Linear Amino Acid Sequence Research Articles

"Bits" and Pieces

Many protein-protein interactions involved in signal transduction occur through the interaction of modular protein domains in one molecule with short linear sequences of amino acids ("motifs") in another. Although protein domains are recognized by a variety of computational tools, bioinformatic approaches alone have not been successful in identifying the short sequence motifs to which domains bind. A new approach, applying motif-determining algorithms to smaller subproteomic collections of proteins that are already known to associate with each other in high-throughput protein-protein interaction screens, now appears to be capable of capturing a reasonably large number of low-affinity core motif sequences. Application of this approach to the genomes of yeast, fruit flies, nematodes, and humans has doubled the number of known or suspected protein-protein interaction motifs.

From nuclear structure concepts to protein folding and non-conventional drug design

Some of the paradigms emerging from the study of the phenomena of phase transitions in finite many-body systems, like e.g. the atomic nuclues, can be used at profit to solve the protein folding problem (how does a linear sequence of amino acids, immersed in the solvent, code for a unique, biological active, three dimensional native structure of the protein?), within the framework of simple (although not oversimplified) models. Also to design non-conventional drugs which do not create resistance (do not induce mutations in the virus or bacteria expressing the protein). The application of these concepts to the design of inhibitors of the HIV-1-PR, an enzyme which plays a central role in the life cycle of the HIV virus will be illustrated in terms of all-atom simulations and in vitro experimental results.

Regulation of Epithelial Na+ Channel Activity by Conserved Serine/Threonine Switches within Sorting Signals

The PY and YXXphi motifs are canonical sorting signals involved in trafficking. Nedd4-2 and the mu(2)-subunit of the AP-2 complex target these motifs to facilitate internalization. Epithelial Na(+) channel (ENaC) subunits contain both motifs in their cytosolic COOH termini where they overlap ((S/T)PPPXYX(S/T)phi). Just preceding the PY and embedded within the YXXphi motifs are conserved serine/threonine. We test here whether these conserved Ser/Thr modulate ENaC activity by influencing the function of the internalization domains. We find that co-expression of dominant-negative dynamin (K44A) with ENaC increases channel activity. Conversely, co-expression of Nedd4-2 and epsin with ENaC decrease activity. Alanine substitution of the conserved Thr(628) preceding the PY motif in gamma-mENaC had no effect on basal activity. Channels with this mutation, however, responded to K44A and epsin but not Nedd4-2. Similarly, mutation of the proline repeat in the PY motif of gamma-mENaC disrupted only Nedd4-2 regulation having no effect on regulation by K44A and epsin. Alanine substitution of the conserved Thr within the YXX motif of gamma-mENaC (T635A) increased basal activity. Channels containing this mutation responded to Nedd4-2 but not K44A and epsin. Channels containing the T635(D/E) substitution in gamma-mENaC did not have increased basal activity and responded to Nedd4-2 but not K44A. The double mutant T628A,T635A did not respond to Nedd4-2 or K44A. Mutation of Thr(628) and Thr(635) also disrupted ENaC precipitation with the mu(2)-subunit of the AP-2 complex. Moreover, the YXXphi motif, independent of the PY motif, was sufficient to target degradation with T635A disrupting this effect. These results demonstrate that the overlapping PY and YXXphi motifs in ENaC are, in some instances, capable of independent function and that the Ser/Thr just preceding and within these domains impact this function.

The favorable features of tryptophan synthase for proving Beadle and Tatum's one gene-one enzyme hypothesis.

