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Drawing upon the combined expertise of the international "who's who" in amino acid research, this series is a real benchmark for amino acid chemistry, providing a comprehensive discussion of the occurrence, uses and applications of amino acids and, by extension, their polymeric forms, peptides and proteins. The practical value of each volume is heightened by the inclusion of experimental procedures. Clearly structured in three main sections, this second volume in the series begins with the synthesis and chemistry of modified amino acids.

The second part deals with the catalysis of reactions by amino acids, while the final section is devoted to enzymes, including proteases as catalysts, semisynthetic enzymes, catalysis by peptide-based enzyme models, substrate and protein recognition, as well as mammalian and insect peptide hormones.

Three conceptually different approaches are available for the design of protein-binding site mimetic peptides. These approaches are based on one or more of the following information about the proteins of interest: structure, sequence, and function. In random combinatorial methods that are based solely on protein function, such as phage display Li and Caberoy, and synthetic peptide combinatorial libraries Houghten et al. A strategy termed peptide scanning is based on the synthesis of the entire protein sequence — or large parts of it — in the shape of short, overlapping peptides, which are then individually tested for binding to the respective partner protein Frank, , enabling the identification of protein-binding sites.

Structure-based design, finally, involves the design and generation of protein-binding site mimics based on the 3D structure of the protein—protein complexes Eichler, Types of protein-binding sites illustrated by the HIV-1 envelope protein gp The epitope V3-loop tip, pink is located in a single sequence stretch and can be reproduced in a single peptide. The binding site is located in three sequentially discontinuous segments of the protein sequence yellow, green, and red. In a mimetic peptides, these three fragments are presented through a molecular scaffold. Here, we review strategies for the use of synthetic peptides as protein mimics.

Focusing on structure-based design, the potential of such peptides as drugs against diseases, such as viral and bacterial infections, cancer, as well as autoimmune diseases, are discussed. While the recombinant synthesis of proteins containing non-proteinogenic amino acids is possible only through alternative codon usage Mehl et al. This opens the door to improved biological activity and peptide stability, as well as structural modifications.

Peptides composed of these amino acids are stable against proteolysis in vitro and in vivo , as well as metabolism and degradation by microbial colonies Seebach et al. Building blocks for chemical peptide synthesis. A Amino acid derivatives with modified backbone length and side-chain orientation. B Amino acid derivatives with modified aromatic side chains. C Scaffolds for multivalent or discontinuous peptide presentation. Another possibility is the use of d -amino acids. While recombinantly synthesized peptides and proteins are typically composed entirely of l -amino acids, chemical peptide synthesis can also use d -amino acids, which has been shown to increase the proteolytic stability while maintaining biolocical activity when d -amino acids are introduced at defined positions of an antimicrobial peptide Hong et al.

At other positions, on the other hand, using d -amino acids instead of l -amino acids had the opposite effect due to structural damage to the peptide Hong et al. Furthermore, oligomers of N -alkyl glycine monomers, termed peptoids, have been introduced as proteolytically stable peptide derivatives Simon et al. As the amide hydrogen is missing in peptoids, the typical backbone hydrogen bonds present in proteins and peptides cannot be formed, altering the conformational preferences of these molecules.

Peptoids have been used as mimics of antimicrobial peptides termed as ampetoids Chongsiriwatana et al. In addition to alteration of the peptide backbone, the use of non-proteinogenic amino acids enables the introduction of chemical moieties that are not presented by the proteinogenic amino acids, and which can be used to dissect the binding mode of peptides. Functionalized and orthogonally protected amino acids are often used for chemo selective ligation strategies Tornoe et al. In addition, lysine, among other amino acids, can be used for the synthesis of branched peptides Franke et al. Furthermore, a range of scaffold molecules, such as trimesic acid derivatives Berthelmann et al.

These secondary structures are stabilized by hydrogen bonds between amide nitrogen and carbonyl oxygen atoms. Bullock et al. The various strategies of mimicking protein-binding sites through secondary structure mimics have also been extensively reviewed recently Pelay-Gimeno et al. Replacing hydrogen bonds by salt bridges has been reported by Otaka et al. Woods et al. The relatively rigid structure of Hao-containing peptides preserves the structure of the recognition strand, and at the time serves as a template for the recognition strand.

