Excision of a proposed electron transfer pathway in cytochrome c peroxidase and its replacement by a ligand-binding channel

A previously proposed electron transfer (ET) pathway in the heme enzyme cytochrome c peroxidase has been excised from the structure, leaving an open ligand-binding channel in its place. Earlier studies on cavity mutants of this enzyme have revealed structural plasticity in this region of the molecule. Analysis of these structures has allowed the design of a variant in which the specific section of protein backbone representing a previously proposed ET pathway is accurately extracted from the protein. A crystal structure verified the creation of an open channel that overlays the removed segment, extending from the surface of the protein to the heme at the core of the protein. A number of heterocyclic cations were found to bind to the proximal-channel mutant with affinities that can be rationalized based on the structures. It is proposed that small ligands bind more weakly to the proximal-channel mutant than to the W191G cavity due to an increased off rate of the open channel, whereas larger ligands are able to bind to the channel mutant without inducing large conformational changes. The structure of benzimidazole bound to the proximal-channel mutant shows that the ligand accurately overlays the position of the tryptophan radical center that was removed from the wild-type enzyme and displaces four of the eight ordered solvent molecules seen in the empty cavity. Ligand binding also caused a small rearrangement of the redesigned protein loop, perhaps as a result of improved electrostatic interactions with the ligand. The engineered channel offers the potential for introducing synthetic replacements for the removed structure, such as sensitizer-linked substrates. These installed "molecular wires" could be used to rapidly initiate reactions, trap reactive intermediates, or answer unresolved questions about ET pathways.     

The Png1-Rad23 complex regulates glycoprotein turnover

Misfolded proteins in the endoplasmic reticulum (ER) are destroyed by a pathway termed ER-associated protein degradation (ERAD). Glycans are often removed from glycosylated ERAD substrates in the cytosol before substrate degradation, which maintains the efficiency of the proteasome. Png1, a deglycosylating enzyme, has long been suspected, but not proven, to be crucial in this process. We demonstrate that the efficient degradation of glycosylated ricin A chain requires the Png1-Rad23 complex, suggesting that this complex couples protein deglycosylation and degradation. Rad23 is a ubiquitin (Ub) binding protein involved in the transfer of ubiquitylated substrates to the proteasome. How Rad23 achieves its substrate specificity is unknown. We show that Rad23 binds various regulators of proteolysis to facilitate the degradation of distinct substrates. We propose that the substrate specificity of Rad23 and other Ub binding proteins is determined by their interactions with various cofactors involved in specific degradation pathways.     

Zinc-substituted cytochrome P450cam: characterization of protein conformers F420 and F450 by photoinduced electron transfer

Metal substitution of heme proteins is widely applied in the study of biologically relevant electron transfer (ET) reactions. It has been shown that many modified proteins remain in their native conformation and can provide useful insights into the molecular mechanism of electron transfer between the native protein and its substrates. We investigated ET reactions between zinc-substituted cytochrome P450(cam) and small organic compounds such as quinones and ferrocene, which are capable of accessing the protein's hydrophobic channel and binding close to the active site, like its native substrate, camphor. Following the substitution method developed by Gunsalus and co-workers [Wagner, G. C., et al. (1981) J. Biol. Chem. 256, 6262-6265], we have identified two dominant forms of the zinc-substituted protein, F450 and F420, that exhibit different photophysical and photochemical properties. The ET behavior of F420 suggests that hydrophobic redox-active ligands are able to penetrate the hydrophobic channel and place themselves in the direct vicinity of the Zn-porphyrin. In contrast, the slower ET quenching rates observed in the case of F450 indicate that the association is weak and occurs outside of the protein channel. Therefore, we conclude that F420 corresponds to the open structure of the native cytochrome P450(cam) while F450 has a closed or partially closed channel that is characteristic of the camphor-containing cytochrome P450(cam). The existence of two distinct conformers of Zn-bound P450(cam) is consistent with the findings of Goodin and co-workers [Lee, Y.-T., et al. (2010) Biochemistry 49, 3412-3419] and has significant consequences for future electron transfer studies on this popular metalloenzyme.     