LAST year we celebrated the 100th anniversary of the birth of George Beadle. He, with Edward Tatum, in 1941, proposed one of the major conceptual advances in biology in the 20th century: the existence of a one gene–one enzyme relationship (Beadle and Tatum 1941).2 Unfortunately, like many biologists proposing exciting hypotheses, they could not provide the experimental evidence that would prove they were correct. At that time, the existing knowledge about gene and enzyme structure was inadequate to permit the relationship's verification. George Beadle was aware of this and undoubtedly regretted his inability to provide support for their hypothesis. In a retrospective article written for Phage and the Origins of Molecular Biology, Beadle (1966)(p. 30) states that at the Cold Spring Harbor Symposium of 1951 “the number whose faith in one gene–one enzyme remained steadfast could be counted on the fingers of one hand—with a couple of fingers left over.” Why? Why was their proposed basic relationship not accepted? Why could they not provide proof for their hypothesis? Beadle and Tatum's principal difficulty was that in the 1940s we knew relatively little about the chemical nature of genetic material. The prevailing view then was that genetic material might be protein. Existing information on DNA structure suggested that it consisted of repetitive nucleotide sequences. If correct, it would be unlikely that DNA could specify the many different proteins present in each organism. Also, it was not yet firmly established that polypeptides consist of linear sequences of amino acids. Therefore, during the early years following Beadle and Tatum's proposal of the one gene–one enzyme relationship, our understanding of the structural features of genes and enzymes was insufficient to allow formulation of a finite molecular relationship. Advances in the 1950s dealt with most of these issues and suggested a plausible experimental strategy that could be applied in comparing the structural features of a gene with that of its corresponding protein. One of the earliest relevant findings, by Avery et al. (1944), suggested that the pneumococcal transforming principle—presumably the genetic material of the organism—was very likely DNA (see Lederberg 1994). Unfortunately, the significance of this finding was not appreciated initially; questions were raised about the purity of the transforming principle, our understanding of the transformation process was incomplete, and doubts existed that pneumococcal genetic material would be structurally comparable to the genetic material of higher organisms. Somewhat later, in the early 1950s, Al Hershey and Martha Chase presented their isotopic labeling studies with bacteriophage-infected Escherichia coli. Their results provided what was considered convincing evidence that phage genetic material is in fact DNA (Hershey and Chase 1952; see Stahl 1998). Most importantly, in 1953, Jim Watson and Francis Crick described the double-helical structure of DNA (Watson and Crick 1953). Their contribution changed forever how genetic material would be viewed by everyone. The realization that each gene probably consists of a linear sequence of nucleotides prompted subsequent thought on the existence of a genetic code, an essential consideration with a direct bearing on the gene-enzyme relationship. During this period Matt Meselson and Frank Stahl established that DNA is replicated by synthesis of complementary DNA strands, revealing the elegant features of DNA structure that provide for its exact replication (Meselson and Stahl 1958). Also of great importance, Fred Sanger showed that an insulin polypeptide consists of a unique linear sequence of amino acids (Sanger 1952; Stretton 2002). The significance of the contributions of Sanger, Erwin Chargaff, and others are described in an article by Horace Judson (Judson 1993). The discoveries cited, and observations by others supporting their conclusions, redefined the gene–enzyme relationship of Beadle and Tatum as the gene-protein colinearity hypothesis. Despite these advances in the 1950s, the technology that would have allowed either isolation of a gene or determination of its nucleotide sequence was lacking. Furthermore, since the concept of the genetic code was yet to be developed, knowledge of Watson and Crick's double-helical structure of DNA was initially of little help to those wishing to experimentally address the structural relationship between a gene and the enzyme it was proposed to specify. In the late 1950s Seymour Benzer demonstrated that one could construct a linear fine-structure genetic map by crossing mutants with genetically separable alterations in the same genetic region (Benzer 1957). His findings were consistent with the interpretation that a gene consists of a specific linear sequence of nucleotides. At approximately the same time, Vernon Ingram identified the amino acid change in the hemoglobin of humans with sickle cell anemia, and he developed the protein “fingerprinting” technique, a method that could be applied in detecting the single amino acid change in any mutant protein (Ingram 1958; see Ingram 2004). These two advances provided an excellent pair of experimental approaches that could be applied immediately in testing the gene–protein colinearity concept.

