Chemical synthesis and semisynthesis of membrane proteins

Total chemical synthesis and semisynthesis of proteins have become widely used tools to alter and control the chemical structure of soluble proteins, Thus, offering unique possibilities to understand protein function in vitro and in vivo. However, these approaches rely on our ability to produce and chemoselectively link peptide segments with each other or with recombinantly produced protein segments. Access to integral membrane and membrane-associated proteins via these approaches has been hampered by the fact that integral membrane peptides or lipid-modified peptides are difficult to obtain mostly due to incomplete amino acid coupling reactions and their poor handling properties. This article will highlight the advances in the total chemical synthesis and semisynthesis of small viral as well as bacterial ion channels. Recent synthesis approaches for membrane-associated proteins will be discussed as well.     

Combinatorial SNARE complexes modulate the secretion of cytoplasmic granules in human neutrophils

Mobilization of human neutrophil granules is critical for the innate immune response against infection and for the outburst of inflammation. Human neutrophil-specific and tertiary granules are readily exocytosed upon cell activation, whereas azurophilic granules are mainly mobilized to the phagosome. These cytoplasmic granules appear to be under differential secretory control. In this study, we show that combinatorial soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes with vesicle-associated membrane proteins (VAMPs), 23-kDa synaptosome-associated protein (SNAP-23), and syntaxin 4 underlie the differential mobilization of granules in human neutrophils. Specific and tertiary granules contained VAMP-1, VAMP-2, and SNAP-23, whereas the azurophilic granule membranes were enriched in VAMP-1 and VAMP-7. Ultrastructural, coimmunoprecipitation, and functional assays showed that SNARE complexes containing VAMP-1, VAMP-2, and SNAP-23 mediated the rapid exocytosis of specific/tertiary granules, whereas VAMP-1 and VAMP-7 mainly regulated the secretion of azurophilic granules. Plasma membrane syntaxin 4 acted as a general target SNARE for the secretion of the distinct granule populations. These data indicate that at least two SNARE complexes, made up of syntaxin 4/SNAP-23/VAMP-1 and syntaxin 4/SNAP-23/VAMP-2, are involved in the exocytosis of specific and tertiary granules, whereas interactions between syntaxin 4 and VAMP-1/VAMP-7 are involved in the exocytosis of azurophilic granules. Our data indicate that quantitative and qualitative differences in SNARE complex formation lead to the differential mobilization of the distinct cytoplasmic granules in human neutrophils, and a higher capability to form diverse SNARE complexes renders specific/tertiary granules prone to exocytosis.     

Enzymatic semisynthesis of [LeuB30] insulin

Experimental conditions for the preparation of [LeuB30] insulin by coupling of des-AlaB30 insulin with Leu-OBu(t) were determined using Achromobacter protease I and trypsin as catalysts. Successful coupling required a large excess of the amine component (0.8 M), a high concentration of organic cosolvent (35-50%) and neutral pH of the reaction mixture. The coupling yield of Achromobacter protease I after 24 h at 37 degrees C was almost the same or a little higher than that at 25 degrees C. With trypsin, the coupling yield at 37 degrees C after 24 h was considerably lower than at 25 degrees C. This was partly ascribed to the difference in concentration of organic cosolvent at 37 degrees C and 25 degrees C; 35% and 50%, respectively, or possibly of enzyme stability at these temperatures. The maximum product yield was about 90% with both enzymes under optimal conditions. A preparative scale experiment was performed with Achromobacter protease I; the yield of [LeuB30] insulin was 51% using porcine insulin as the starting material. This semisynthetic insulin was identified by HPLC and amino acid analysis. No difference was observed in CD spectra between [LeuB30] insulin and human insulin.     

Design and semisynthesis of spermine-sensitive Ribonuclease S'.

Spermine-sensitive stabilization of semisynthetic Ribonuclease S' was successfully carried out by sequence specific incorporation of a poly-anion domain into alpha-helix region of S-peptide.     

Enzymatic semisynthesis of human insulin: an update

Peptide bond formation can be enzymatically catalysed by the reverse reaction of proteases. Application is seen in the industrial production of human insulin. Human insulin derivative can be enzymatically prepared using either porcine insulin or the single chain B(1-29)-A(1-21) insulin precursor as the starting material. This is accomplished by either (1) digesting the starting material at Lys29 with Achromobacter lyticus protease I (Ach) and then coupling with Thr-X (X = blocking residue) (two-step reaction) or (2) subjecting Ala-B30 of porcine insulin or Gly-A1 of the single chain insulin precursor to transpeptidation with Thr-X (one-step reaction). Trypsin and Ach can be used for either type of reaction and, in the immobilized form, for the two-step reaction. Since the single chain insulin precursor can be produced by gene technology (yeast), use of immobilized trypsin or Ach and the two-step reaction using the single chain insulin precursor as the starting material ensures the continuous production of human insulin making it a feasible method for industrial manufacture.     