Protein unfolding and degradation by the AAA+ Lon protease

AAA+ proteases employ a hexameric ring that harnesses the energy of ATP binding and hydrolysis to unfold native substrates and translocate the unfolded polypeptide into an interior compartment for degradation. What determines the ability of different AAA+ enzymes to unfold and thus degrade different native protein substrates is currently uncertain. Here, we explore the ability of the E. coli Lon protease to unfold and degrade model protein substrates beginning at N-terminal, C-terminal, or internal degrons. Lon has historically been viewed as a weak unfoldase, but we demonstrate robust and processive unfolding/degradation of some substrates with very stable protein domains, including mDHFR and titin(I27) . For some native substrates, Lon is a more active unfoldase than related AAA+ proteases, including ClpXP and ClpAP. For other substrates, this relationship is reversed. Thus, unfolding activity does not appear to be an intrinsic enzymatic property. Instead, it depends on the specific protease and substrate, suggesting that evolution has diversified rather than optimized the protein unfolding activities of different AAA+ proteases.     

泛素活化酶Ube1活性中心疏水区关键苯丙氨酸位点的丙氨酸突变对泛素传递活性的影响

泛素化系统调节真核细胞中几乎所有的生命活动,参与细胞内绝大多数蛋白质的降解,而疾病的发生往往伴随着相关蛋白质的异常。研究与疾病相关蛋白质的泛素化途径,通过该途径促进或抑制其活性对疾病的研究和治疗具有重大的意义。然而,细胞内的泛素传递网络错综复杂,天然条件下很难确认某一特定蛋白质的泛素化途径。我们前期工作建立的正交泛素传递途径中的泛素和泛素化酶含有特殊的突变,是鉴定某一特定E3下游蛋白质底物的有效手段。但E1与E2结合位点的突变设计仍有待完善。本研究重新审视了E1-E2的相互作用,着眼于泛素活化酶Ube1活性中心内的疏水区,突变了该区上3个关键的苯丙氨酸残基为丙氨酸,即F637A、F729A、F741A,并设置合理的实验对照来探究此突变对泛素从E1向E2传递活性的影响。结果表明,突变体m Ube1较好地阻断了与UbcH7的识别及Ub的传递,提示本研究涉及的3个关键苯丙氨酸位点对E1-E2互相识别的重要性,及其作为新的正交泛素传递途径中E1突变位点的合理性。     

Description of the torcetrapib series of cholesteryl ester transfer protein inhibitors, including mechanism of action

We have identified a series of potent cholesteryl ester transfer protein (CETP) inhibitors, one member of which, torcetrapib, is undergoing phase 3 clinical trials. In this report, we demonstrate that these inhibitors bind specifically to CETP with 1:1 stoichiometry and block both neutral lipid and phospholipid (PL) transfer activities. CETP preincubated with inhibitor subsequently bound both cholesteryl ester and PL normally; however, binding of triglyceride (TG) appeared partially reduced. Inhibition by torcetrapib could be reversed by titration with both native and synthetic lipid substrates, especially TG-rich substrates, and occurred to an equal extent after long or short preincubations. The reversal of TG transfer inhibition using substrates containing TG as the only neutral lipid was noncompetitive, suggesting that the effect on TG binding was indirect. Analysis of the CETP distribution in plasma demonstrated increased binding to HDL in the presence of inhibitor. Furthermore, the degree to which plasma CETP shifted from a free to an HDL-bound state was tightly correlated to the percentage inhibition of CE transfer activity. The finding by surface plasmon resonance that torcetrapib increases the affinity of CETP for HDL by approximately 5-fold likely represents a shift to a binding state that is nonpermissive for lipid transfer. In summary, these data are consistent with a mechanism whereby this series of inhibitors block all of the major lipid transfer functions of plasma CETP by inducing a nonproductive complex between the transfer protein and HDL.     

Preferential binding of yeast tRNA ligase to pre-tRNA substrates.

Joining of tRNA halves during splicing in extracts of Saccharomyces cerevisiae requires each of the three enzymatic activities associated with the tRNA ligase polypeptide. Joining is most efficient for tRNA as opposed to oligonucleotide substrates and is sensitive to single base changes at a distance from splice sites suggesting considerable specificity. To examine the basis for this specificity, binding of ligase to labeled RNA substrates was measured by native gel electrophoresis. Ligase bound tRNA halves with an association constant 1600-fold greater than that for a nonspecific RNA. Comparison of binding of a series of tRNA processing intermediates revealed that tRNA-structure, particularly in the region around the splice sites, contributes to specific binding. Finally, the ligase was shown to form multiple, discrete complexes with tRNA substrates. The basis for recognition by ligase and its role in a tRNA processing pathway are discussed.     

