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Nathan A Schnarr

Publications and source records attributed to Nathan A Schnarr.

12 recordsLinked to original sources
PubMed2006

Extender unit and acyl carrier protein specificity of ketosynthase domains of the 6-deoxyerythronolide B synthase.

Polyketide synthases (PKSs) catalyze the production of numerous biologically important natural products via repeated decarboxylative condensation reactions. Modular PKSs, such as the 6-deoxyerythronolide B synthase (DEBS), consist of multiple catalytic modules, each containing a unique set of covalently linked catalytic domains. To better understand the engineering opportunities of these assembly lines, the extender unit and acyl carrier protein (ACP) specificity of keto synthase (KS) domains from modules 3 and 6 of DEBS were analyzed. These studies were undertaken with a newly developed didomain [KS][AT] construct, which lacks its own ACP domain and can therefore be interrogated with homologous or heterologous ACP or acyl-ACP substrates. By substituting the natural methylmalonyl extender unit with a malonyl group, a modest role was demonstrated for the KS in recognition of the nucleophilic substrate. The KS domain from module 3 of DEBS was found to exhibit a distinct ACP-recognition profile from the KS domain of module 6. On the basis of the above kinetic insights, a hybrid module was constructed ([KS3][AT3][KR5][ACP5][TE]) which displayed substrate recognition and elongation capabilities consistent with the natural module 3 protein. Unlike module 3, however, which lacks a ketoreductase (KR) domain, the hybrid module was able to catalyze reduction of the beta-ketothioester product of chain elongation. The high expression level and functionality of this hybrid protein demonstrates the usefulness of kinetic analysis for hybrid module design.

Acyl Carrier Protein↗
PubMed2005

Analysis of covalently bound polyketide intermediates on 6-deoxyerythronolide B synthase by tandem proteolysis-mass spectrometry.

Polyketide natural products are biosynthesized via successive chain-elongation events mediated by elaborate protein assemblies. Facile detection of protein-bound intermediates in these systems will increase our understanding of enzyme reactivity and selectivity. We have developed a tandem proteolysis/mass spectrometric method for monitoring substrate loading and elongation in 6-deoxyerythronolide B synthase (DEBS), responsible for production of the macrolide precursor to erythromycin. Information regarding ketosynthase loading and polyketide unit elongation is readily acquired without need for complex protein or small molecule labels. A panel of structurally related substrates is evaluated through competition experiments and kinetic assays using LC-MS to resolve closely related species. Strong stereochemical effects are observed for ketosynthase substrate specificity. Semiquantitative kinetic analyses allow the resolution of the effects of structural and stereochemical changes on the individual ketosynthase-catalyzed steps of acyl-enzyme formation and polyketide chain extension.

Acyl Carrier Protein↗
PubMed2005

Stereochemical assignment of intermediates in the rifamycin biosynthetic pathway by precursor-directed biosynthesis.

Natural and semisynthetic rifamycins are clinically important inhibitors of bacterial DNA-dependent RNA polymerase. Although the polyketide-nonribosomal peptide origin of the naphthalene core of rifamycin B is well established, the absolute and relative configuration of both stereocenters introduced by the first polyketide synthase module is obscured by aromatization of the naphthalene ring. To decode the stereochemistry of the rifamycin polyketide precursor, we synthesized all four diastereomers of the biosynthetic substrate for module 2 of the rifamycin synthetase in the form of their N-acetylcysteamine (SNAC) thioester. Only one diastereomer was turned over in vivo into rifamycin B, thus establishing the absolute and relative configuration of the native biosynthetic intermediates.

Actinomycetales↗
PubMed2005

pH-Switchable strand orientation in peptide assemblies.

[structure: see text] The design of antiparallel coiled-coil heterotrimers with singly mismatched electrostatic interfaces is reported. The new complexes exhibit expected properties for well-formed coiled-coils and have stabilities comparable to those of parallel analogues. The mismatched interface facilitates switching from a parallel to antiparallel complex by pH-triggered strand exchange.

Amino Acid Sequence↗
PubMed2004

Strand orientation by steric matching: a designed antiparallel coiled-coil trimer.

The design of an antiparallel coiled-coil 1:1:1 heterotrimer is described. Control of strand orientation results from proper alignment of sterically matched hydrophobic core side chains. Matched core layers position one cyclohexylalanine side chain against two alanine ones. Substitution of three consecutive heptad a positions with all permutations of two alanines and one cyclohexylalanine (AAX, AXA, AAX, where A = alanine, X = cyclohexylalanine) affords a parallel 1:1:1 heterotrimer, as previously reported. Here, we report that moving the substitution sites in one strand to d rather than a positions affords a new peptide that can form an antiparallel complex with the other original components. The new assembly is characterized by circular dichroism spectroscopy ([theta;](222) = -30 317 deg cm(2) dmol(-1), T(m) = 77 degrees C, DeltaG(unf) = 17.1 kcal/mol), and its stoichiometry and aggregation number are confirmed by nickel tag affinity analysis and analytical ultracentrifugation. Disulfide exchange data support the preference for an antiparallel arrangement. Examination of the functionally identical parallel complex demonstrates that the antiparallel structure is comparably stable, as confirmed by a direct competition assay that established an equilibrium 55:45 ratio of each assembly.