Kinetic characterization of carboxypeptidase-Y-catalyzed peptide semisynthesis Prediction of yields

Summary Carboxypeptidase-Y-catalyzed peptide semisynthesis has been characterized at pH 7.5, 25°C from initial rate steady state kinetic and progress reaction studies of hydrolysis and aminolysis of-N-benzoyl-L-tyrosine 4-nitro-anilide using the natural L-amino acids and their amides as nucleophiles. The reaction mechanism previously shown to account for carboxypeptidase-Y-catalyzed aminolysis reactions (Christensen et al., 1992) was found also to account for all of the reactions studied here. It involves in addition to the classical serine proteinase mechanism: i) complex formation between the free enzyme and the nucleophile, an interaction characterized by the competitive inhibition constant,K _i, and ii) reaction of the nucleophile with the acylated enzyme forming a complex of enzyme and aminolysis product, characterized by the aminolysis kinetic parameter,K _N.A competitive inhibitory effect showing binding to the free enzyme is seen mainly with large hydrophobic amino acids and their amides i.e. the same residues as those preferred on either side of the scissile bond in carboxypeptidase-Y substrates. The stoichiometry of the inhibition is 1 : 1 and the actual binding position most likely is that of the leaving group of substrates,S ₁.Aminolysis effects are obtained with a wide range of amino acids and amino acid amides, exceptions are Pro and, probably due to their low solubility, Tyr, Trp, Asp and Glu. TheK _N-values show relatively little dependence on the chemical nature of the side groups, but a marked difference between the amino acid and its amide. The amides interact more strongly. The kinetic parameter,k _c/K_m, of the hydrolysis of the aminolysis products is another important factor in peptide semisynthesis. Thek _c/K_m-values obtained of the amidated aminolysis products are much less than those of the products formed with free amino acids. All in all this leads to rather efficient aminolysis with the L-amino acid amides and poor aminolysis with the L-amino acids.Abbreviations BzTyrNHPhNO₂ -N-benzoyl-L-tyrosinyl 4-nitro-aniline - Xaa L-amino acids - Xaaa L-amino acid amides - Z-Phe Carbobenzoxy-L-phenylalanine - Z-Met Carbobenzoxy-L-methionine - BzTyr -N-benzoyl-L-tyrosine - AlaVal L-alanyl-L-valine - ValAla L-valyl-L-alanine     

An approach to the semisynthesis of acyl carrier protein

A scheme has been devised for the preparation of semisynthetic derivatives of acyl carrier protein (ACP). Acetylated synthetic ACP_1–6 is coupled via its activated pentachlorophenol ester to native ACP (7–77), which had previously been acetylated and converted to the S-5′-dithiobis(2-nitrobenzoate)(DTNB) derivative. Removal of the DTNB moiety after the coupling yielded active ACP in good yield.     

Enzyme-assisted semisynthesis of polypeptide active esters and their use.

A method is described for the preparation of polypeptides activated uniquely at the C-terminus. The polypeptide is incubated in a concentrated solution of an amino acid active ester, the latter having its amino group free but adequately protected by protonation. The amino acid ester is coupled via its amino group to the C-terminus of the polypeptide by enzymic catalysis (reverse proteolysis). The resulting polypeptide C-terminal active ester is then isolated and coupled to a suitable amino component (generally a polypeptide) in a subsequent chemical coupling. The method appears to be generally applicable; fragments of horse heart cytochrome c, and porcine insulin, are used as examples. Two new analogues of cytochrome c have been prepared by using this method, with yields of up to 60% in the final coupling. Scope and limitations of the method are discussed.     

The preparation of protected fragments of lysozyme for semisynthesis.

This paper reports the development of methods for preparing tryptic fragments of hen's-egg lysozyme in an appropriate state of protection for use in the chemical synthesis of modified polypeptides. 1. We describe the cleavage of the disulphide bridges of the enzyme and the simulatneous protection of the liberated thiol groups by S-sulphonation. Lysozyme resisted the usual conditions for this reaction. We have confirmed the stability of the S-sulphonyl group to the conditions met in peptide synthesis. 2. We describe the reversible protection of the amino groups of the enzyme by reaction with various anhydrides of 1,2-dicarboxylic acids. We conclude that 2-methylmaleic anhydride and exo-cis-3,6-endoxo-delta4-tetrahydrophthalic anhydride are unsuitable for our purpose but that maleic anhydride can, in spite of certain drawbacks, be used. 3. We describe the tryptic cleavage of the thiol- and amino-protected protein and the separation of the fragments. 4. We describe the reversible protection of the carboxylic acid groups (including the specific deprotection of the alpha-carboxyl group), the imidazolyl group and the aloph-amino groups of the fragments. Several alternative groups have been evaluated for most of these purposes. The side-chain amides did not present any serious problem of libility, 5. We describe experiments on the stability of the side chain of tryptophan, both protected by formylation and unprotected, to the acid conditions needed for the deprotection of the other functional groups in the peptide. We conclude that protection of tryptophan is unnecessary. We suggest that most of the methods described are of general application in peptide semisynthesis by fragment condensation. An Appendix is included to which points 6-ll appertain...     