Some peptides are able to be structurally rearranged in response to external stimuli, such as temperature, pH, ionic strength, and presence of special ions and light. In , Mart et al. In addition, special applications in medicine, such as drug delivery, tissue engineering, tissue regeneration, wound healing, and nerve cell regrowth rely upon stimuli-responsive peptides. In this review, two selected examples are presented. One example is the use of an azobenzene moiety as light-sensitive switch Woolley, ; Renner and Moroder, To make this approach more feasible for in vivo application, longer wavelengths should be used for azobenzene isomerization, considering UV-light scattering through cells and tissues.

Samanta et al. Stimuli responsive peptides. B Transition of a random coil peptide upon temperature stimulus Pochan et al. Pochan et al. Other peptides undergo non-reversible hydrogelation when heated Max1, Max2 Pochan et al. Because of their biocompatibility, biodegradability, weak immunogenicity and selectivity, peptidic hydrogels can serve as potential cancer drugs and antimicrobials, as well as for wound healing Mart et al.

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Current drug discovery and development approaches are focused on three different types of molecules Craik et al. The traditional approach of using small molecules as drugs is still widely used. While small molecules have been shown to be excellent tools to block the catalytic site of enzymes, as well as the ligand binding sites of numerous receptors, they are less promising for the inhibition of protein—protein interactions, which often involve larger interfaces, which typically cannot be adequately addressed by small molecules.

Therefore, protein-based drugs, so-called Biologics, are increasingly used as inhibitors of protein—protein interactions. Many proteins, however, have additional effector functions or binding sites for other ligands, causing problems in in vivo applications. Furthermore, proteins can be immunogenic, resulting in immunological clearance before reaching their target site.

As an alternative to both small molecule and protein-based drugs, peptides are becoming more relevant as drug candidates, as documented by an increasing number of peptide drugs approved for clinical use Fosgerau and Hoffmann, Due to their potential for highly specific binding, combined with low immunogenicity, peptides are promising candidates as inhibitors of protein—protein interactions. Specific protein—protein interactions are involved in the pathogenesis of numerous diseases.

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The design and generation of peptides that mimic the respective protein-binding site, as potential inhibitors of the interactions, is therefore a promising therapeutic strategy. Such mimetic molecules are typically designed based on the 3D structure of the protein—protein complex, which yields information on the location of the binding sites within the proteins, as well as the hot spot amino acids directly involved in the intermolecular interaction Eichler, This general strategy will be illustrated here using examples of the various protein—protein interactions, which are involved in the entry of the human immunodeficiency virus type 1 HIV-1 into cells.

Furthermore, a range of protein-mimicking peptides used in the treatment of cancer and as antibiotics or anti-inflammatory compounds, will be reviewed. The highly active antiretroviral therapy HAART has been a breakthrough in the treatment of HIV-1 infection, leading to an effective reduction of morbidity and mortality through drastic suppression of viral replication and, hence, reduction of plasma HIV-1 viral load. HAART consists of a mixture of at least three different drugs with at least two different molecular targets [for details see Arts and Hazuda ].

Almost all of these drugs are small molecules that address intracellular targets. Due to the high genetic variability of HIV-1, the virus is able to rapidly become resistant against drugs. Therefore, there is an ongoing need for new therapeutic strategies against HIV One of these strategies is the prevention of HIV-1 entry into its host cell by blocking the interactions between viral and host proteins that are involved in the entry process. This can be achieved by using peptides, which mimic the binding sites of the involved proteins.

Entry of HIV-1 into its host cells is initiated by a cascade of protein—protein interactions between the viral and host cell proteins. These interactions involve the trimeric viral spike, composed of glycoproteins gp and gp41, as well as the primary receptor CD4 and corecptors CCR5 and CXCR4 on the host cell Wilen et al.

In contrast to the generally high genetic variability of HIV-1, the CD4-binding site of gp is highly conserved. Peptides mimicking the CD4-binding site are therefore promising candidates as HIV-1 entry inhibitors. Furthermore, as the epitopes of various broadly neutralizing anti-HIV-1 antibodies have been shown to overlap the CD4-binding site, this part of gp is an immunogen candidate for the generation of HIV-1 neutralizing antibodies.

Based on the X-ray structure of gp in complex with CD4 Kwong et al. While the triazacyclophane scaffold peptide did not affect HIV-1 infection Chamorro et al. Peptide mimics of the CD4-binding site of gp Highlighted in orange, blue, and green are the fragments forming the discontinuous CD4 binding site. B CD4bs-M Franke et al. C CD4-binding site mimic with triazacyclophane scaffold Chamorro et al. Understanding the molecular and structural details of the interaction of antibodies with their viral antigens is an important step in the quest for a still elusive HIV-1 vaccine Burton et al.

