Spectroscopic evidence for backbone desolvation of helical peptides by 2,2,2-trifluoroethanol: an isotope-edited FTIR study

2,2,2-Trifluoroethanol (TFE) is widely used to induce helix formation in peptides and proteins, but the mechanism behind this effect is still poorly understood. Several recent papers have proposed that TFE acts by selectively desolvating the peptide backbone groups of the helix state. Infrared (IR) spectroscopy of the amide I band of polypeptides can be used to probe both secondary structure and backbone solvation, making this technique well suited for addressing the effect of TFE on polypeptide conformation. In this paper, we report the IR spectra as a function of TFE concentration for an alanine-rich peptide based on the repeat (AAKAA)(n)(). The IR spectra confirm that TFE desolvates the helical state of the peptide to a greater extent than the random coil state. Moreover, using a series of specifically (13)C-labeled peptides, the precise residues desolvated in the presence of TFE were identified. The residues most desolvated by TFE are the alanines located at position i - 4 in the sequence, where i is a lysine residue. This pattern of desolvation is consistent with molecular dynamics simulations which predict strong interactions between the lysine side chain at position n and the backbone carbonyl of the alanine at position i - 4. This is the first direct spectroscopic evidence of specific desolvation of helix backbone atoms in model alanine-rich peptides.     

Evolutionary Dynamics of Recently Duplicated Genes: Selective Constraints on Diverging Paralogs in the <Emphasis Type="Italic">Drosophila pseudoobscura</Emphasis> Genome

Duplicated genes produce genetic variation that can influence the evolution of genomes and phenotypes. In most cases, for a duplicated gene to contribute to evolutionary novelty it must survive the early stages of divergence from its paralog without becoming a pseudogene. I examined the evolutionary dynamics of recently duplicated genes in the Drosophila pseudoobscura genome to understand the factors affecting these early stages of evolution. Paralogs located in closer proximity have higher sequence identity. This suggests that gene conversion occurs more often between duplications in close proximity or that there is more genetic independence between distant paralogs. Partially duplicated genes have a higher likelihood of pseudogenization than completely duplicated genes, but no single factor significantly contributes to the selective constraints on a completely duplicated gene. However, DNA-based duplications and duplications within chromosome arms tend to produce longer duplication tracts than retroposed and inter-arm duplications, and longer duplication tracts are more likely to contain a completely duplicated gene. Therefore, the relative position of paralogs and the mechanism of duplication indirectly affect whether a duplicated gene is retained or pseudogenized. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The α-Helix of Ribonuclease T1as an Independent Stability Unit: Direct Comparison of Peptide and Protein Stability

Measurements of the change in conformational stability, Δ(ΔG), upon mutation of two acidic residues at the C terminus of the helix of ribonuclease T₁have recently been reported. Here, we investigate peptides based on the sequence of the helix with the same mutations: Glu28 replaced with Gln, Asp29 replaced with Asn, and the double mutant. In addition, the mutant Lys25 to Gln was studied. Changes in helix content of the peptides with pH confirm the conclusion found in the intact protein, that the charged forms of the acidic residues destabilize the protein by destabilizing the helix. The pH-dependence of the change in confor mational free energy for the peptides and mutant proteins show fair correspondence for D29N and the double mutant. The mutants E28Q and K25Q, on the other hand, give striking agreement between the protein and peptide systems. This agreement suggests that the helix of ribonuclease T₁behaves as an independently stabilized structural unit of the intact protein and that stabilization of the helical form of the peptide is mirrored in the protein.     

Molecular dynamics simulations predict a tilted orientation for the helical region of dynorphin A(1-17) in dimyristoylphosphatidylcholine bilayers

