Entire Protein Chain Research Articles

Chemical Synthesis of Microtubule-Associated Protein Tau.

Deposits of the microtubule-associated protein Tau (MAPT) serve as a hallmark of neurodegenerative diseases known as tauopathies. Numerous studies have demonstrated that in diseases such as Alzheimer's disease (AD), Tau undergoes extensive remodeling. The attachment of post-translational modifications distributed throughout the entire sequence of the protein correlates with clinical presentation. A systematic examination of these protein alterations can shed light on their roles in both healthy and diseased states. However, the ability to access these modifications in the entire protein chain is limited as Tau can only be produced recombinantly or through semisynthesis. In this article, we describe the first chemical synthesis of the longest 2N4R isoform of Tau, consisting of 441 amino acids. The 2N4R Tau was divided into 3 major segments and a total of 11 fragments, all of which were prepared via solid-phase peptide synthesis. The successful chemical strategy has relied on the strategic use of two cysteine sites (C291 and C322) for the native chemical ligations (NCLs). This was combined with modern preparative protein chemistries, such as mercaptothreonine ligation (T205), diselenide-selenoester ligation (D358), and mutations of mercaptoamino acids into native residues via homogeneous radical desulfurization (A40, A77, A119, A157, A246, and A390). The successful completion of the synthesis has established a robust and scalable route to the native protein in multimilligram quantities and high purity. In broader terms, the presented strategy can be applied to the preparation of other shorter isoforms of Tau as well as to introduce all post-translational modifications that are characteristic of tauopathies such as AD.

Bioengineering of Novel Non-Replicating mRNA (NRM) and Self-Amplifying mRNA (SAM) Vaccine Candidates Against SARS-CoV-2 Using Immunoinformatics Approach.

Presently, the world needs safe and effective vaccines to overcome the COVID-19 pandemic. Our work has focused on formulating two types of mRNA vaccines that differ in capacity to copy themselves inside the cell. These are non-amplifying mRNA (NRM) and self-amplifying mRNA (SAM) vaccines. Both the vaccine candidates encode an engineered viral replicon which can provoke an immune response. Hence we predicted and screened twelve epitopes from the spike glycoprotein of SARS-CoV-2. We used five CTL, four HTL, and three B-cell-activating epitopes to formulate each mRNA vaccine. Molecular docking revealed that these epitopes could combine with HLA molecules that are important for boosting immunogenicity. The B-cell epitopes were adjoined with GPGPG linkers, while CTL and HTL epitopes were linked with KK linkers. The entire protein chain was reverse translated to develop a specific NRM-based vaccine. We incorporate gene encoding replicase in the upstream region of CDS encoding antigen to design the SAM vaccine. Subsequently, signal sequences were added to human mRNA to formulate vaccines. Both vaccine formulations translated to produce the epitopes in host cells, initiate a protective immune cascade, and generate immunogenic memory, which can counter future SARS-CoV-2 viral exposures before the onset of infection.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12033-021-00432-6.

A Global Machine Learning Based Scoring Function for Protein Structure Prediction

We present a knowledge-based function to score protein decoys based on their similarity to native structure. A set of features is constructed to describe the structure and sequence of the entire protein chain. Furthermore, a qualitative relationship is established between the calculated features and the underlying electromagnetic interaction that dominates this scale. The features we use are associated with residue-residue distances, residue-solvent distances, pairwise knowledge-based potentials and a four-body potential. In addition, we introduce a new target to be predicted, the fitness score, which measures the similarity of a model to the native structure. This new approach enables us to obtain information both from decoys and from native structures. It is also devoid of previous problems associated with knowledge-based potentials. These features were obtained for a large set of native and decoy structures and a back-propagating neural network was trained to predict the fitness score. Overall this new scoring potential proved to be superior to the knowledge-based scoring functions used as its inputs. In particular, in the latest CASP (CASP10) experiment our method was ranked third for all targets, and second for freely modeled hard targets among about 200 groups for top model prediction. Ours was the only method ranked in the top three for all targets and for hard targets. This shows that initial results from the novel approach are able to capture details that were missed by a broad spectrum of protein structure prediction approaches. Source codes and executable from this work are freely available at http://mathmed.org/#Software and http://mamiris.com/.