Robinson et al. Coupling of such a stabilized V3-loop mimic to a lipopeptide carrier, which self-assembles into virus-like particles Ghasparian et al. Phage display peptide libraries Smith, have often been used to identify peptides that bind to antibodies and thus mimic their epitopes mimotopes. Mimotopes of the broadly neutralizing HIV-1 antibody b12 have been found Boots et al. As the viral spike proteins gp and gp41 are presented as trimers, Schellinger et al. This trimeric peptide bound to b12 substantially better than the monomeric mimotope, illustrating the importance of trimeric presentation, which was achieved using the so-called click reaction Rostovtsev et al.

Peptide mimics of turn structures. C V3-loop mimic, stabilized via d -Proline and l -Proline Riedel et al. Trimeric presentation of a b12 mimotope in conjunction with a T-helper cell epitope Schellinger et al. In addition to gp mimetic peptides, peptides that present parts of gp41 are also intensively researched Cai et al. In particular this applies to peptides that mimic a six-helix bundle, consisting of a three-stranded coiled-coil structures formed by an N-terminal NHR and a C-terminal CHR heptad repeat of gp41 Chan et al. Peptides presenting parts of the six-helical bundle are thought to be able to interfere with its correct formation and, consequently, inhibit virus-cell fusion.

Already in , Wild et al. Furthermore, the peptide exhibited a strong anti-HIV-1 activity, which could be further enhanced through dimerization. Trimers of the NHR-mimetic peptide were later found to be better HIV-1 entry inhibitors than the respective monomeric peptide Nakahara et al. Covalent stabilization of such peptide trimers through inter-chain disulfide bridges dramatically increased the antiviral potency Bianchi et al.

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Peptide mimics of HIV-1 gp Similar to the NHR mimics, peptides mimicking the CHR region of gp41 were developed to inhibit the formation of the six-helical bundle. In , Wild et al.

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Later on, the first and so far only HIV-1 fusion inhibitor approved for clinical use Enfuvirtide was developed based on this peptide Kilby and Eron, ; Lalezari et al. Another fusion inhibitor, called Sifuvirtide, was developed based on the 3D structure of HIV-1 gp41 and computer modeling He et al. Sifuvirtide could effectively block six-helical bundle formation and was active even against Enfuvirtide-resistant HIV-1 strains.

Otaka et al. Trimeric presentation of a CHR mimetic peptide on a C 3 -symmetric scaffold dramatically increased the antiviral activity of the peptide Nomura et al. Cellular receptors play important roles in signal transduction pathways, as well as in viral entry. As discussed in the previous chapter, HIV-1 contacts two receptors on the host cell surface prior to fusion with the cell membrane. Peptides that mimic these receptors are useful tools to explore the details of virus infection mechanism, as well as to develop new drugs against HIV In , Drakopoulou et al.

To retain the native structure of the CDR H2-like loop, the peptide was transferred onto a scorpion toxin, which served as a structural scaffold. Optimizing CD4M led to a variant withfold increased affinity to gp, as well as infection-inhibitory activity Vita et al. Based on the X-ray structure of CD4 in complex with gp Kwong et al. This peptide was able to bind to gp at low nanomolar concentrations, inhibit binding of CD4 to gp, as well as to induce conformational changes in gp similar to those triggered by CD4, from which it was derived.

The importance of conformational stability of CD4 mimetic peptides could be further confirmed by Meier et al. Peptides that present the binding site of CD4 for gp were covalently stabilized in their loop structure by cyclization through a disulfide bond between the N- and C-terminus. Using alanine and d -phenylalanine substitution analogs, the importance of the hot spot amino acid phenylalanine 43 could be confirmed at the peptide level. These results were further confirmed by molecular dynamics simulations. The concept of mimicking protein-binding sites through complex synthetic peptides has recently been extended to peptides that mimic the extracellular domains of seven transmembrane G protein-coupled receptors GPCRs , which is composed of the N-terminus NT and the three extracellular loops ECLs.

Although the 3D structures of both receptors are available Wu et al. Therefore, peptides that mimic the binding site of these receptors for gp could be useful tools for the exploration of HIVcoreceptor interaction at the molecular level. Peptide mimics of cellular receptors. The extracellular loops are highlighted in orange, red, and green.

The N-terminus is depicted in blue. These sequence stretches are presented in the CRF1 mimetic peptide Pritz et al. In a similar approach, Pritz et al. Improving the scaffold for the presentation of the ECLs and N-terminus, as well as increasing the overall yields through synthesis optimization, enabled structural analysis of the receptor mimic — ligand interaction through NMR spectroscopy Abel et al.