Combinatorial SNARE complexes with VAMP7 or VAMP8 define different late endocytic fusion events

Both heterotypic and homotypic fusion events are required to deliver endocytosed macromolecules to lysosomes and remodel late endocytic organelles. A trans-SNARE complex consisting of Q-SNAREs syntaxin 7, Vti1b and syntaxin 8 and the R-SNARE VAMP8 has been shown by others to be responsible for homotypic fusion of late endosomes. Using antibody inhibition experiments in rat liver cell-free systems, we confirmed this result, but found that the same Q-SNAREs can combine with an alternative R-SNARE, namely VAMP7, for heterotypic fusion between late endosomes and lysosomes. Co-immunoprecipitation demonstrated separate syntaxin 7 complexes with either VAMP7 or VAMP8 in solubilized rat liver membranes. Additionally, overexpression of the N-terminal domain of VAMP7, in cultured fibroblastic cells, inhibited the mixing of a preloaded lysosomal content marker with a marker delivered to late endosomes. These data show that combinatorial interactions of SNAREs determine whether late endosomes undergo homotypic or heterotypic fusion events.     

Combinatorial discovery of new asymmetric cis platinum anticancer complexes is made possible with solid-phase synthetic methods

To efficiently access asymmetric cis platinum (II) complexes for biological evaluation, a new solid-phase synthesis was designed. This synthesis was used for the preparation of a small library of platinum compounds. Several compounds from this library revealed promising activity during a cytotoxicity screen. Two active compounds were, therefore, synthesised on a larger scale and tested more extensively against a larger panel of cell-lines, confirming their high potential as antitumour compounds. The work presented illustrates how a combination of a new methodology and established techniques can speed up the search for platinum complexes with improved cytotoxic profiles compared to cisplatin.     

Combinatorial depletion analysis to assemble the network architecture of the SAGA and ADA chromatin remodeling complexes

Despite the availability of several large‐scale proteomics studies aiming to identify protein interactions on a global scale, little is known about how proteins interact and are organized within macromolecular complexes. Here, we describe a technique that consists of a combination of biochemistry approaches, quantitative proteomics and computational methods using wild‐type and deletion strains to investigate the organization of proteins within macromolecular protein complexes. We applied this technique to determine the organization of two well‐studied complexes, Spt–Ada–Gcn5 histone acetyltransferase (SAGA) and ADA, for which no comprehensive high‐resolution structures exist. This approach revealed that SAGA/ADA is composed of five distinct functional modules, which can persist separately. Furthermore, we identified a novel subunit of the ADA complex, termed Ahc2, and characterized Sgf29 as an ADA family protein present in all Gcn5 histone acetyltransferase complexes. Finally, we propose a model for the architecture of the SAGA and ADA complexes, which predicts novel functional associations within the SAGA complex and provides mechanistic insights into phenotypical observations in SAGA mutants.     

组合生物催化

自然界最有效的分子是由酶催化的反应所产生,并对这些产物进行自然选择,使其具有优化的生理活性,组合生物催化（Combinatorial Biocatalysis)利用酶反应的多样性,完成有机库（Organic Library)的反复合成,这些反复的反应,可以用分离的酶或全细胞,在天然或非天然的环境中、在溶液或固相中与底物进行反应。组合生物催化是组合方法的在药物发现和发展中产生和优化先导化合物（LeadCompound)的一个有力补充。     

Combinatorial biocatalysis

The published applications of combinatorial biocatalysis have continued to expand at a growing rate. This is exemplified by the variety of enzyme catalysts and whole-cell catalysts used for the creation of libraries through a wide range of biocatalytic reactions, including acylation, glycosylation, halogenation, oxidation and reduction. These biocatalytic methods add the capability to perform unique chemistries or selective reactions with complex or labile reagents when integrated with classical combinatorial synthesis methods. Thus, applications towards the production of libraries de novo, the expansion of chemically derived combinatorial libraries, and the generation of novel combinatorial reagents for library synthesis can be achieved. Theoretically, these results illustrate what is already evident from nature: that complex, biologically active, structurally diverse compound libraries can be generated through the application of biocatalysis alone or in combination with classical organic synthesis approaches.     

Enhancing protein engineering capabilities by combining mutagenesis and semisynthesis.

If site-directed mutagenesis could be used to facilitate protein semisynthesis, then structural engineering goals should be achieved that are unattainable by either technique alone. We tested this possibility by mutating Ser65 of yeast cytochrome c to methionine, creating a new site for CNBr cleavage. Fragments obtained by cleaving there were found to refold cooperatively, bringing together the breakpoint termini and leading to efficient autocatalytic peptide bond synthesis. Structurally modified fragments may be substituted for natural ones. Generally, naturally occurring sites are unsuitable for autocatalytic religation, for reasons briefly discussed, and thus the power of this new approach lies in the freedom to choose sites, including enzymatic ones, that are appropriate to the semisynthetic goals.