A global machine learning based scoring function for protein structure prediction

ABSTRACTWe present a knowledge‐based function to score protein decoys based on their similarity to native structure. A set of features is constructed to describe the structure and sequence of the entire protein chain. Furthermore, a qualitative relationship is established between the calculated features and the underlying electromagnetic interaction that dominates this scale. The features we use are associated with residue–residue distances, residue–solvent distances, pairwise knowledge‐based potentials and a four‐body potential. In addition, we introduce a new target to be predicted, the fitness score, which measures the similarity of a model to the native structure. This new approach enables us to obtain information both from decoys and from native structures. It is also devoid of previous problems associated with knowledge‐based potentials. These features were obtained for a large set of native and decoy structures and a back‐propagating neural network was trained to predict the fitness score. Overall this new scoring potential proved to be superior to the knowledge‐based scoring functions used as its inputs. In particular, in the latest CASP (CASP10) experiment our method was ranked third for all targets, and second for freely modeled hard targets among about 200 groups for top model prediction. Ours was the only method ranked in the top three for all targets and for hard targets. This shows that initial results from the novel approach are able to capture details that were missed by a broad spectrum of protein structure prediction approaches. Source codes and executable from this work are freely available at http://mathmed.org/#Software and http://mamiris.com/. Proteins 2014; 82:752–759. © 2013 Wiley Periodicals, Inc.

An Evaluation of Explicit Receptor Flexibility in Molecular Docking Using Molecular Dynamics and Torsion Angle Molecular Dynamics

Incorporating receptor flexibility into molecular docking should improve results for flexible proteins. However, the incorporation of explicit all-atom flexibility with molecular dynamics for the entire protein chain may also introduce significant error and "noise" that could decrease docking accuracy and deteriorate the ability of a scoring function to rank native-like poses. We address this apparent paradox by comparing the success of several flexible receptor models in cross-docking and multiple receptor ensemble docking for p38α mitogen-activated protein (MAP) kinase. Explicit all-atom receptor flexibility has been incorporated into a CHARMM-based molecular docking method (CDOCKER) using both molecular dynamics (MD) and torsion angle molecular dynamics (TAMD) for the refinement of predicted protein-ligand binding geometries. These flexible receptor models have been evaluated, and the accuracy and efficiency of TAMD sampling is directly compared to MD sampling. Several flexible receptor models are compared, encompassing flexible side chains, flexible loops, multiple flexible backbone segments, and treatment of the entire chain as flexible. We find that although including side chain and some backbone flexibility is required for improved docking accuracy as expected, docking accuracy also diminishes as additional and unnecessary receptor flexibility is included into the conformational search space. Ensemble docking results demonstrate that including protein flexibility leads to to improved agreement with binding data for 227 active compounds. This comparison also demonstrates that a flexible receptor model enriches high affinity compound identification without significantly increasing the number of false positives from low affinity compounds.

Nicked apomyoglobin: a noncovalent complex of two polypeptide fragments comprising the entire protein chain.

Limited proteolysis of the 153-residue chain of horse apomyoglobin (apoMb) by thermolysin results in the selective cleavage of the peptide bond Pro88-Leu89. The N-terminal (residues 1-88) and C-terminal (residues 89-153) fragments of apoMb were isolated to homogeneity and their conformational and association properties investigated in detail. Far-UV circular dichroism (CD) measurements revealed that both fragments in isolation acquire a high content of helical secondary structure, while near-UV CD indicated the absence of tertiary structure. A 1:1 mixture of the fragments leads to a tight noncovalent protein complex (1-88/89-153, nicked apoMb), characterized by secondary and tertiary structures similar to those of intact apoMb. The apoMb complex binds heme in a nativelike manner, as given by CD measurements in the Soret region. Second-derivative absorption spectra in the 250-300 nm region provided evidence that the degree of exposure of Tyr residues in the nicked species is similar to that of the intact protein at neutral pH. Also, the microenvironment of Trp residues, located in positions 7 and 14 of the 153-residue chain of the protein, is similar in both protein species, as given by fluorescence emission data. Moreover, in analogy to intact apoMb, the nicked protein binds the hydrophobic dye 1-anilinonaphthalene-8-sulfonate (ANS). Taken together, our results indicate that the two proteolytic fragments 1-88 and 89-153 of apoMb adopt partly folded states characterized by sufficiently nativelike conformational features that promote their specific association and mutual stabilization into a nicked protein species much resembling in its structural features intact apoMb. It is suggested that the formation of a noncovalent complex upon fragment complementation can mimic the protein folding process of the entire protein chain, with the difference that the folding of the complementary fragments is an intermolecular process. In particular, this study emphasizes the importance of interactions between marginally stable elements of secondary structure in promoting the tertiary contacts of a native protein. Considering that apoMb has been extensively used as a paradigm in protein folding studies for the past few decades, the novel fragment complementing system of apoMb here described appears to be very useful for investigating the initial as well as late events in protein folding.

