Synthetic peptides as models for intrinsic membrane proteins

There are many ways in which lipids can modulate the activity of membrane proteins. Simply a change in hydrophobic thickness of the lipid bilayer, for example, already can have various consequences for membrane protein organization and hence for activity. By using synthetic transmembrane peptides, it could be established that these consequences include peptide oligomerization, tilt of transmembrane segments, and reorientation of side chains, depending on the specific properties of the peptides and lipids used. The results illustrate the potential of the use of synthetic model peptides to establish general principles that govern interactions between membrane proteins and surrounding lipids.     

Mass spectrometric evaluation of synthetic peptides as primary structure models for peptide and protein deamidation.

Solid-phase peptide synthesis and deamidation measurements using a novel mass spectrometric technique were carried out for 94 model asparaginyl peptides from 3 to 13 residues in length. Deamidation rates of these peptides in pH 7.4, 37.0 degrees C, 0.15 M Tris-HCl buffer were measured and evaluated. It was found that they validate the use of pentapeptide models as surrogates for the primary sequence dependence of peptide and protein deamidation rates and the discovery by difference of secondary, tertiary and quaternary structure effects. Deamidation of the pentapeptide models, compared with that of longer peptides of more intricate structure, is discussed, and the application of this technique to deamidation measurement of intact proteins is demonstrated.     

Low-frequency motions in protein molecules. Beta-sheet and beta-barrel.

Low-frequency internal motions in protein molecules play a key role in biological functions. Based on previous work with alpha-helical structure, the quasi-continuum model is extended to the beta-structure, another fundamental element in protein molecules. In terms of the equations derived here, one can easily calculate the low-frequency wave number of a beta-sheet in an accordionlike motion, and the low-frequency wave number of a beta-barrel in a breathing motion. The calculated results for immunoglobulin G and concanavalin A agree well with the observations. These findings further verify that the observed low-frequency motion (or the so-called dominant low-frequency mode) in a protein molecule is essentially governed by the collective fluctuations of its weak bonds, especially hydrogen bonds, and the internal displacement of the massive atoms therein, as described by the quasi-continuum model.     

A model of morphogenesis for protein secondary structures.

This paper proposes a model for the expected probability distribution for a certain class of biological structures. In particular, a model is derived for the distribution of lengths of helices, sheets, turns, and coils as a function of the length of the structure divided by the length of the protein it is contained in. A fit between the derived lognormal function and the structures for some proteins whose three-dimensional structure is known was significant. The fit produces fundamental parameters particular to each structure type that are related to the underlying structure and its morphogenesis. The importance of the result is that a universal mathematical distribution can be used to explain certain protein morphogeneses. Also, these fundamental parameters can be used as an aid in predicting whether a given sequence is a particular secondary structure or not, without a knowledge of its three-dimensional structure.     

Peptide models for the study of coupled conformational properties of protein secondary structures

This letter describes the construction and use of molecular models designed for the investigation of the coupled conformational properties of polypeptide structures. The models incorporate several degrees of conformational freedom that are not present in other varieties of models, and are in addition furnished with angle dials so that changes in torsional bond angles accompanying motion of the structure may be read out directly. The models are particularly useful in establishing the qualitative mechanical behavior of coupled structures. Once the mechanical behavior of the system is known, it becomes relatively straight-forward to compute the energetics of the system under study.     

Synthetic peptide substrates for a tyrosine protein kinase

Immunoprecipitates containing the transforming protein of the avian sarcoma virus, Y73, together with its associated tyrosine-specific protein kinase, have an activity which will phosphorylate the synthetic peptide Lys-Leu-Ile-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg at the tyrosine residue. This peptide corresponds to 10 out of 11 amino acids surrounding the phosphorylated tyrosine in both pp60src and P90, the transforming proteins of Rous sarcoma virus and Y73 virus, respectively. The apparent Km for phosphorylation of the peptide was about 5 mM. A second peptide with the sequence Lys-Leu-Ile-Asp-Asn-Glu-Tyr-Thr-ala-Arg differing from the first peptide only by the absence of the glutamic acid 4 residues from the tyrosine was also phosphorylated, but the apparent Km for the reaction was 40 mM. Several sites of tyrosine phosphorylation in viral transforming proteins have been found to have one or more glutamic acids close to the phosphorylated tyrosine on the NH2-terminal side. Taken together with our in vitro phosphorylation studies, this suggests that the primary sequence surrounding target tyrosines may play a role in recognition of substrates by tyrosine protein kinases, and in particular, that glutamic acid residues on the NH2-terminal side may be important.     