Alanine↗
PubMed2004

Coiled-coil surface presentation: an efficient HIV gp41 binding interface mimic.

An efficient mimic of the gp41 N-terminal coiled-coil trimer is described. The native protein mediates fusion of viral and cellular membranes, and its function is critical for infectivity. A central event in this process is formation of a "trimer of hairpins" structure in which a C-terminal gp41 sequence binds to a hydrophobic groove on the N-terminal trimer surface. Inhibition of this interaction is a promising therapeutic strategy, but the isolated trimer is not a convenient screening target, since the exposed hydrophobic pocket causes aggregation and precipitation. The problem has been circumvented in several ways such as attachment of auxiliary scaffolding elements or covalent subunit tethering. Here we report a more efficient approach, in which purely peptidic systems comparable in size to the native trimer display the expected specificity for the C-terminal ligand. Steric matching of 2:1 alanine/cyclohexylalanine core layers promotes formation of a 1:1:1 heterotrimer, whose surface interhelical interfaces can be uniquely controlled. Two of these interfaces contain solubilizing Glu/Lys pairs, while the third presents the gp41 interface. The model system binds the C-terminal peptide, while a control complex with only half the interface does not. A variety of biophysical methods are used to characterize the complex. The ability to control complex stoichiometry with only interior core residues should permit formation of any such interface, and extension to other viral systems is underway.

Amino Acid Sequence↗
PubMed2003

Sequential and specific exchange of multiple coiled-coil components.

The capacity for sequential and specific exchange of single peptides from coiled-coil heterotrimers is investigated. Dual hydrophobic-hydrophilic interface systems permit iterative cycles of pH-triggered strand exchange that can specifically replace one, two, or even all three initial trimer components. The resultant new complexes are either resistant to or capable of further exchange. Control experiments demonstrate that background exchange among different complexes is negligible. When triggered, however, selective displacement of the same peptide from only one of two distinct heterotrimers is feasible. Previously documented peptidic cross-linking strategies remain operative in these more intricate environments.

Alanine↗
PubMed2003

pH-triggered strand exchange in coiled-coil heterotrimers.

The capacity for pH-triggered strand exchange in designed coiled-coil heterotrimers is demonstrated. Systems employing both hydrophobic core (steric matching) and hydrophilic interface (electrostatic matching) design principles assemble into specific 1:1:1 heterotrimers. Alteration of pH creates electrostatic mismatches, inducing strand exchange in the presence of a suitable replacement peptide. Complexes with one Lys/Lys interface, favored at neutral to high pH, can be transformed to ones with a Glu/Glu contact by lowering pH and adding an appropriate new binding partner. The need to simultaneously maintain matched core alignments enforces specificity in this exchange, such that only a single specific peptide is replaced. These principles have subsequently been applied to the design of dynamically triggered cross-linked structures, in which a bifunctional disulfide-tethered peptide can cross-link two heterotrimers. Both formation and disruption of the cross-link are under pH control.

Amino Acid Sequence↗
PubMed2003

Specific control of peptide assembly with combined hydrophilic and hydrophobic interfaces.

Designed coiled-coil heterotrimers are described whose assembly is governed by both hydrophobic and hydrophilic forces. Sterically matched hydrophobic core side-chain packing of alanine and cyclohexylalanine has been shown to promote formation of a 1:1:1 heterotrimer. Manipulation of hydrophilic glutamic acid (Glu)/lysine (Lys) pairs at each of three helical contact interfaces provides a secondary recognition mechanism. Peptides with matched cores and hydrophilic contacts form stable heterotrimers (DeltaG(unf) at 25 degrees C = 17.93 kcal/mol; MW(app) = 11362 vs 11563 calcd for trimer), as do those with a single Lys/Lys (but not Glu/Glu) interface. The additional specificity engendered by simultaneous operation of two interfaces was used to design a system in which six different peptides are mixed to form three specific and independent heterotrimers in the same solution.

Alanine↗
PubMed2002

Peptide tic-tac-toe: heterotrimeric coiled-coil specificity from steric matching of multiple hydrophobic side chains.

Specific coiled-coil heterotrimers result from steric matching of hydrophobic core side chains. A 2:1 heterotrimer is formed by peptides containing alanine or cyclohexylalanine, respectively, at a central core residue. Detailed thermodynamic analysis reveals that the designed complex is considerably more stable than the corresponding alanine homotrimer (deltaT(m) = 25 degrees C, deltadeltaG(unf) = 4.5 kcal/mol), while control complexes with naphthylalanine or cyclopropylalanine peptides are much less stable. However, the cyclohexylalanine homotrimer is of comparable stability to the 2:1 complex, prompting an investigation of multiply substituted peptides. A specific 1:1:1 heterotrimer is formed from three independent peptide strands, each bearing one large (cyclohexylalanine) and two small (alanine) side chains at the same three core positions but in different order. The combined impact of three substitutions improves specificity to the point where each pure peptide and all pairwise equimolar mixtures form significantly less stable complexes (deltaTm = 22-24 degrees C). The capacity for specific complex formation governed by multiple unnatural core side chains should facilitate design of numerous new peptide assemblies.

Alanine↗