Binding and folding: in search of intramolecular chaperone-like building block fragments.

We propose an intramolecular chaperone which catalyzes folding and neither dissociates nor is cleaved. This uncleaved foldase is an intramolecular chain-linked chaperone, which constitutes a critical building block of the structure. Macroscopically, all molecular chaperones facilitate folding reactions and manifest similar energy landscapes. However, microscopically they differ. While intermolecular chaperones catalyze folding by unfolding misfolded conformations or prevent misfolding, the chain-linked cleaved (proregion) and uncleaved intramolecular chaperone-like building blocks suggested here, catalyze folding by binding to, stabilizing and increasing the populations of native conformations of adjacent building block fragments. In both, the more stable the intramolecular chaperone fragment region, the faster is the folding rate. Hence, mechanistically, intramolecular chaperones and chaperone-like segments are similar. Both play a dual role, in folding and in protein function. However, while the functional role of the proregions is inhibitory, necessitating their cleavage, the function of the uncleaved intramolecular chaperone-like building blocks does not require their subsequent removal. On the contrary, it requires that they remain in the structure. This may lead to the difference in the type of control they are under: proteins folding with the assistance of the proregion have been shown to be under kinetic control. It has been suggested that kinetically controlled folding reactions, with the proregion catalyst removed, lend longevity under harsh conditions. On the other hand, proteins with uncleaved intramolecular chaperone-like building blocks, with their 'foldases' still attached, are largely under thermodynamic control, consistent with the control observed in most protein folding reactions. We propose that an uncleaved intramolecular chaperone-like fragment occurs frequently in proteins. We further propose that such proteins would be prone to changing conditions and in particular, to mutations in this critical building block region. We describe the features qualifying it for its proposed chaperone-like role, compare it with inter- and intramolecular chaperones and review current literature in this light. We further propose a mechanism showing how it lowers the barrier heights, leading to faster folding reaction rates. Since these fragments constitute an intergal part of the protein structure, we call these critical building blocks intramolecular, chaperone-like fragments, to clarify, distinguish and adhere to the definition of the transiently associating chaperones. The new mechanism presented here differs from the concept of 'folding nuclei'. While the concept of folding nuclei focuses on a non-sequential distribution of the folding information along the entire protein chain, the chaperone-like building block fragments proposition focuses on a segmental distribution of the folding information. This segmental distribution controls the distributions of the populations throughout the hierarchical folding processes.

Cysteine to serine replacements in 6-hydroxy-D-nicotine oxidase. Consequences for enzyme activity, cofactor incorporation, and formation of high molecular weight protein complexes with molecular chaperones (GroEL)