Tertiary motifs as building blocks for the design of protein‐binding peptides

Despite advances in protein engineering, the de novo design of small proteins or peptides that bind to a desired target remains a difficult task. Most computational methods search for binder structures in a library of candidate scaffolds, which can lead to designs with poor target complementarity and low success rates. Instead of choosing from pre‐defined scaffolds, we propose that custom peptide structures can be constructed to complement a target surface. Our method mines tertiary motifs (TERMs) from known structures to identify surface‐complementing fragments or “seeds.” We combine seeds that satisfy geometric overlap criteria to generate peptide backbones and score the backbones to identify the most likely binding structures. We found that TERM‐based seeds can describe known binding structures with high resolution: the vast majority of peptide binders from 486 peptide‐protein complexes can be covered by seeds generated from single‐chain structures. Furthermore, we demonstrate that known peptide structures can be reconstructed with high accuracy from peptide‐covering seeds. As a proof of concept, we used our method to design 100 peptide binders of TRAF6, seven of which were predicted by Rosetta to form higher‐quality interfaces than a native binder. The designed peptides interact with distinct sites on TRAF6, including the native peptide‐binding site. These results demonstrate that known peptide‐binding structures can be constructed from TERMs in single‐chain structures and suggest that TERM information can be applied to efficiently design novel target‐complementing binders.     

Crystal structures of complexes of bacterial DD-peptidases with peptidoglycan-mimetic ligands: the substrate specificity puzzle

The X-ray crystal structures of covalent complexes of the Actinomadura R39 dd-peptidase and Escherichia coli penicillin-binding protein (PBP) 5 with β-lactams bearing peptidoglycan-mimetic side chains have been determined. The structure of the hydrolysis product of an analogous peptide bound noncovalently to the former enzyme has also been obtained. The R39 dd-peptidase structures reveal the presence of a specific binding site for the d-α-aminopimelyl side chain, characteristic of the stem peptide of Actinomadura R39. This binding site features a hydrophobic cleft for the pimelyl methylene groups and strong hydrogen bonding to the polar terminus. Both of these active site elements are provided by amino acid side chains from two separate domains of the protein. In contrast, no clear electron density corresponding to the terminus of the peptidoglycan-mimetic side chains is present when these β-lactams are covalently bound to PBP5. There is, therefore, no indication of a specific side-chain binding site in this enzyme. These results are in agreement with those from kinetics studies published earlier and support the general prediction made at the time of a direct correlation between kinetics and structural evidence. The essential high-molecular-mass PBPs have demonstrated, to date, no specific reactivity with peptidoglycan-mimetic peptide substrates and β-lactam inhibitors and, thus, probably do not possess a specific substrate-binding site of the type demonstrated here with the R39 dd-peptidase. This striking deficiency may represent a sophisticated defense mechanism against low-molecular-mass substrate-analogue inhibitors/antibiotics; its discovery should focus new inhibitor design.     

Binding of Zn-chlorin to a synthetic four-helix bundle peptide through histidine ligation

We have used a two histidine-containing synthetic peptide (Sharp et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 10465-10470) as a scaffold to bind Zn(II) chlorin e6 (ZnCe6) through histidine ligation. Protocols for the preparation and purification of the peptide using an Escherichia coli expression system are presented. Size-exclusion chromatography and circular dichroism measurements indicate that the peptide self-assembles into a four-helix bundle protein. Two variants of the peptide lacking either one or both of the histidine residues were used to demonstrate the stoichiometry of ZnCe6 binding. Comparison of the titration profiles determined by UV-vis spectroscopy for the purified one- and two-histidine peptides suggests that the two-histidine peptide can bind two ZnCe6. The binding stoichiometry of ZnCe6 was verified by gel chromatography and native gel electrophoresis using the peptide variant lacking histidine residues as the control. Like many other chlorophyll analogue molecules, ZnCe6 can be photooxidized. The light-induced electron transfer between the ZnCe6-peptide complex and the added phenyl-p-benzoquinone was measured using time-resolved EPR spectroscopy and shown to be faster and have a higher yield than the electron transfer between unbound ZnCe6 and quinone. The implications of constructing a ZnCe6-peptide complex in terms of artificial photosynthesis are discussed.     