The structural properties of the endogenous opioid peptide dynorphin A(1-17) (DynA), a potential analgesic, were studied with molecular dynamics simulations in dimyristoylphosphatidylcholine bilayers. Starting with the known NMR structure of the peptide in dodecylphosphocholine micelles, the N-terminal helical segment of DynA (encompassing residues 1-10) was initially inserted in the bilayer in a perpendicular orientation with respect to the membrane plane. Parallel simulations were carried out from two starting structures, systems A and B, that differ by 4 A in the vertical positioning of the peptide helix. The complex consisted of approximately 26,400 atoms (dynorphin + 86 lipids + approximately 5300 waters). After >2 ns of simulation, which included >1 ns of equilibration, the orientation of the helical segment of DynA had undergone a transition from parallel to tilted with respect to the bilayer normal in both the A and B systems. When the helix axis achieved a approximately 50 degrees angle with the bilayer normal, it remained stable for the next 1 ns of simulation. The two simulations with different starting points converged to the same final structure, with the helix inserted in the bilayer throughout the simulations. Analysis shows that the tilted orientation adopted by the N-terminal helix is due to specific interactions of residues in the DynA sequence with phospholipid headgroups, water, and the hydrocarbon chains. Key elements are the "snorkel model"-type interactions of arginine side chains, the stabilization of the N-terminal hydrophobic sequence in the lipid environment, and the specific interactions of the first residue, Tyr. Water penetration within the bilayer is facilitated by the immersed DynA, but it is not uniform around the surface of the helix. Many water molecules surround the arginine side chains, while water penetration near the helical surface formed by hydrophobic residues is negligible. A mechanism of receptor interaction is proposed for DynA, involving the tilted orientation observed from these simulations of the peptide in the lipid bilayer.     

ERVK9, transposons and the evolution of MHC class I duplicons within the alpha-block of the human and chimpanzee

The genomic sequences within the alpha-block (approximately 288-310 kb) of the human and chimpanzee MHC class I region contains ten MHC class I genes and three MIC gene fragments grouped together within alternating duplicated genomic segments or duplicons. In this study, the chimpanzee and human genomic sequences were analyzed in order to determine whether the remnants of the ERVK9 and other retrotransposon sequences are useful genomic markers for reconstructing the evolutionary history of the duplicated MHC gene families within the alpha-block. A variety of genes, pseudogenes, autologous DNA transposons and retrotransposons such as Alu and ERVK9 were used to categorize the ten duplicons into four distinct structural groups. The phylogenetic relationship of the ten duplicons was examined by using the neighbour joining method to analyze transposon sequence topologies of selected Alu members, LTR16B and Charlie9. On the basis of these structural groups and the phylogeny of the duplicated transposon sequences, a duplication model was reconstructed involving four multipartite tandem duplication steps to explain the organization and evolution of the ten duplicons within the alpha-block of the chimpanzee and human. The phylogenetic analysis and inferred duplication history suggests that the Patr/HLA-F was the first MHC class I gene to have been fixed and not required as a precursor for further duplication within the alpha-block of the ancestral species.     

Solution NMR structure of S100B bound to the high-affinity target peptide TRTK-12

The solution NMR structure is reported for Ca(2+)-loaded S100B bound to a 12-residue peptide, TRTK-12, from the actin capping protein CapZ (alpha1 or alpha2 subunit, residues 265-276: TRTKIDWNKILS). This peptide was discovered by Dimlich and co-workers by screening a bacteriophage random peptide display library, and it matches exactly the consensus S100B binding sequence ((K/R)(L/I)XWXXIL). As with other S100B target proteins, a calcium-dependent conformational change in S100B is required for TRTK-12 binding. The TRTK-12 peptide is an amphipathic helix (residues W7 to S12) in the S100B-TRTK complex, and helix 4 of S100B is extended by three or four residues upon peptide binding. However, helical TRTK-12 in the S100B-peptide complex is uniquely oriented when compared to the three-dimensional structures of other S100-peptide complexes. The three-dimensional structure of the S100B-TRTK peptide complex illustrates that residues in the S100B binding consensus sequence (K4, I5, W7, I10, L11) are all involved in the S100B-peptide interface, which can explain its orientation in the S100B binding pocket and its relatively high binding affinity. A comparison of the S100B-TRTK peptide structure to the structures of apo- and Ca(2+)-bound S100B illustrates that the binding site of TRTK-12 is buried in apo-S100B, but is exposed in Ca(2+)-bound S100B as necessary to bind the TRTK-12 peptide.     