A Three-Dimensional Molecular Model of Lipid-Free Apolipoprotein A-I Determined by Cross-Linking/Mass Spectrometry and Sequence Threading

Apolipoprotein (apo) A-I, a 243-residue, 28.1-kDa protein is a major mediator of the reverse cholesterol transport (RCT) pathway, a process that may reduce the risk of cardiovascular disease in humans. In plasma, a small fraction of lipid-free or lipid-poor apoA-I is likely a key player in the first step of RCT. Therefore, a basic understanding of the structural details of lipid-free apoA-I will be useful for elucidating the molecular details of the pathway. To address this issue, we applied the combined approach of cross-linking chemistry and high-resolution mass spectrometry (MS) to obtain distance constraints within the protein structure. The 21 lysine residues within apoA-I were treated with homo bifunctional chemical cross-linkers capable of covalently bridging two lysine residues residing within a defined spacer arm length. After trypsin digestion of the sample, individual peptide masses were identified by MS just after liquid chromatographic separation. With respect to the linear amino acid sequence, we identified 5 short-range and 12 long-range cross-links within the monomeric form of lipid-free apoA-I. Using the cross-linker spacer arm length as a constraint for identified Lys pairs, a molecular model was built for the lipid-free apoA-I monomer based on homology with proteins of similar sequence and known three-dimensional structures. The result is the first detailed model of lipid-free apoA-I. It depicts a helical bundle structure in which the N- and C-termini are in close proximity. Furthermore, our data suggest that the self-association of lipid-free apoA-I occurs via C- and N-termini of the protein based on the locations of six cross-links that are unique to the cross-linked dimeric form of apoA-I.

A major allergen gene-fusion protein for potential usage in allergen-specific immunotherapy

Specific immunotherapy is a common treatment of allergic diseases and could potentially be applied to other immunologic disorders. Despite its use in clinical practice, more defined and safer allergy vaccine preparations are required. Differences between epitopes of IgE that recognize the 3-dimensional structure of allergens and T cells that recognize linear amino acid sequences provide a suitable tool for novel vaccine development for specific immunotherapy. The aim of the study was to delete B-cell epitopes and prevent IgE crosslinking, but to preserve T-cell epitopes by fusion of 2 major allergens of bee venom because of a change in the conformation. By genetic engineering, we produced a fusion protein composed of the 2 major bee venom allergens: phospholipase A 2 (Api m 1) and hyaluronidase (Api m 2). The Api m [1/2] fusion protein induced T-cell proliferation and both T H 1-type and T H 2-type cytokine responses. In contrast, IgE reactivity was abolished, and profoundly reduced basophil degranulation and type 1 skin test reactivity was observed. Pretreatment of mice with Api m [1/2] fusion protein significantly suppressed the development of specific IgE as well as other antibody isotypes after immunization with the native allergen. The novel fusion protein of 2 major allergens bypasses IgE binding and mast cell/basophil IgE FcepsilonRI crosslinking and protects from IgE development.

Modulation of the Folding Energy Landscape of Cytochrome c with Salt

The folding reaction of acid-unfolded cytochrome c in the presence of various amounts of KCl was investigated with Trp fluorescence and resonance Raman spectroscopies. It was found that the too-early-too-much polypeptide chain collapse induced by KCl yields some stable folding intermediates, which need to overcome a higher energy barrier to fold into their native conformation. We propose that the charge distribution on the polypeptide chain is part of the folding codon encoded in the linear amino acid sequence. The charge screening effect introduced by KCl alters the shape of the energy landscape by raising the slope of the upper rim and introduces a rugged energy surface toward the bottom of the folding funnel.

The evolution of molecular biology.