6-Hydroxy-D-nicotine oxidase, an enzyme with FAD covalently attached to the protein, contains 6 cysteine residues in positions 45 (Cys1), 59 (Cys2), 136 (Cys3), 173 (Cys4), 260 (Cys5) and 433 (Cys6). Cys2, 3, 5, and 6 were replaced with serine by site-directed mutagenesis. The effects of these exchanges on enzyme activity, the autocatalytic incorporation of the cofactor, and the interaction of the mutant proteins with molecular chaperones were analyzed. The flavinylation of 6-hydroxy-D-nicotine oxidase is dependent on the presence of allosteric effectors, e.g. glycerol 3-phosphate or other phosphorylated tricarbon compounds. Replacement of Cys2 or Cys5 abolished this dependence. Covalent incorporation of FAD was reduced to an undetectable level in the Cys3 and Cys5 mutants. Replacement of Cys6 by Ser had no significant effect on enzyme activity and cofactor attachment. Deletion of two amino acids, Phe and Arg, situated 12 and 11 amino acid residues, respectively, from the carboxyl terminus of the protein, resulted in an inactive enzyme with no covalently bound FAD. This result indicates that almost the entire protein chain has to be synthesized before the cofactor can be incorporated, making a cotranslational flavinylation step rather unlikely. The distribution of the 6-hydroxy-D-nicotine oxidase polypeptide between the high molecular weight complexes and the free soluble form was analyzed by gel filtration on Sephacryl S-200. The wild-type holoenzyme as well as the wild-type apoenzyme were recovered in the eluent fraction of the column while the mutant proteins were retained in high molecular weight complexes, predominantly in those associated with GroEL, as revealed by immunoprecipitation. The extent of complex formation with this molecular chaperone depended on the position of the mutated Cys residue within the protein. Complex formation was highest with protein from the mutants Cys2 and Cys3, less with the Cys5, and absent with the Cys6 mutant protein. Thus, alterations in the amino-terminal part of the 6-hydroxy-D-nicotine oxidase appear more important for the interaction with molecular chaperones than alterations situated in the carboxyl-terminal part of the protein.

T-cell recognition and antigen presentation of lysozyme.

Several years ago, this laboratory introduced a comprehensive strategy for the systematic localization of all the continuous sites on a protein that are involved in B- and T-cell recognition. The strategy depends on the synthesis of consecutive overlapping peptides that together account for the entire protein chain. Using this approach, the full submolecular profile of continuous regions on hen egg lysozyme recognized by T cells (T sites) were localized. Four major T-cell recognition sites, three of which were subject to individual genetic control, were localized in the six mouse strains examined. In addition to these four continuous T sites, T-cell recognition of lysozyme also involved the three previously defined discontinuous antibody binding sites as demonstrated with lysozyme-specific long-term T cell cultures. Contrary to a long held impression, T-cell recognition, therefore, is not restricted only to sequence features, but can also be directed to protein discontinuous surface areas of high conformational dependency. More recently, we have examined in two mouse strains the proliferative response to peptides and to native protein of lymph node cells from mice primed with synthetic overlapping peptides either individually or as a mixture. It was found that the pattern of T-cell recognition observed after priming with peptides differs from that obtained when the native protein is used as the immunogen. If antigen processing proceeds via fragmentation, then only those regions containing T sites would be expected to be effective in priming for a T-cell response to the intact protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Haemoglobin binding with haptoglobin. Localization of the haptoglobin-binding sites on the beta-chain of human haemoglobin by synthetic overlapping peptides encompassing the entire chain.

A synthetic approach was employed to identify the haptoglobin-binding sites on the beta-chain of human haemoglobin. This approach consists of the synthesis of a series of consecutive overlapping peptides that, together, systematically represent the entire protein chain. Fourteen synthetic peptides (beta 1-15, beta 11-25 etc.) were examined for their ability to bind human haptoglobin by quantitative solid-phase radiometric titrations of 125I-labelled haptoglobin. Of these 14 peptides only peptides beta 11-25 and beta 131-146 bound haptoglobin significantly; peptide beta 21-35 exhibited a small binding activity as a consequence of the overlap with peptide beta 11-25. On this basis and by examination of the three-dimensional structure of haemoglobin, it was concluded that the beta-chain of haemoglobin has two binding sites for haptoglobin that reside in, but do not necessarily encompass all of, the regions beta 11-25 and beta 131-146.