Biological and physical properties of a beta-endorphin analog containing only D-amino acids in the amphiphilic helical segment 13-31

Our approach to the modeling of beta-endorphin has been based on the proposal that three basic structural units can be distinguished in the natural peptide hormone: a highly specific opiate recognition sequence at the N terminus (residues 1-5) connected via a hydrophilic link (residues 6-12) to a potential amphiphilic helix in the C-terminal residues 13-31. Our previous studies showed the validity of this approach and have demonstrated the importance of the amphiphilic helical structure in the C terminus of beta-endorphin. The present model, peptide 5, has been designed in order to evaluate further the requirements of the amphiphilic secondary structure as well as to determine the importance of this basic structural element as compared to more specific structural features which might occur in the C-terminal segment. For these reasons, peptide 5 retains the three structural units previously postulated for beta-endorphin; the major difference with regard to previous models is that the whole C-terminal segment, residues 13-31, has been built using only D-amino acids. In aqueous buffered solutions as well as in 2,2,2-trifluoroethanol-containing solutions, the CD spectra of peptide 5 show the presence of a considerable amount of left-handed helical structure. Enzymatic degradation studies employing rat brain homogenate indicate that peptide 5 is stable in this milieu. In delta- and mu-opiate receptor-binding assays, peptide 5 shows a slightly higher affinity than beta-endorphin for both receptors while retaining the same delta/mu selectivity. In opiate assays on the guinea pig ileum, the potency of peptide 5 is twice that of beta-endorphin. In the rat vas deferens assay, which is very specific for beta-endorphin, peptide 5 displays mixed agonist-antagonist activity. Most remarkably, peptide 5 displays a potent opiate analgesic effect when injected intracerebroventricularly into mice. At equal doses, the analgesic effect of peptide 5 is less than that of beta-endorphin (10-15%) but longer lasting. In conjunction with our previous model studies, these results clearly demonstrate that the amphiphilic helical structure in the C terminus of beta-endorphin is of predominant importance with regard to activity in rat vas deferens and analgesic assays. The similarity between the in vitro and in vivo opiate activities of beta-endorphin and peptide 5, when compared to the drastic change in chirality in the latter model, demonstrates that even a left-handed amphiphilic helix formed by D-amino acids can function satisfactorily as a structural unit in a beta-endorphin-like peptide.     

Duplication events and the evolution of segmental identity

Duplication of genes, genomes, or morphological structures (or some combination of these) has long been thought to facilitate evolutionary change. Here we focus on studies of the teleost fishes to consider the conceptual similarities in the evolutionary potential of these three different kinds of duplication events. We review recent data that have confirmed the occurrence of a whole-genome duplication event in the ray-finned fish lineage, and discuss whether this event may have fuelled the radiation of teleost fishes. We then consider the fates of individual duplicated genes, from both a theoretical and an experimental viewpoint, focusing on our studies of teleost Hox genes and their functions in patterning the segmented hindbrain. Finally, we consider the duplication of morphological structures, once again drawing on our experimental studies of the hindbrain, which have revealed that experimentally induced duplicated neurons can produce functionally redundant neural circuits. We posit that the availability of duplicated material, independent of its nature, can lead to functional redundancy, which in turn enables evolutionary change.     

Type I collagen triplet duplication mutation in lethal osteogenesis imperfecta shifts register of alpha chains throughout the helix and disrupts incorporation of mutant helices into fibrils and extracellular matrix

The majority of collagen mutations causing osteogenesis imperfecta (OI) are glycine substitutions that disrupt formation of the triple helix. A rare type of collagen mutation consists of a duplication or deletion of one or two Gly-X-Y triplets. These mutations shift the register of collagen chains with respect to each other in the helix but do not interrupt the triplet sequence, yet they have severe clinical consequences. We investigated the effect of shifting the register of the collagen helix by a single Gly-X-Y triplet on collagen assembly, stability, and incorporation into fibrils and matrix. These studies utilized a triplet duplication in COL1A1 exon 44 that occurred in the cDNA and gDNA of two siblings with lethal OI. The normal allele encodes three identical Gly-Ala-Hyp triplets at aa 868-876, whereas the mutant allele encodes four. The register shift delays helix formation, causing overmodification. Differential scanning calorimetry yielded a decrease in T(m) of 2 degrees C for helices with one mutant chain and a 6 degrees C decrease in helices with two mutant chains. An in vitro binary co-processing assay of N-proteinase cleavage demonstrated that procollagen with the triplet duplication has slower N-propeptide cleavage than in normal controls or procollagen with proalpha1(I) G832S, G898S, or G997S substitutions, showing that the register shift persists through the entire helix. The register shift disrupts incorporation of mutant collagen into fibrils and matrix. Proband fibrils formed inefficiently in vitro and contained only normal helices and helices with a single mutant chain. Helices with two mutant chains and a significant portion of helices with one mutant chain did not form fibrils. In matrix deposited by proband fibroblasts, mutant chains were abundant in the immaturely cross-linked fraction but constituted a minor fraction of maturely cross-linked chains. The profound effects of shifting the collagen triplet register on chain interactions in the helix and on fibril formation correlate with the severe clinical consequences.     

