inc.     

Simultaneous prediction of protein secondary structure and transmembrane spans

Prediction of transmembrane spans and secondary structure from the protein sequence is generally the first step in the structural characterization of (membrane) proteins. Preference of a stretch of amino acids in a protein to form secondary structure and being placed in the membrane are correlated. Nevertheless, current methods predict either secondary structure or individual transmembrane states. We introduce a method that simultaneously predicts the secondary structure and transmembrane spans from the protein sequence. This approach not only eliminates the necessity to create a consensus prediction from possibly contradicting outputs of several predictors but bears the potential to predict conformational switches, i.e., sequence regions that have a high probability to change for example from a coil conformation in solution to an α‐helical transmembrane state. An artificial neural network was trained on databases of 177 membrane proteins and 6048 soluble proteins. The output is a 3 × 3 dimensional probability matrix for each residue in the sequence that combines three secondary structure types (helix, strand, coil) and three environment types (membrane core, interface, solution). The prediction accuracies are 70.3% for nine possible states, 73.2% for three‐state secondary structure prediction, and 94.8% for three‐state transmembrane span prediction. These accuracies are comparable to state‐of‐the‐art predictors of secondary structure (e.g., Psipred) or transmembrane placement (e.g., OCTOPUS). The method is available as web server and for download at www.meilerlab.org . Proteins 2013; 81:1127–1140. © 2013 Wiley Periodicals, Inc.     

Two antimicrobial and nematicidal peptides derived from sequences encoded Picea sitchensis

Two antimicrobial peptides (piceain 1 and 2) derived from sequences encoded Picea sitchensis are identified. Their amino acid sequences are KSLRPRCWIKIKFRCKSLKF and RPRCWIKIKFRCKSLKF, respectively. One intra‐molecular disulfide bridge is formed by these two half‐cysteines in both piceain 1 and 2. Antimicrobial activities of synthesized piceains against several kinds of microorganisms were tested. They showed antimicrobial activities against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and fungus Candida albicans but little antimicrobial activity against Bacillus subtilis. The results of nematicidal test showed they exerted strong nematicidal activities against Caenorhabditis elegans, following exposure for 5 h at concentrations as low as 10 µg/ml. They had weak hemolytic abilities against human and rabbit red cells. At the concentration of 250 µg/ml, they induced red cell hemolysis of less than 5%. Circular dichroism spectra of the two antimicrobial peptides were investigated in several solutions. Their main secondary structure components are β‐sheet and random. The current work provides a novel family of antimicrobial and nematicidal peptides with unique disulfided loop containing nine amino acid residues. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

同义密码子的反常蛋白质二级结构偏好性

统计分析了 119种人蛋白质和 92种大肠杆菌蛋白质的mRNA序列和蛋白质二级结构的关系 .从二肽频数出发 ,研究了同义密码子使用对蛋白质二级结构的影响 ,证明其影响在 10 %到 2 0 %的量级 .对于人和大肠杆菌 ,在 90 %置信水平上 ,4 0 0对二肽中分别有 79对和 6 0对 ,在 95 %置信水平上 ,分别有 4 5对和 36对二肽的相应密码子二联体具有不同于氨基酸的反常二级结构偏好性 ,并且这种反常不能归因于随机涨落     

Structure and Conformational Heterogeneity of a Weak Toxin from the Cobra Naja kaouthiaVenom

Resonances in the two-dimensional ¹H NMR spectra of a weak toxin (WTX) from the venom of cobra Naja kaouthiafor all 65 amino acid residues were assigned. The amino acid sequence of WTX, determined by the sequential assignment of spin systems, was found to be similar to that of the CM-9a toxin from the N. kaouthiavenom. Unlike CM-9a, WTX contains an additional Trp36 residue; Lys50 and Tyr52 are interchanged; and there is a Thr residue in place of Arg2. For some residues of WTX, the presence of two components of approximately equal intensities in the spectra was shown, which is explained by the conformational heterogeneity of the polypeptide owing to the cis–transisomerization of the peptide bond Arg32–Pro33. The data (contacts of the nuclear Overhauser effect, constants of spin–spin coupling of protons, and rates of exchange of amide protons for deuterium of the solvent) made it possible to determine the secondary structure of two forms of WTX, which is characterized by the presence of two antiparallel -sheets, one of which consists of two strands (regions 1–5 and 13–17) and the other, of three strands (regions 23–28, 38–43, and 55–59).