T cell recognition of ragweed allergen Ra3: Localization of the full T cell recognition profile by synthetic overlapping peptides representing the entire protein chain

In the preceding report, we described the systematic localization of the full profile of the continuous antigenic sites on ragweed allergen, Ra3, with antibodies from three different host species using a comprehensive strategy, previously introduced by this laboratory. The strategy consists of studying the immunochemical activity of a series of consecutive synthetic overlapping peptides, of uniform size and overlaps, which encompass the entire protein chain. This study reports the localization of the continuous regions on Ra3 that are recognized by T cells from mice immunized with Ra3. The 10 overlapping peptides encompassing the entire Ra3 molecule were examined in vitro for their ability to stimulate lymph node cells from Ra3-primed BALB/c (H-2d), C3H/He (H-2k) SWR (H-2q) and SJL (H-2s) mice. Several regions of the molecule (T sites) were found to stimulate Ra3-primed lymph node cells. This strategy has enabled the localization of the full submolecular profile of T cell recognition of the Ra3 molecule by these mouse strains. Three of the regions recognized by T cells coincided with regions recognized by antibodies (i.e. B cells). It is noteworthy that in addition to sites recognized by both T and B cells the protein has at least one site which is recognized exclusively by T cells and to which no detectable antibody response is directed.

Antibody recognition of ragweed allergen Ra3: Localization of the full profile of the continuous antigenic sites by synthetic overlapping peptides representing the entire protein chain

A comprehensive synthetic approach, for the localization of the full profile of the continuous antigenic sites on proteins, previously introduced by this laboratory, was applied here to localize the continuous antigenic sites of ragweed allergen, Ra3. The following 10 consecutive peptides, each comprising 15 residues (except for peptide 91-101) and overlapping each of its neighbors by 5 residues, were synthesized and purified: 1-15, 11-25, 21-35, 31-45, 41-55, 51-65, 61-75, 71-85, 81-95 and 91-101. Quantitative radiometric titrations of protein and peptide adsorbents were performed with 125I-labeled anti-Ra3 IgG antibodies from rabbit, outbred mouse and human antisera. The specificity of antibody binding to peptide adsorbents was confirmed by inhibition experiments. These studies established the full profile of antigenic (IgG-binding) sites of Ra3 and permitted comparison with the allergenic (IgE-binding) sites recently localized. It was found that the recognition by IgG antibodies was independent of the host species in which the antibodies were raised. Furthermore, the regions recognized by human IgE antibodies coincided with those recognized by IgG antibodies in three different species. Thus, Ra3 was found to have 4 continuous antigenic sites which occupy the same locations as the allergenic sites.

Antigenic structure of human hemoglobin: Delineation of the antigenic site (site 2) within region 41–65 of the alpha chain by immunochemistry of synthetic peptides

A comprehensive synthetic approach consisting of a series of consecutive, uniform overlapping peptides encompassing the entire protein chain was recently used to determine the full antigenic profile of the α-chain of human hemoglobin (Hb). The peptides synthesized enabled the localization of five major “continuous” antigenic regions within the α chain. The present findings describe the delineation of an antigenic site (site 2) residing within the region 41–65. Ten peptides representing the α-chain regions 41–55, 51–65, 45–54, 45–56, 45–58, 45–60, 48–56, 49–56, 50–56, and 51–56 were synthesized and purified. Quantitative radioimmunoadsorbent titrations were used to determine binding to peptide adsorbents of radioiodinated anti-Hb antibodies that were raised in rabbit, goat, and outbred mouse. In one set of peptides, the N-terminal was fixed while the C-terminal end was increased by increments of two residues from Gln-54 to Lys-60 (i.e., peptides 45–54, 45–45, 45–58, and 45–60). Binding studies revealed that maximum antibody activity resided in peptide 45–45, indicating that Lys-56 marks the C-terminal boundary of the site. In the second set of peptides, the C-terminal was fixed at Lys-56 while the peptides were elongated at their N-terminal by one-residue increments from Gly-51 to Leu-48. Antibody-binding studies with these peptides indicated that Ser-49 defines the N-terminal boundary of the site. Therefore, the antigenic site within region 41–65 of the α chain comprises residues 49–56. The relevance of these findings to the immune recognition of Hb and other proteins is discussed.

Antigen presentation of myoglobin: profiles of T cell proliferative responses following priming with synthetic overlapping peptides encompassing the entire molecule.