An engineered amino-terminal domain of yeast phosphoglycerate kinase with native-like structure.

Previous studies have suggested that the carboxy-terminal peptide (residues 401-415) and interdomain helix (residues 185-199) of yeast phosphoglycerate kinase, a two-domain enzyme, play a role in the folding and stability of the amino-terminal domain (residues 1-184). A deletion mutant has been created in which the carboxy-terminal peptide is attached to the amino-terminal domain (residues 1-184) plus interdomain helix (residues 185-199) through a flexible peptide linker, thus eliminating the carboxy-terminal domain entirely. CD, fluorescence, gel filtration, and NMR experiments indicated that, unlike versions described previously, this isolated N-domain is soluble, monomeric, compactly folded, native-like in structure, and capable of binding the substrate 3-phosphoglycerate with high affinity in a saturable manner. The midpoint of the guanidine-induced unfolding transition was the same as that of the native two-domain protein (Cm approximately 0.8 M). The free energy change associated with guanidine-induced unfolding was one-third that of the native enzyme, in agreement with previous studies that evaluated the intrinsic stability of the N-domain and the contribution of domain-domain interactions to the stability of PGK. These observations suggest that the C-terminal peptide and interdomain helix are sufficient for maintaining a native-like fold of the N-domain in the absence of the C-domain.     

Examination of the requirement for an amphiphilic helical structure in beta-endorphin through the design, synthesis, and study of model peptides

In our approach to beta-endorphin modeling, we have proposed that the biological properties of the natural peptide are determined by the combination of three basic structural units: a highly specific opiate recognition sequence at the NH2 terminus (residues 1-5) connected via a hydrophilic peptide link (residues 6-12) to a potential amphiphilic helix in the COOH-terminal residues 13-31. In the alpha-helical conformation the hydrophobic domain twists around the length of the helix and covers almost one-half of its surface. The other distinctive features of the helix include its basicity and the two aromatic residues Phe18 and Tyr27. In contrast to previous models we have studied, peptide 4 is a "negative" model in the sense that it was designed and examined in order to determine how the lack of a well defined amphiphilic structure affects the biological properties of beta-endorphin. For this purpose, peptide 4 retains the three structural units previously postulated for beta-endorphin, but the amino acids of the 13-31 region are arranged in such a way that no definite continuous hydrophobic zone could be formed in an alpha- or pi-helical conformation of this region. In aqueous buffered solutions, peptide 4 showed almost the same amount of alpha-helical structure as beta-endorphin, with a slight tendency toward less helicity in 50% aqueous 2,2,2-trifluoroethanol. In rat brain homogenate, peptide 4 was degraded slightly slower than beta-endorphin, in contrast to the apparently much higher stability of previous models under the same conditions. With regard to opiate receptor binding, peptide 4 was twice as potent as beta-endorphin in mu-receptor assays but half as potent in delta-receptor assays. The opiate potency of peptide 4 on the guinea pig ileum was higher than that of beta-endorphin. In contrast, in the rat vas deferens assay, which is very specific for beta-endorphin, the potency of peptide 4 was very low and could be shown not to be mediated by the same opiate mechanism or by the same opiate receptor. A comparison of these results with those of previous model peptides provides further evidence for the importance of an amphiphilic helical structure in beta-endorphin residues 13-31, which determines the resistance to proteolysis of the natural molecule and contributes to the delta- and mu-opiate receptor interaction. The amphiphilicity of this helical structure must also be essential for high opiate activity on the rat vas deferens (epsilon-receptors), whereas no such structural requirement appears to be necessary for interaction with the opiate receptors on the guinea pig ileum.     