Biology's various affairs with holism and reductionism, and their contribution to understanding life at the molecular level

89. Isolation of a Close AAV5 Relative from Goat Tissues: Evidence of Host Promiscuity

Top of pageAbstract Preexisting immunity to AAV vectors can potentially limit their clinical usefulness by blocking tissue transduction and/or by causing the elimination of vector-transduced cells. We previously demonstrated that the AAV2-neutralizing capacities of human plasmas (as measured by in vitro assay) are frequently substantial relative to proposed doses of AAV vectors. Out of a group of 50 hemophiliacs, approximately 40% had AAV2-neutralizing capacities exceeding 1e13 viral particles/ml or about 6e16 viral particles/total blood volume. Furthermore, the majority of the group with high anti-AAV2 titers also had significant titers against other AAV serotypes (AAV1, and 3–6). To create improved gene therapy vectors, we are currently screening tissues and adenovirus preparations from a wide variety of non-primate species to isolate AAV capsids that have less preexisting immunity in humans. One of the more surprising results from this screen was the PCR amplification of a close AAV5 relative from a crude goat adenovirus preparation. AAV5 is a human parvovirus, originally isolated from a penile flat condylomatous lesion, with a high sero-prevalence in the post-pubertal population. The goat adenovirus preparation used as the PCR template was produced during routine diagnostic adenovirus screening of a tissue sample from a goat that had died of enteritis. The presence of parvovirus-like particles in the initial stock was demonstrated by electron microscopy and no primate-derived products were used at any point in the viral culture. The 2,805 bp goat AAV PCR fragment, which encodes 603 bp of rep, the central intron, and all of cap, is 94 % homologous to AAV5. The DNA and protein homologies to AAV5 for the partial rep region are 98% and 99%, respectively, and are 93% and 94% for the cap region, respectively. With respect to the linear amino acid sequence, the distribution of the amino acid differences between the AAV5 and goat AAV capsids is highly polar. All of the 44 amino acid differences occur exclusively in the C-terminal hypervariable region of the capsid protein in a scattered fashion. It has been proposed that this region comprises the surface loops of AAV5 by analogy to AAV2. Because of its high homology to AAV5, it is likely that the goat AAV arose from a recent species jump of AAV5 from humans to goats. The fact that all of the amino acid differences between the AAV5 and the goat capsids occur in regions that are probably surface exposed implies that capsid evolution is being driven primarily by the humoral immune system of the new host or by adaptation to ruminant receptors. The lack of amino acid changes in non-surface exposed regions of cap may imply a lack of pressure from the cellular immune response. Interspecies infection, particularly in the case of domestic animals, may be a previously unappreciated but significant mechanism for diversity generation and virus evolution for AAVs. This novel goat AAV capsid has been successfully used to pseudo-type AAV2 vector genomes and is currently being evaluated for tissue tropism and resistance to human preexisting humoral immunity.

Sequences required for the flagellar targeting of an integral membrane protein.

Previous studies have established that the ISO1 glucose transporter of Leishmania enriettii resides primarily in the flagellar membrane, whereas the ISO2 glucose transporter is located in the pellicular plasma membrane surrounding the cell body. This pronounced difference in subcellular targeting is conferred by the NH2-terminal domain of the transporters, since this is the only region of the two permeases that differs in sequence. Analysis of the 130 residue NH2-terminal domain of ISO1 using multiple terminal deletion mutants and various internal deletion mutants established that a sequence located between amino acids 84 and 100 of this domain is required for flagellar trafficking. In addition, chimeras between ISO1 and ISO2 indicated that the region between residues 110 and 118 of ISO1 is also required for flagellar targeting. These results imply that flagellar targeting information for this integral membrane protein does not constitute a simple linear sequence of amino acids but is at least bipartite in structure.