Recently, the regions of myoglobin, which are recognized by T cells (T sites), were localized by a comprehensive synthetic strategy in which uniform synthetic overlapping peptides encompassing the entire protein chain were examined for stimulation of T cell proliferative activity. In this study, we report about the proliferative response to these peptides, as well as to the native protein, of lymph node cells from mice primed with the overlapping peptides either individually or in a mixture. Some, but not all, of the T site-containing peptides were effective in priming for an anti-myoglobin T cell response. Further, several peptides, which were highly immunogenic as free synthetic peptides, were not associated with any of the known T sites in this protein. Thus, the pattern of T cell recognition following priming with the overlapping peptides differs from the pattern observed when the native protein is the priming antigen. If antigen processing proceeds via fragmentation, then only those regions containing T sites would be expected to be effective in priming for a T cell response to the intact protein and, conversely, highly immunogenic peptides would correspond to T sites of the protein. Therefore, these findings indicate that the current concept of antigen fragmentation as a prerequisite for its presentation must be reappraised. We suggest that, in the presentation of a protein antigen, the protein is recognized predominantly intact and that the crucial aspects of presentation are determined by interaction with the cell membrane which trigger cellular activating events.

T cell recognition of lysozyme. IV. Localization and genetic control of the continuous T cell recognition sites by synthetic overlapping peptides representing the entire protein chain.

Recently, this laboratory has developed a comprehensive strategy for the systematic localization of all the 'continuous' antigenic (as well as other binding) sites of complex multivalent protein antigens involved in B and T cell recognition. The strategy depends on the synthesis of consecutive overlapping peptides that together account for the entire protein chain. This strategy was applied here for the localization of the 'continuous' T cell recognition sites of hen egg lysozyme. Eight overlapping peptides encompassing the entire protein chain of lysozyme were synthesized and examined for their ability to stimulate in vitro proliferation of T cells from several mouse strains (A/J, H-2a; BALB/c and DBA/2, H-2d; B10.BR, H-2k; DBA/1, H-2q; SJL, H-2s) that had been primed with native lysozyme. This approach enabled the identification of a full profile of in vitro active lysozyme peptides and the localization of four major T cell recognition sites, three of which were subject to individual control.

T cell recognition of myoglobin. Localization of the sites stimulating T cell proliferative responses by synthetic overlapping peptides encompassing the entire molecule.

A comprehensive strategy for the systematic localization of all continuous antigenic sites within a protein has previously been introduced by this laboratory. The strategy consists of studying the immunochemical activity of a series of consecutive synthetic peptides that encompass the entire protein chain and that are uniform in size and in overlap at their N- and C-terminals with neighbouring peptides. By application of this strategy to sperm whale myoglobin, we have been able to delineate the continuous sites of T cell recognition of myoglobin in three high responder mouse strains. Thirteen 17-residue peptides that encompass the entire myoglobin chain and overlap by five residues at both ends were synthesized, purified and characterized. The peptides were examined in vitro for their ability to stimulate lymph node cells from myoglobin-primed DBA/2 (H-2d), BALB/c (H-2d) and SJL (H-2s) mice as well as long-term cultures of myoglobin-specific T cells. Several regions of the molecule (T sites) were found to stimulate myoglobin-primed lymph node cells and myoglobin-specific longterm T cell cultures. This strategy has enabled the localization of the full profile of dominant sites of T cell recognition in myoglobin for these mouse strains. Of these T sites, one region, residues 107-125, was clearly immunodominant in these strains and was found to coincide with the antigenic (i.e. antibody binding) site 4 of myoglobin. Also, other regions stimulated T cells and appeared to coincide with previously known antigenic sites. It is noteworthy that, in addition to sites recognized by both T and B cells, the protein has other sites which are recognized exclusively by T cells and to which no detectable antibody response is directed.

A novel approach for localization of the continuous protein antigenic sites by comprehensive synthetic surface scanning: antibody and T-cell activity to several influenza hemagglutinin synthetic sites.