Orientation and helical conformation of a tissue-specific hunter-killer peptide in micelles

Hunter-killer peptides are chimeric synthetic peptides that selectively target specific cell types for an apoptotic death. These peptides, which are models for potential therapeutics, contain a homing sequence for receptor-mediated interactions and a pro-apoptotic sequence. Homing domains have been designed to target angiogenic tumor cells, prostate cells, arthritic tissue and, most recently, adipose tissue. After a receptor-mediated internalization, the apoptotic sequence, which contains D-enantiomer amino acids, initiates apoptosis through mitochondrial membrane disruption. We have begun structure and functional studies on a peptide (HKP1) that specifically targets angiogenic tumor cells for apoptosis. As a model for mitochondrial membrane disruption, we have examined peptide-induced leakage of a calcein fluorophore from large unilamellar vesicles. These experiments demonstrate more potent leakage activity by HKP1 than the peptide lacking the homing domain. Circular dichroism and 2D homonuclear NMR experiments demonstrate that this tumor-specific HKP adopts a left-handed amphipathic helix in association with dodecylphosphorylcholine micelles in a parallel orientation to the lipid-water interface with the homing domain remaining exposed to solvent. The amphipathic helix of the apoptotic domain orients with nonpolar leucine and alanine residues inserting most deeply into the lipid environment.     

The collagen-like peptide (GER)15GPCCG forms pH-dependent covalently linked triple helical trimers

A collagen-like peptide with the sequence (GER)(15) GPCCG was synthesized to study the formation of a triple helix in the absence of proline residues. This peptide can form a triple helix at acidic and basic pH, but is insoluble around neutral pH. The formation of a triple helix can be used to covalently oxidize the cysteine residues into a disulfide knot. Three disulfide bonds are formed between the three chains as has been found at the carboxyl-terminal end of the type III collagen triple helix. This is a new method to covalently link collagen-like peptides with a stereochemistry that occurs in nature. The peptide undergoes a reversible, cooperative triple helix coil transition with a transition midpoint (T(m)) of 17 to 20 degrees C at acidic pH and 32 to 37 degrees C at basic pH. At acidic pH there was little influence of the T(m) on the salt concentration of the buffer. At basic pH increasing the salt concentration reduced the T(m) to values comparable to the stability at acidic pH. These experiments show that the tripeptide unit GER which occurs frequently in collagen sequences can form a triple helical structure in the absence of more typical collagen-like tripeptide units and that charge-charge interactions play a role in the stabilization of the triple helix of this peptide.     

The triple helical structure and stability of collagen model peptide with 4(S)-hydroxyprolyl-Pro-Gly units

Extensive studies on the structure of collagen have revealed that the hydroxylation of Pro residues in a variety of model peptides with the typical (X-Y-Gly)(n) repeats (X and Y: Pro and its analogues) represents one of the major factors influencing the stability of triple helices. While(2S,4R)-hydroxyproline (Hyp) at the position Y stabilizes the triple helix, (2S,4S)-hydroxyproline (hyp) at the X-position destabilizes the helix as demonstrated that the triple helix of (hyp-Pro-Gly)(15) is less stable than that of (Pro-Pro-Gly)(15) and that a shorter peptide (hyp-Pro-Gly)(10) does not form the helix. To clarify the role of the hydroxyl group of Pro residues to play in the stabilization mechanism of the collagen triple helix, we synthesized and crystallized a model peptide (Pro-Hyp-Gly)(4) -(hyp-Pro-Gly)(2) -(Pro-Hyp-Gly)(4) and analyzed its structure by X-ray crystallography and CD spectroscopy. In the crystal, the main-chain of this peptide forms a typical collagen like triple helix. The majority of hyp residues take down pucker with exceptionally shallow angles probably to relieve steric hindrance, but the remainders protrude the hydroxyl group toward solvent with the less favorable up pucker to fit in a triple helix. There is no indication of the existence of an intra-molecular hydrogen bond between the hydroxyl moiety and the carbonyl oxygen of hyp supposed to destabilize the triple helix. We also compared the conformational energies of up and down packers of the pyrrolidine ring in Ac-hyp-NMe(2) by quantum mechanical calculations.     