A cysteine-rich protein in the Theromyzon (Annelida: Hirudinea) cocoon membrane

The aquatic leech, Theromyzon rude, secretes a flexible, proteinaceous cocoon that is resistant to a broad range of denaturing conditions (e.g. heat, denaturing chemicals). We have partially solubilized the Theromyzon cocoon membrane in 10% acetic acid and identified two major protein fragments. Microsequencing of both Theromyzon cocoon protein (Tcp) fragments generated an identical stretch of the amino-terminal sequence that was used to clone the corresponding gene. The predicted linear amino acid sequence of the resulting cDNA contained an unusually high cysteine content (17.8%). Sequence analysis identified six internal repeats, each comprising 12 ordered Cys residues in a ∼62 amino acid repeating unit. Sequence comparisons identified homology with undescribed, Cys-rich repeats across animal phyla (i.e. Arthropod, Nematoda).

Advances in B-cell epitope analysis of autoantigens in connective tissue diseases

The characterization of autoantibody specificities in rheumatic diseases is important in both diagnostic and basic research areas. Identification of the epitopes recognized by autoantibodies and their clinical and biological significance is not a trivial task. Epitopes may range in complexity from simple linear sequences of amino acids to complex quaternary structures. In addition to this structural complexity the frequency with which an autoantigen and its epitopes are recognized in a patient population may be useful in diagnosis, defining disease subgroups, and may offer information on disease prognosis. In this review recent advances in the epitope mapping of autoantigens in connective tissue diseases are discussed, with particular emphasis placed on the methodologies used to identify epitopes and the classification of the structural features of epitopes. To illustrate the identification of epitope structure, clinically relevant autoantigens, including CENP-A, PM/Scl-100, fibrillarin, filaggrin, Ro-52, and dsDNA, are discussed as examples of each type of epitope.

Ab initio Tertiary Structure Prediction of Proteins

A daunting challenge in the area of computational biology has been to develop a method to theoretically predict the correct three-dimensional structure of a protein given its linear amino acid sequence. The ability to surmount this challenge, which is known as the protein folding problem, has tremendous implications. We introduce a novel ab initio approach for the protein folding problem. The accurate prediction of the three-dimensional structure of a protein relies on both the mathematical model used to mimic the protein system and the technique used to identify the correct structure. The models employed are based solely on first principles, as opposed to the myriad of techniques relying on information from statistical databases. The framework integrates our recently proposed methods for the prediction of secondary structural features including helices and strands, as well as β-sheet and disulfide bridge formation. The final stage of the approach, which culminates in the tertiary structure prediction of a protein, utilizes search techniques grounded on the foundations of deterministic global optimization, powerful methods which can potentially guarantee the correct identification of a protein's structure. The performance of the approach is illustrated with bovine pancreatic trypsin inhibitor protein and the immunoglobulin binding domain of protein G.

Mechanisms of Protein Folding: Molecular Chaperones and Their Application in Biotechnology

AbstractFor Abstract see ChemInform Abstract in Full Text.

Using Secondary Structure Information for Protein Folding in CLP( [formula omitted

The protein folding problem is the problem of predicting the 3D structure of a protein when the linear sequence of aminoacids identifying it is known. In this paper we present a declarative implementation in Constrain & Generate style in CLP( FD) of the protein folding problem, for models based on face-centered cubes. We use information concerning secondary structure (and other heuristics) to sensibly prune the search space. Preliminary results on real proteins are encouraging.

Expression, purification, and characterization of minimized chicken riboflavin carrier protein from a synthetic gene in Escherichia coli

Minimized proteins have long been used to elicit immune response to particular regions of a protein antigen. Most efforts to derive minimized proteins have employed synthetic peptide fragments. Here we describe molecular cloning and production of a minimized chicken riboflavin carrier protein (mini-RCP) sequence that harbours all the four neutralizing epitopes but lacks the sequences that otherwise elicit undesirable antibodies. The gene encoding mini-RCP is engineered by contiguous alignment of nucleotide sequences coding for selected epitopes of chicken RCP separated by leucyl alanine residues. The gene has been constructed from eight oligonucleotides by employing overlapping PCR strategy and expressed in Escherichia coli, using the T7 promoter system. The recombinant protein could be purified to homogeneity by a single step Ni 2+ affinity chromatography. Western blot experiments using epitope specific antisera confirm that the corresponding linear amino acid sequences are available for immunorecognition in the engineered protein. This methodology enables continuous production and purification in bulk amounts of the minimized RCP as a source of candidate immunocontraceptive vaccine in mammals.