Nanowiring of a redox enzyme by metallized peptides

A molecular assembly consisting of a redox enzyme, NADH peroxidase, a metallized double-helical peptide, and a gold nanoparticle immobilized onto a gold wire derivatized with a benzenedithiol compound, initiated and conducted redox signals in the presence of H(2)O(2) and NADH. The current generated by the binding of NADH, the electron donor, was transduced through the molecular assembly with apparently little loss of signal to the solution. The currents measured correlate to an electron transfer rate constant on the order of 3,000 s(-1) within each assembly. This electron transfer rate is two orders of magnitude higher than the endogenous electron transfer rate from NADH to the native enzyme, 27 s(-1). This rate indicates that the metallized peptide is in a conformation conducive for electron transfer and, in conjunction with the redox enzyme, can form effective conduits of electrical signals. This work demonstrates the feasibility of utilizing designed and highly efficient biomolecular assemblies for the production of ultra-sensitive, in-situ biosensors.     

Biochemical and kinetic characterization of BACE1: investigation into the putative species-specificity for beta- and beta'-cleavage sites by human and murine BACE1

Beta-amyloid peptides (Abeta) are produced by a sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. The lack of Abeta production in beta-APP cleaving enzyme (BACE1)(-/-) mice suggests that BACE1 is the principal beta-secretase in mammalian neurons. Transfection of human APP and BACE1 into neurons derived from wild-type and BACE1(-/-) mice supports cleavage of APP at the canonical beta-secretase site. However, these studies also revealed an alternative BACE1 cleavage site in APP, designated as beta', resulting in Abeta peptides starting at Glu11. The apparent inability of human BACE1 to make this beta'-cleavage in murine APP, and vice versa, led to the hypothesis that this alternative cleavage was species-specific. In contrast, the results from human BACE1 transgenic mice demonstrated that the human BACE1 is able to cleave the endogenous murine APP at the beta'-cleavage site. To address this discrepancy, we designed fluorescent resonance energy transfer peptide substrates containing the beta- and beta'-cleavage sites within human and murine APP to compare: (i) the enzymatic efficiency; (ii) binding kinetics of a BACE1 active site inhibitor LY2039911; and (iii) the pharmacological profiles for human and murine recombinant BACE1. Both BACE1 orthologs were able to cleave APP at the beta- and beta'-sites, although with different efficiencies. Moreover, the inhibitory potency of LY2039911 toward recombinant human and native BACE1 from mouse or guinea pig was indistinguishable. In summary, we have demonstrated, for the first time, that recombinant BACE1 can recognize and cleave APP peptide substrates at the postulated beta'-cleavage site. It does not appear to be a significant species specificity to this cleavage.     

Deconstructing the DGAT1 Enzyme: Membrane Interactions at Substrate Binding Sites

Diacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in the triacylglyceride synthesis pathway. Bovine DGAT1 is an endoplasmic reticulum membrane-bound protein associated with the regulation of fat content in milk and meat. The aim of this study was to evaluate the interaction of DGAT1 peptides corresponding to putative substrate binding sites with different types of model membranes. Whilst these peptides are predicted to be located in an extramembranous loop of the membrane-bound protein, their hydrophobic substrates are membrane-bound molecules. In this study, peptides corresponding to the binding sites of the two substrates involved in the reaction were examined in the presence of model membranes in order to probe potential interactions between them that might influence the subsequent binding of the substrates. Whilst the conformation of one of the peptides changed upon binding several types of micelles regardless of their surface charge, suggesting binding to hydrophobic domains, the other peptide bound strongly to negatively-charged model membranes. This binding was accompanied by a change in conformation, and produced leakage of the liposome-entrapped dye calcein. The different hydrophobic and electrostatic interactions observed suggest the peptides may be involved in the interactions of the enzyme with membrane surfaces, facilitating access of the catalytic histidine to the triacylglycerol substrates.     

Exploration of the One-Bead One-Compound Methodology for the Design of Prolyl Oligopeptidase Substrates

Here we describe the design, synthesis and evaluation of the first solid-phase substrates for prolyl oligopeptidase (POP), a cytosolic serine peptidase associated with schizophrenia, bipolar affective disorder and related neuropsychiatric disorders. This study seeks to contribute to the future design of a one-bead one-compound (OBOC) peptide library of POP substrates, based on an intramolecular energy transfer substrate. Unexpectedly, the enzymatic evaluation of the substrates attached on solid-phase by means of the HMBA linker were cleaved through the ester bond, thereby suggesting an unknown esterase activity of POP, in addition to its known peptidase activity. By performing multiple activity assays, we have confirmed the esterase activity of this enzyme and its capacity to process the substrates on solid-phase. Finally, we tested a new linker, compatible with both the solid-phase peptide-synthesis used and the enzymatic assay, for application in the future design of an OBOC library.     