The design and production of semisynthetic ribonucleases with increased thermostability by incorporation of S-peptide analogues with enhanced helical stability

Recent work has shown that, with synthetic analogues of C-peptide (residues 1-13 of ribonuclease A), the stability of the peptide helix in H2O depends strongly on the charge on the N-terminal residue. We have asked whether, in semisynthetic ribonuclease S reconstituted from S-protein plus an analogue of S-peptide (1-15), the stability of the peptide helix is correlated with the Tm of the reconstituted ribonuclease S. Six peptides have been made, which contain Glu9----Leu, a blocked alpha-COO- group (-CONH2), and either Gln11 or Glu11. The N-terminal residue has been varied; its charge varies from +2 (Lys) to -1 (succinyl-Ala). We have measured the stability of the peptide helix, the affinity of the peptide for S-protein (by C.D. titration), and the thermal stability of the reconstituted ribonuclease S. All six peptide analogues show strongly enhanced helix formation compared to either S-peptide (1-15) or (1-19), and the helix content increases as the charge on the N-terminal residue changes from +2 to -1. All six peptides show increased affinity for S-protein compared to S-peptide (1-19), and all six reconstituted ribonucleases S show an increase in Tm compared to the protein with S-peptide (1-19). The Tm increases as the charge on residue 1 changes from +2 to -1. The largest increment in Tm is 6 degrees. The results suggest that the stability of a protein can be increased by enhancing the stability of its secondary structure.     

Triple-resonance multidimensional NMR study of calmodulin complexed with the binding domain of skeletal muscle myosin light-chain kinase: indication of a conformational change in the central helix

Heteronuclear 3D and 4D NMR experiments have been used to obtain 1H, 13C, and 15N backbone chemical shift assignments in Ca(2+)-loaded calmodulin complexed with a 26-residue synthetic peptide (M13) corresponding to the calmodulin-binding domain (residues 577-602) of rabbit skeletal muscle myosin light-chain kinase. Comparison of the chemical shift values with those observed in peptide-free calmodulin [Ikura, M., Kay, L. E., & Bax, A. (1990) Biochemistry 29, 4659-4667] shows that binding of M13 peptide induces substantial chemical shift changes that are not localized in one particular region of the protein. The largest changes are found in the first helix of the Ca(2+)-binding site I (E11-E14), the N-terminal portion of the central helix (M72-D78), and the second helix of the Ca(2+)-binding site IV (F141-M145). Analysis of backbone NOE connectivities indicates a change from alpha-helical to an extended conformation for residues 75-77 upon complexation with M13. This conformational change is supported by upfield changes in the C alpha and carbonyl chemical shifts of these residues relative to M13-free calmodulin and by hydrogen-exchange experiments that indicate that the amide protons of residues 75-82 are in fast exchange (kexch greater than 10 s-1 at pH 7, 35 degrees C) with the solvent. No changes in secondary structure are observed for the first helix of site I or the C-terminal helix of site IV. Upon complexation with M13, a significant decrease in the amide exchange rate is observed for residues T110, L112, G113, and E114 at the end of the second helix of site III.     

CD and NMR investigations on trifluoroethanol-induced step-wise folding of helical segment from scorpion neurotoxin.

A 14 amino acid residue peptide from the helical region of Scorpion neurotoxin has been structurally characterized using CD and NMR spectroscopy in different solvent conditions. 2,2,2-Trifluoroethanol (TFE) titration has been carried out in 11 steps from 0 to 90% TFE and the gradual stabilization of the conformation to form predominantly alpha-helix covering all of the 14 residues has been studied by 1H and 13C NMR spectroscopy. Detailed information such as coupling constants, chemical shift indices, NOESY peak intensities and amide proton temperature coefficients at each TFE concentration has been extracted and analysed to derive the step-wise preferential stabilization of the helical segments along the length of the peptide. It was found that there is a finite amount of the helical conformation in the middle residues 5-11 even at low TFE concentrations. It was also observed that > 75% TFE (v/v) is required for the propagation of the helix to the N and C termini and for correct packing of the side chains of all of the residues. These observations are significant to understanding the folding of this segment in the protein and may throw light on the inherent preferences and side chain interactions in the formation of the helix in the peptide.     