Synthetic peptide arrays for investigating protein interaction domains

Synthetic peptide array technology was first developed in the early 1990s by Ronald Frank. Since then the technique has become a powerful tool for high throughput approaches in biology and biochemistry. Here, we focus on peptide arrays applied to investigate the binding specificity of protein interaction domains such as WW, SH3, and PDZ domains. We describe array-based methods used to reveal domain networks in yeast, and briefly review rules as well as ideas about the synthesis and application of peptide arrays. We also provide initial results of a study designed to investigate the nature and evolution of SH3 domain interaction networks in eukaryotes.     

Hydrogen bond connectivity patterns and hydrophobic interactions in crystal structures of small, acyclic peptides.

Crystal structures of all available unblocked linear peptides with two to five residues were retrieved from the Cambridge Structural Database and their intermolecular contacts and packing modes studied using molecular graphics. This survey reveals that interactions between hydrophobic portions of the molecules are critically important in determining the overall features of their crystal packing patterns. Distinct hydrophobic columns or layers are observed in almost all crystal structures. Analyses of the relationships between these interactions and crystal growth properties of small peptides are given. It is suggested that needle growth is promoted by hydrophobic packing, usually along a short crystallographic axis (4.6-6.0 angstroms). Also contributing to these morphologic characteristics are entropic factors associated with hydrophobic aggregation as well as tightly bound water molecules on hydrophobic faces. The paper also provides a comprehensive overview of hydrogen bond patterns in acyclic peptide crystals. It is demonstrated that one of their primary roles is to provide a scaffolding within which hydrophobic groups can aggregate. Even though there is a high density of hydrogen bonds in the crystals, often with complex patterns and networks, certain motifs are found to recur in a number of structures indicating specific hydrogen bond preferences. Water, for example, is an integral part of the hydrogen bond networks in these crystals, usually acting as the primary donor for main-chain carboxylate groups in peptide hydrates.     

Analyzing protein circular dichroism spectra for accurate secondary structures.

We have developed an algorithm to analyze the circular dichroism of proteins for secondary structure. Its hallmark is tremendous flexibility in creating the basis set, and it also combines the ideas of many previous workers. We also present a new basis set containing the CD spectra of 22 proteins with secondary structures from high quality X-ray diffraction data. High flexibility is obtained by doing the analysis with a variable selection basis set of only eight proteins. Many variable selection basis sets fail to give a good analysis, but good analyses can be selected without any a priori knowledge by using the following criteria: (1) the sum of secondary structures should be close to 1.0, (2) no fraction of secondary structure should be less than -0.03, (3) the reconstructed CD spectrum should fit the original CD spectrum with only a small error, and (4) the fraction of alpha-helix should be similar to that obtained using all the proteins in the basis set. This algorithm gives a root mean square error for the predicted secondary structure for the proteins in the basis set of 3.3% for alpha-helix, 2.6% for 3(10)-helix, 4.2% for beta-strand, 4.2% for beta-turn, 2.7% for poly(L-proline) II type 3(1)-helix, and 5.1% for other structures when compared with the X-ray structure.     

Synthetic peptide models for the redox-active disulfide loop of glutaredoxin. Conformational studies

Two cyclic peptide disulfides (Sequence: see text). (X = L-Tyr or L-Phe) have been synthesized as models for the 14-membered redox-active disulfide loop of glutaredoxin. 1H NMR studies at 270 MHz in chloroform solutions establish a type I beta-turn conformation for the Pro-X segment in both peptides, stabilized by a 4----1 hydrogen bond between the Cys(1) CO and Cys(4) NH groups. Nuclear Overhauser effects establish that the aromatic ring in the X = Phe peptide is oriented over the central peptide unit. In dimethyl sulfoxide solutions two conformational species are observed in slow exchange on the NMR time scale, for both peptides. These are assigned to type I and type II beta-turn structures with -Pro-Tyr(Phe)- as the corner residues. The structural assignments are based on correlation of NMR parameters with model 14-membered cyclic cystine peptides with Pro-X spacers. Circular dichroism studies based on the -S-S- n-omega* transition suggest a structural change in the disulfide bridge with changing solvent polarity, establishing conformational coupling between the peptide backbone and the disulfide linkage in these systems.     

Conformationally constrained alpha-helical peptide models for protein ion channels.

Recently we described the design, synthesis, and characterization of some simple amphiphilic alpha-helical models for protein ion channels. These peptides, composed of only Leu and Ser residues, are hypothesized to form helical bundles capable of passing ions across phospholipid bilayers. In an effort to demonstrate that the peptides are, in fact, helical in their active ion-conducting state, the conformationally constrained amino acid, C alpha, C alpha-dimethylglycine (alpha-aminoisobutyric acid, Aib), was introduced simultaneously at three positions into one of the model peptides, H2N-(Leu-Ser-Leu-Leu-Leu-Ser-Leu)3-CONH2, giving H2N-(Leu-Ser-Leu-Aib-Leu-Ser-Leu)3-CONH2. Examination of a tetrameric model for the channel suggested that this substitution should have a minimal effect on conductance. CD spectroscopy of the Aib-modified and original peptide in phospholipid vesicles indicated that both were highly alpha-helical. Furthermore, the Aib-containing peptide formed proton channels nearly identical in conductance to the original peptide.     