The use of ionisation constants of amino acids for protein signal analysis within the resonant recognition model--application to proteases

Biological functions of proteins and their active 3D structures are determined by the linear sequences of amino acids. The resonant recognition model (RRM) is a physico-mathematical model developed for structure/function analysis of protein and DNA sequences. Here, we are comparing results of the RRM analysis [1,2] of protease proteins using the electron-ion interaction potential (EIIP) and ionisation constant (IC) of amino acids. The results obtained reveal that the IC parameter can be successfully used to determine the characteristic patterns of different functional protease subgroups.

Prediction of Human Protein Function from Post-translational Modifications and Localization Features

We have developed an entirely sequence-based method that identifies and integrates relevant features that can be used to assign proteins of unknown function to functional classes, and enzyme categories for enzymes. We show that strategies for the elucidation of protein function may benefit from a number of functional attributes that are more directly related to the linear sequence of amino acids, and hence easier to predict, than protein structure. These attributes include features associated with post-translational modifications and protein sorting, but also much simpler aspects such as the length, isoelectric point and composition of the polypeptide chain.

Importance of M-protein C terminus as substrate antigen for serodetection of equine arteritis virus infection.

Equine arteritis virus (EAV), an enveloped positive-stranded RNA virus, is the prototype of the arterivirus group. In a previous paper (A. Kheyar, S. Martin, G. St.-Laurent, P. J. Timoney, W. H. McCollum, and D. Archambault, Clin. Diagn. Lab. Immunol. 4:648-652, 1997), we have shown that the unglycosylated membrane (M) protein, which is composed of 162 amino acids (aa), is a major target of equine antibody to EAV. In order to determine the antigenic regions of the M protein, the cDNA encoding the M protein of EAV was inserted into the procaryotic expression vector pGEX-4T-1 to produce recombinant glutathione S-transferase-M fusion protein. Various deletion mutant clones, which covered the entire sequence of the M protein, were then generated by inverse PCR and expressed in Escherichia coli to examine, by a Western blot assay, the antigenic reactivity of the clone-derived truncated M proteins with sera from horses either experimentally or naturally infected with EAV. Deletion of the hydrophobic N-terminal 87 aa did not abolish immune reactivity of the protein with serum antibodies to EAV, thereby demonstrating the antigenicity of the C-terminal region (aa 88 to 162) of the M protein. Further truncations of the M-protein C-terminal domain defined particular linear epitope-containing amino acid sequence regions. However, only the M-protein C-terminal region was readily recognized by all EAV-specific horse antisera tested in this study. Based on these findings, only the M-protein C-terminal polypeptide composed of aa 88 to 162 is necessary to identify horse serum antibodies specific to the EAV M protein. Thus, this polypeptide might be useful for serodetection of EAV-infected animals.

A protein sequence that can encode native structure by disfavoring alternate conformations.

The linear sequence of amino acids contains all the necessary information for a protein to fold into its unique three-dimensional structure. Native protein sequences are known to accomplish this by promoting the formation of stable, kinetically accessible structures. Here we describe a Pro residue in the center of the third transmembrane helix of the cystic fibrosis transmembrane conductance regulator that promotes folding by a distinct mechanism: disfavoring the formation of a misfolded structure. The generality of this mechanism is supported by genome-wide transmembrane sequence analyses. Furthermore, the results provide an explanation for the increased frequency of Pro residues in transmembrane alpha-helices. Incorporation by nature of such 'negative folding determinants', aimed at preventing the formation of off-pathway structures, represents an additional mechanism by which folding information is encoded within the evolved sequences of proteins.