Computational, spectroscopic, and resonant mirror biosensor analysis of the interaction of adrenodoxin with native and tryptophan-modified NADPH-adrenodoxin reductase

In steroid hydroxylation system in adrenal cortex mitochondria, NADPH-adrenodoxin reductase (AR) and adrenodoxin (Adx) form a short electron-transport chain that transfers electrons from NADPH to cytochromes P-450 through FAD in AR and [2Fe-2S] cluster in Adx. The formation of [AR/Adx] complex is essential for the electron transfer mechanism in which previous studies suggested that AR tryptophan (Trp) residue(s) might be implicated. In this study, we modified AR Trps by N-bromosuccinimide (NBS) and studied AR binding to Adx by a resonant mirror biosensor. Chemical modification of tryptophans caused inhibition of electron transport. The modified protein (AR*) retained the native secondary structure but showed a lower affinity towards Adx with respect to AR. Activity measurements and fluorescence data indicated that one Trp residue of AR may be involved in the electron transferring activity of the protein. Computational analysis of AR and [AR/Adx] complex structures suggested that Trp193 and Trp420 are the residues with the highest probability to undergo NBS-modification. In particular, the modification of Trp420 hampers the correct reorientation of AR* molecule necessary to form the native [AR/Adx] complex that is catalytically essential for electron transfer from FAD in AR to [2Fe-2S] cluster in Adx. The data support an incorrect assembly of [AR*/Adx] complex as the cause of electron transport inhibition.     

Structural characteristics of channels and pathways in photosystem II including the identification of an oxygen channel

Here we use crystal structures to investigate and review channels and pathways for the transfer of substrates (water, plastoquinone (PQ)) and products (electrons, protons, oxygen, reduced PQ (PQH(2))) to, and from, the redox active catalytic sites of photosystem II (PSII). A putative oxygen channel has been identified which is about 21A in length, leading from the water splitting site to the lumen. This channel follows a path along the lumenal surface of CP43, passing across the interface of the large extrinsic loop which joins the fifth and sixth transmembrane helices of this chlorophyll binding protein. In so doing it seems to minimise interactions with the excited states of chlorophylls bound within the PSII complex, especially those that constitute the primary electron donor, P680. Two additional channels leading from the water splitting site, and also exiting at the lumen, were also identified. Their hydrophilic nature suggests that they probably facilitate the delivery of water to, and protons from, the catalytic site. Also discussed are unique features in the electron transfer pathway of PSII, as compared with those of purple photosynthetic bacteria, and structural implications of the PSII Q(B)-site in terms of PQ protonation and PQ/PQH(2) diffusion.     

Mass spectrometric mapping of ion channel proteins (porins) and identification of their supramolecular membrane assembly

Mass spectrometric peptide mapping, particularly by matrix-assisted laser desorption-ionization (MALDI-MS), has recently been shown to be an efficient tool for the primary structure characterization of proteins. In combination with in situ proteolytic digestion of proteins separated by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), mass spectrometric peptide mapping permits identification of proteins from complex mixtures such as cell lysates. In this study we have investigated several ion channel membrane proteins (porins) and their supramolecular assembly in mitochondrial membranes by peptide mapping in solution and upon digestion in the gel matrix. Porins are integral membrane proteins serving as nonspecific diffusion pores or as specific systems for the transport of substrates through bacterial and mitochondrial membranes. The well-characterized porin from Rhodobacter capsulatus (R.c.-porin) has been found to be a native trimeric complex by the crystal structure and was used as a model system in this study. R.c.-porin was characterized by MALDI-MS peptide mapping in solution, and by direct in situ-gel digestion of the trimer. Furthermore, in this study we demonstrate the direct identification of the noncovalent complex between a mitochondrial porin and the adenine nucleotide translocator from rat liver, by MALDI-MS determination of the specific peptides due to both protein sequences in the SDS-PAGE gel band. The combination of native gel electrophoresis and mass spectrometric peptide mapping of the specific gel bands should be developed as a powerful tool for the molecular identification of protein interactions. Proteins Suppl. 2:63–73, 1998. © 1998 Wiley-Liss, Inc.     