Effects of detergents on the secondary structures of prion protein peptides as studied by CD spectroscopy.

Pathogenic prion proteins (PrP(Sc)) are thought to be produced by alpha-helical to beta-sheet conformational changes in the normal cellular prion proteins (PrP(C)) located solely in the caveolar compartments. In order to inquire into the possible conformational changes due to the influences of hydrophobic environments within caveolae, the secondary structures of prion protein peptides were studied in various kinds of detergents by CD spectra. The peptides studied were PrP(129-154) and PrP(192-213); the former is supposed to assume beta-sheets and the latter alpha-helices, in PrP(Sc). The secondary structure analyses for the CD spectra revealed that in buffer solutions, both PrP(129-154) and PrP(192-213) mainly adopted random-coils (approximately 60%), followed by beta-sheets (30%-40%). PrP(129-154) showed no changes in the secondary structures even in various kinds of detergents such as octyl-beta-D-glucopyranoside (OG), octy-beta-D-maltopyranoside (OM). sodium dodecyl sulfate (SDS), Zwittergent 3-14 (ZW) and dodecylphosphocholine (DPC). In contrast, PrP(192-213) changed its secondary structure depending on the concentration of the detergents. SDS, ZW, OG and OM increased the alpha-helical content, and decreased the beta-sheet and random-coil contents. DPC also increased the alpha-helical content, but to a lesser extent than did SDS, ZW, OG or OM. These results indicate that PrP(129-154) has a propensity to adopt predominantly beta-sheets. On the other hand, PrP(192-213) has a rather fickle propensity and varies its secondary structure depending on the environmental conditions. It is considered that the hydrophobic environments provided by these detergents may mimic those provided by gangliosides in caveolae, the head groups of which consist of oligosaccharide chains containing sialic acids. It is concluded that PrP(C) could be converted into a nascent PrP(Sc) having a transient PrP(Sc) like structureunder the hydrophobic environments produced by gangliosides.     

Hidden Markov models that use predicted secondary structures for fold recognition.

There are many proteins that share the same fold but have no clear sequence similarity. To predict the structure of these proteins, so called "protein fold recognition methods" have been developed. During the last few years, improvements of protein fold recognition methods have been achieved through the use of predicted secondary structures (Rice and Eisenberg, J Mol Biol 1997;267:1026-1038), as well as by using multiple sequence alignments in the form of hidden Markov models (HMM) (Karplus et al., Proteins Suppl 1997;1:134-139). To test the performance of different fold recognition methods, we have developed a rigorous benchmark where representatives for all proteins of known structure are matched against each other. Using this benchmark, we have compared the performance of automatically-created hidden Markov models with standard-sequence-search methods. Further, we combine the use of predicted secondary structures and multiple sequence alignments into a combined method that performs better than methods that do not use this combination of information. Using only single sequences, the correct fold of a protein was detected for 10% of the test cases in our benchmark. Including multiple sequence information increased this number to 16%, and when predicted secondary structure information was included as well, the fold was correctly identified in 20% of the cases. Moreover, if the correct secondary structure was used, 27% of the proteins could be correctly matched to a fold. For comparison, blast2, fasta, and ssearch identifies the fold correctly in 13-17% of the cases. Thus, standard pairwise sequence search methods perform almost as well as hidden Markov models in our benchmark. This is probably because the automatically-created multiple sequence alignments used in this study do not contain enough diversity and because the current generation of hidden Markov models do not perform very well when built from a few sequences.     

Multiscale simulations of protein folding: application to formation of secondary structures

A multiscale simulation method of protein folding is proposed, using atomic representation of protein and solvent, combing genetic algorithms to determine the key protein structures from a global view, with molecular dynamic simulations to reveal the local folding pathways, thus providing an integrated landscape of protein folding. The method is found to be superior to previously investigated global search algorithms or dynamic simulations alone. For secondary structure formation of a selected peptide, RN24, the structures and dynamics produced by this method agree well with corresponding experimental results. Three most populated conformations are observed, including hairpin, β-sheet and α-helix. The energetic barriers separating these three structures are comparable to the kinetic energy of the atoms of the peptide, implying that the transition between these states can be easily triggered by kinetic perturbations, mainly through electrostatic interactions between charged atoms. Transitions between α-helix and β-sheet should jump over at least two energy barriers and may stay in the energetic trap of hairpin. It is proposed that the structure of proteins should be jointly governed by thermodynamic and dynamic factors; free energy is not the exclusive dominant for stability of proteins.     