The determination in this laboratory of the complete antigenic structures of several proteins initially relied on a multi-approach complex chemical strategy and revealed that antigenic sites are surface locations which could be either 'continuous' or 'discontinuous' in architecture. More recently, we introduced a simplified comprehensive synthetic approach for the localization of the continuous antigenic sites of a protein. The approach depends on the synthesis of consecutive overlapping peptides, of uniform size and overlaps, and encompass the entire protein chain, from the beginning to the end. The latter approach is rather costly and labor-intensive, especially when applied to large protein molecules. All these studies showed, however, that protein antigenic sites occupy surface areas on a protein molecule. In order to render the determination of protein antigenic sites more feasible within a reasonable period of time, we considered that only the protein surface needs to be examined. Thus, for a protein of known three-dimensional structure, the protein surface can be readily screened for the continuous antigenic sites by the systematic synthesis and examination of immunochemical activity of all exposed segments of the protein. We have applied this approach here to influenza A virus hemagglutinin. Twelve peptides (11 reported for the first time here, and one reported previously), representing continuous surface segments of the molecule, have so far been synthesized, purified, characterized and their immunochemical activity studied. The peptides were found to bind anti-viral antibodies raised in outbred mice and antibodies in human sera from individuals that had suffered a recent influenza A infection. In one mouse strain (Balb/c; H-2d) so far examined, several of the peptides stimulated an in vitro proliferative response of T cells from virus (X-31)- primed mice. Finally, antisera to the peptides were raised in mice and, as expected, were found to bind to intact virus. In most cases, anti-peptide antibodies, did not bind disrupted virus. These studies indicate that protein 'continuous' antigenic sites can be localized by systematic synthetic scanning of the surface. It is emphasized that the approach is useful only for the localization of 'continuous' sites. The results also reveal that the antigenic structure of influenza virus hemagglutinin is more complex than has hitherto been suspected.

Molecular localization of the full profile of the continuous regions recognized by myoglobin-primed T-cells using synthetic overlapping peptides encompassing the entire molecule.

The molecular localization of the full antigenic profile for T-cell recognition of a complex multi-determinant protein antigen has not to date been accomplished. Previously, this laboratory has introduced a comprehensive strategy for the systematic localization of all continuous antigenic sites within a protein. This strategy depends on the synthesis of a series of overlapping peptides that together account for the entire structure of a protein. Such a strategy has been applied, in this report, to the delineation of the continuous sites of T-cell recognition of sperm whale myoglobin. Thirteen peptides, accounting for the entire protein chain, were synthesized and subsequently examined in vitro for their ability to stimulate lymph node cells from myoglobin primed DBA/2 (H-2d) mice, a known high responder. This strategy has enabled for the first time the localization of the full profile of the protein regions which contain the sites of T-cell recognition. Three regions gave a high response (one being immunodominant and coinciding with antigenic, i.e. antibody binding, site 4 of myoglobin). At least three regions appear to coincide with previously known antigenic (antibody binding) sites. Noteworthy is the finding of regions that are recognized by T-cells but to which no detectable antibody response is directed.

Haemoglobin binding with haptoglobin. Localization of the haptoglobin-binding site on the alpha-chain of human haemoglobin.

A synthetic approach was employed to identify the haptoglobin-binding site on the alpha-chain of human haemoglobin. This approach cosists of the synthesis of a series of consecutive overlapping peptides that, together, systematically represent the entire protein chain. Fourteen peptides were synthesized (alpha 1-15, alpha 11-25, alpha 21-35, alpha 31-45, alpha 41-55, alpha 51-65, alpha 61-75, alpha 71-85, alpha 81-95, alpha 91-105, alpha 101-115, alpha 111-125, alpha 121-135 and alpha 131-141), and their ability bind human haptoglobin was studied, Only peptide alpha 121-135 bound haptoglobin significantly. On this basis we conclude that the haptoglobin-binding site on the alpha-chain of haemoglobin resides within, but does not necessarily encompass all of, the region alpha 121-135.

Immunochemical studies of fragments of bovine serum albumin

Antigenic properties of 14 fragments of bovine albumin were measured using antisera to albumin and to two of its fragments. All seven fragments larger than 21,000 daltons formed immune precipitates. Although immune precipitates were not formed with smaller fragments, inhibition tests indicated the presence of antigenic sites on several of these fragments as well. The results predict the occurrence of six or more antigenic determinants and allow assignment of their positions in the parent molecule. These sites are distributed along the entire protein chain, with the sites of greatest antibody affinity situated in the COOH-terminal region. Evidence is presented that some sites are homologous, reacting with the same populations of antibodies, and that other sites are unique, binding to an exclusive population of antibodies.