Characterization of the biologically important interaction between troponin C and the N-terminal region of troponin I

The N-terminal regulatory region of Troponin I, residues 1-40 (TnI 1-40, regulatory peptide) has been shown to have a biologically important function in the interactions of troponin I and troponin C. Truncated analogs corresponding to shorter versions of the N-terminal region (1-30, 1-28, 1-26) were synthesized by solid-phase methodology. Our results indicate that residues 1-30 of TnI comprises the minimum sequence to retain full biological activity as measured in the acto-S1-TM ATPase assay. Binding of the TnI N-terminal regulatory peptides (TnI 1-30 and the N-terminal regulatory peptide (residues 1-40) labeled with the photoprobe benzoylbenzoyl group, BBRp) were studied by gel electrophoresis and photochemical cross-linking experiments under various conditions. Fluorescence titrations of TnI 1-30 were carried out with TnC mutants that carry a single tryptophan fluorescence probe in either the N- or C-domain (F105W, F105W/C domain (88-162), F29W and F29W/N domain (1-90)) (Fig. 1). Low Kd values (Kd < 10(-7) M) were obtained for the interaction of F105W and F105W/C domain (88-162) with TnI 1-30. However, there was no observable change in fluorescence when the fluorescence probe was located at the N-domain of the TnC mutant (F29W and F29W/N domain (1-90)). These results show that the regulatory peptide binds strongly to the C-terminal domain of TnC.     

Evidence for transit peptide acquisition through duplication and subsequent frameshift mutation of a preexisting protein gene in rice

Many proteins synthesized in the cytosol are delivered to their appropriate compartments in the cell by specific targeting signals. Here, we provide new insight into the generation of the chloroplast-targeting signal (called the transit peptide) in rice. First, we identified the mitochondrial ribosomal protein L13 (mt rpl13) gene on chromosome 5. Downstream of the gene, we identified a DNA fragment of 266 bp: a segment within a duplication of mt rpl13. The duplicated region was transcribed and found to encode an open reading frame (ORF) of 160 amino acids (aa) (orf160). The orf160 gene comprises C-terminal 60 aa derived from the mt rpl13 gene and N-terminal 100 aa derived from another duplicated fragment of a pentatricopeptide repeat (ppr)564 gene that encodes 564 aa with ppr motifs on chromosome 1. Examination of the localization of the ORF160 protein tagged with green fluorescent protein (GFP) showed that it is targeted to the chloroplasts. As such, ORF160 clearly contains a transit peptide. Interestingly, this was translated from the alternative reading frame of the duplicated fragment of ppr564. To confirm this, the reading frame of the ppr564 gene was shifted according to that of the orf160 gene, and the frameshifted ppr564 sequence was fused to the gene for GFP. The expressed GFP-fused protein was also located in the chloroplasts. These results provide clear evidence for the generation of the transit peptide through duplication and subsequent frameshifting of a reading frame of a preexisting protein gene. We also demonstrate the importance of sequence redundancy and frameshift mutation in this evolutionary process.     

Sequence dependence of renucleation after a Gly mutation in model collagen peptides

Missense mutations in the collagen triple helix that replace one Gly residue in the (Gly-X-Y)(n) repeating pattern by a larger amino acid have been shown to delay triple helix folding. One hypothesis is that such mutations interfere with the C- to N-terminal directional propagation and that the identity of the residues immediately N-terminal to the mutation site may determine the delay time and the degree of clinical severity. Model peptides are designed to clarify the role of tripeptide sequences N-terminal to the mutation site, with respect to length, stability, and nucleation propensity, to complete triple helix folding. Two sets of peptides with different N-terminal sequences, one with the natural sequence alpha1(I) 886-900, which is just adjacent to the Gly(901) mutation, and one with a GPO(GAO)(3) sequence, which occurs at alpha1(I) 865-879, are studied by CD and NMR. Placement of the five tripeptides of the natural alpha1(I) collagen sequence N-terminal to the Gly to Ala mutation site results in a peptide that is folded only C-terminal to the mutation site. In contrast, the presence of the Hyp-rich sequence GPO(GAO)(3) N-terminal to the mutation allows complete refolding in the presence of the mutation. The completely folded peptide contains an ordered central region with unusual hydrogen bonding while maintaining standard triple helix structure at the N- and C-terminal ends. These peptide results suggest that the location and sequences of downstream regions favorable for renucleation could be the key factor in the completion of a triple helix N-terminal to a mutation.