Synthetic beta-turn peptides as substrates for a tyrosine protein kinase

An attempt has been made at defining the secondary structural requirement for phosphorylation of substrates of a protein tyrosine kinase from the leukemia virus-transformed LSTRA cell line. An examination of the sites of phosphorylation of substrates of protein tyrosine kinases indicated a relatively high probability of the beta-turn as the secondary structural feature at these sites. We have, therefore, synthesized three tyrosine peptides: Ala-Pro-Tyr-Gly-NHCH3, Leu-Pro-Tyr-Ala-NHCH3, and Pro-Gly-Ala-Tyr-NH2, of which the first two peptides, but not the third, would be expected to contain the tyrosine residue in a beta-turn. Circular dichroism and infrared spectral data on the peptides confirmed this expectation. Phosphorylation data on the peptides by the tyrosine kinase showed that the two beta-turn peptides were phosphorylated with Vmax and Km values comparable to those of the 13-residue-long arginine-containing synthetic peptide substrate having a sequence homologous to the autophosphorylation site of the LSTRA kinase. The peptides used here contain the shortest sequence length among the reported synthetic peptide substrates for protein tyrosine kinases. Their preference for the beta-turn indicated that this conformation may serve as the recognition site for tyrosine phosphorylation.     

Elucidating protein secondary structures using alpha-carbon recurrence quantifications.

Secondary structures of proteins were studied by recurrence quantification analysis (RQA). High-resolution, 3-dimensional coordinates of alpha-carbon atoms comprising a set of 68 proteins were downloaded from the Protein Data Bank. By fine-tuning four recurrence parameters (radius, line, residue, separation), it was possible to establish excellent agreement between percent contribution of alpha-helix and beta-sheet structures determined independently by RQA and that of the DSSP algorithm (Define Secondary Structure of Proteins). These results indicate that there is an equivalency between these two techniques, which are based upon totally different pattern recognition strategies. RQA enhances qualitative contact maps by quantifying the arrangements of recurrent points of alpha carbons close in 3-dimensional space. For example, the radius was systematically increased, moving the analysis beyond local alpha-carbon neighborhoods in order to capture super-secondary and tertiary structures. However, differences between proteins could only be detected within distances up to about 6-11 A, but not higher. This result underscores the complexity of alpha-carbon spacing when super-secondary structures appear at larger distances. Finally, RQA-defined secondary structures were found to be robust against random displacement of alpha carbons upwards of 1 A. This finding has potential import for the dynamic functions of proteins in motion.     

A simple motif for protein recognition in DNA secondary structures

DNA in a single-stranded form (ssDNA) exists transiently within the cell and comprises the telomeres of linear chromosomes and the genomes of some DNA viruses. As with RNA, in the single-stranded state, some DNA sequences are able to fold into complex secondary and tertiary structures that may be recognized by proteins and participate in gene regulation. To better understand how such DNA elements might fold and interact with proteins, and to compare recognition features to those of a structured RNA, we used in vitro selection to identify ssDNAs that bind an RNA-binding peptide from the HIV Rev protein with high affinity and specificity. The large majority of selected binders contain a non-Watson-Crick G.T base-pair and an adjacent C:G base-pair and both are essential for binding. This GT motif can be presented in different DNA contexts, including a nearly perfect duplex and a branched three-helix structure, and appears to be recognized in large part by arginine residues separated by one turn of an alpha-helix. Interestingly, a very similar GT motif is necessary also for protein binding and function of a well-characterized model ssDNA regulatory element from the proenkephalin promoter.     

Implications of aromatic–aromatic interactions: From protein structures to peptide models

With increasing structural information on proteins, the opportunity to understand physical forces governing protein folding is also expanding. One of the significant non‐covalent forces between the protein side chains is aromatic–aromatic interactions. Aromatic interactions have been widely exploited and thoroughly investigated in the context of folding, stability, molecular recognition, and self‐assembly processes. Through this review, we discuss the contribution of aromatic interactions to the activity and stability of thermophilic, mesophilic, and psychrophilic proteins. Being hydrophobic, aromatic amino acids tend to reside in the protein hydrophobic interior or transmembrane segments of proteins. In such positions, it can play a diverse role in soluble and membrane proteins, and in α‐helix and β‐sheet stabilization. We also highlight here some excellent investigations made using peptide models and several approaches involving aryl–aryl interactions, as an increasingly popular strategy in protein and peptide engineering. A recent survey described the existence of aromatic clusters (trimer, tetramer, pentamer, and higher order assemblies), revealing the self‐associating property of aryl groups, even in folded protein structures. The application of this self‐assembly of aromatics in the generation of modern bionanomaterials is also discussed.