Evidence for lipid packaging in the crystal structure of the GM2-activator complex with platelet activating factor

GM2-activator protein (GM2-AP) is a lipid transfer protein that has the ability to stimulate the enzymatic processing of gangliosides as well as T-cell activation through lipid presentation. Our previous X-ray crystallographic studies of GM2-AP have revealed a large lipid binding pocket as the central overall feature of the structure with non-protein electron density within this pocket suggesting bound lipid. To extend these studies, we present here the 2A crystal structure of GM2-AP complexed with platelet activating factor (PAF). PAF is a potent phosphoacylglycerol whose toxic patho-physiological effects can be inhibited by GM2-AP. The structure shows an ordered arrangement of two bound lipids and a fatty acid molecule. One PAF molecule binds in an extended conformation within the hydrophobic channel that has an open and closed conformation, and was seen to contain bound phospholipid in the low pH apo structure. The second molecule is submerged inside the pocket in a U-shaped conformation with its head group near the single polar residue S141. It was refined as lyso-PAF as it lacks electron density for the sn-2 acetate group. The alkyl chains of PAF interact through van der Waals' contacts, while the head groups bind in different environments with their phosphocholine moieties in contact with aromatic rings (Y137, F80). The structure has revealed further insights into the lipid binding properties of GM2-AP, suggesting an unexpected unique mode of lipid packaging that may explain the efficiency of GM2-AP in inhibiting the detrimental biological effects of PAF.     

Development and characterization of synthetic antibodies binding to the cystic fibrosis conductance regulator

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel in the apical surface of epithelial cells in the airway and gastrointestinal tract, and mutation of CFTR is the underlying cause of cystic fibrosis. However, the precise molecular details of the structure and function of CFTR in native and disease states remains elusive and cystic fibrosis researchers are hindered by a lack of high specificity, high affinity binding reagents for use in structural and biological studies. Here, we describe a panel of synthetic antigen-binding fragments (Fabs) isolated from a phage-displayed library that are specific for intracellular domains of CFTR that include the nucleotide-binding domains (NBD1 and NBD2), the R-region, and the regulatory insertion loop of NBD1. Binding assays performed under conditions that promote the native fold of the protein demonstrated that all Fabs recognized full-length CFTR. However, only the NBD1-specific Fab recognized denatured CFTR by western blot, suggesting a conformational epitope requirement for the other Fabs. Surface plasmon resonance experiments showed that the R-region Fab binds with high affinity to both the phosphorylated and unphosphorylated R-region. In addition, NMR analysis of bound versus unbound R-region revealed a distinct conformational effect upon Fab binding. We further defined residues involved with antibody recognition using an overlapping peptide array. In summary, we describe methodology complementary to previous hybridoma-based efforts to develop antibody reagents to CFTR, and introduce a synthetic antibody panel to aid structural and biological studies.     

Substrate specificity of recombinant dengue 2 virus NS2B-NS3 protease: influence of natural and unnatural basic amino acids on hydrolysis of synthetic fluorescent substrates

A recombinant dengue 2 virus NS2B-NS3 protease (NS means non-structural virus protein) was compared with human furin for the capacity to process short peptide substrates corresponding to seven native substrate cleavage sites in the dengue viral polyprotein. Using fluorescence resonance energy transfer peptides to measure kinetics, the processing of these substrates was found to be selective for the Dengue protease. Substrates containing two or three basic amino acids (Arg or Lys) in tandem were found to be the best, with Abz-AKRRSQ-EDDnp being the most efficiently cleaved. The hydrolysis of dipeptide substrates Bz-X-Arg-MCA where X is a non-natural basic amino acid were also kinetically examined, the best substrates containing aliphatic basic amino acids. Our results indicated that proteolytic processing by dengue NS3 protease, tethered to its activating NS2B co-factor, was strongly inhibited by Ca2+ and kosmotropic salts of the Hofmeister's series, and significantly influenced by substrate modifications between S4 and S6'. Incorporation of basic non-natural amino acids in short peptide substrates had significant but differential effects on Km and k(cat), suggesting that further dissection of their influences on substrate affinity might enable the development of effective dengue protease inhibitors.