The epidermal growth factor receptor EGFR , which is a key protein of cell proliferation and differentiation Yarden and Sliwkowski, , has also been subject to structure-based design of receptor mimetic peptides. As the receptor forms dimers or even oligomers, Hanold et al. This peptide was stabilized via a triazole crosslink to increase proteolytic stability, while retaining the native structure, resulting in inhibition of EGFR dimerization and, consequently, a reduction of cell viability.

Sequence and functional optimization of EGFR mimetic peptides may be useful for the development of novel cancer drugs addressing EGFR overexpression in tumors. The uncontrolled growth and spread of cells into tumor tissue Vogelstein and Kinzler, defines cancer as one of the main fatal diseases worldwide. Therefore, a major focus in peptide drug development is on oncology Kaspar and Reichert, ; Fosgerau and Hoffmann, Apart from using peptides directly as anticancer drugs Thundimadathil, , they can also serve as targeting agents to direct highly toxic chemotherapeutics to their respective targets, reducing the systemic toxicity of these drugs [for details see Kaspar and Reichert ].

Structure-based approaches are often used in the design of anticancer peptides, such as the inhibitor of cell migration and invasion published by Bifulco et al. The urokinase-type plasminogen activator receptor uPAR , which plays a critical role in cancer cell growth, survival, invasion and metastasis, contains a five amino acid sequence SRSRY between two of its three domains, which is exposed through ligand binding, and mediates chemotactic properties of uPAR. An important target for the therapy of pancreatic, gastritic, and colorectal tumors is gastrin, a peptide hormone, whose activity can be blocked by antibodies that recognize gastrin as their epitope, delaying tumor growth Watson et al.

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Detailed analysis of the antibody epitopes through alanine scanning of gastrin Barderas et al. With the aim to shrink these antibodies to the size of peptidomimetics, Timmerman et al. In most cases, the activity of the obtained peptides was much lower compared to the parent antibodies. To confirm that the Fmoc protecting groups are removed, a kaiser test is performed. Then another Fmoc amino acid is attached by activation of its carboxyl group Coupling.

See examples of the coupling agents at Fig. A kaiser test is needed to confirm the complete coupling. The process is repeated through a cycle of deprotection, coupling and washing until the peptide is completely synthesized. Then the synthesized peptide is cleaved from the resin and side chain protection groups are removed. The synthetic peptide purification is usually including the peptide precipitation from the cleavage reaction mixture by the addition of diethyl ether.

The peptide and resin mixture can be suspended in water or aqueous acid and filtered to remove the resin. Further purification can be by gel-filtration, ion exchange chromatography and reversed-phase HPLC. Many types of research require peptides of 80— amino acids. The unprotected peptide chains react chemo-selectively in aqueous solution. The most common form of chemical ligation involves a peptide thioester that reacts with a terminal cysteine residue. Most synthetic strategies may yield crude products that cannot be purified by standard chromatographic protocols.

The PeptideSynTM technology allows selective coupling of unprotected peptide fragments.

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Subsequent purification involves only the removal of unreacted fragments. This technology is routine at LifeTein in the synthesis of long peptides for research purposes.

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LifeTein has already succeeded in producing very long and complex peptides that could not be produced elsewhere. Peptide Purification Solid-phase peptide synthesis involves only a limited number of undesirable components—by-products. However, the identification and removal of these undesirable components can be problematic. Acidolytic cleavage yields a crude product containing both the desired peptide and impurities, such as deletion peptides, truncated peptides, incompletely deprotected peptides, modified peptides, scavengers, and by-products derived from the cleaved protecting groups.

All these contaminants have to be removed. The impurities are very structurally similar to the desired peptide product. This necessitates high-performance methods such as UV peak detection and reversed phase high-performance liquid chromatography RP-HPLC , which uses Cmodified silica as the stationary phase. The purification of relatively small numbers of peptides requires a universal HPLC material. The process must be very chemically stable so that the same column can be for the acidic, basic, and neutral eluents. The pores must be large enough to allow for good mass transfer characteristics for the largest of the synthetic peptides but not so large so that the loadings are low for the smaller peptides.

The particles should be spherical so that they can be packed using dynamic axial compression and should have a narrow particle size distribution to promote permeability and packed bed stability. The properties of an individual peptide depend on the composition and sequence of amino acids. The target peptide and impurities are retained by the stationary phase depending on their hydrophobicity. Very polar contaminants are eluted at the beginning of the process, with aqueous 0.

Then the polarity of the eluent is gradually reduced by continuously increases of the proportion of acetonitrile.