Physical characterization of the rack effect and hydrogen bond networks in blue copper proteins

A summary of previous research is presented that indicates that the purpose of a blue copper protein's fold and hydrogen bond network, aka, the rack effect, enforce a copper(II) geometry around the copper(I) ion in the metal site. In several blue copper proteins, the C-terminal histidine ligand becomes protonated and detaches from the copper in the reduced forms. Mutants of amicyanin from Paracoccus denitrificans were made to alter the hydrogen bond network and quantify the rack effect by pK_a shifts.

The pK_a's of mutant amicyanins have been measured by pH-dependent electrochemistry. P94F and P94A mutations loosen the Northern loop, allowing the reduced copper to adopt a relaxed conformation: the ability to relax drives the reduction potentials up. The measured potentials are 265 (wild type), 380 (P94A), and 415 (P94F) mV vs. NHE. The measured pK_a's are 7.0 (wild type), 6.3 (P94A), and 5.0 (P94F). The additional hydrogen bond to the thiolate in the mutants is indicated by a red-shift in the blue copper absorption and an increase in the parallel hyperfine splitting in the EPR spectrum. This hydrogen bond is invoked as the cause for the increased stability of the C-terminal imidazole.

Melting curves give a measure of the thermal stability of the protein. A thermodynamic intermediate with pH-dependent reversibility is revealed. Comparisons with the electrochemistry and apoamicyanin suggest that the intermediate involves the region of the protein near the metal site. This region is destabilized in the P94F mutant; coupled with the evidence that the imidazole is stabilized under the same conditions confirms an original concept of the rack effect: a high energy configuration is stabilized at a cost to the rest of the protein.

Two-body photodisintegration of the deuteron at intermediate energy

We have measured the differential cross section for two-body deuteron photodisintegration at center-of-mass angles of 90°, 53° and 37° with photon energies from 1.6 Ge V to 2.8 Ge V. Additional data were taken at θ* = 37° and E_γ = 4.2 GeV. Invariant cross sections at θ* =90° and 53° appear to follow a simple scaling law predicted by constituent counting rules of perturbative QCD, while the cross section at θ* = 37° shows a slower fall-off with photon energy. Angular distributions show increasing forward peaking at higher energies. Agreement with various theoretical predictions based on pQCD and meson-exchange models is discussed.

Capturing protein dynamics with time-resolved luminescence spectroscopy

The presented doctoral research utilizes time-resolved spectroscopy to characterize protein dynamics and folding mechanisms. We resolve millisecond-timescale folding by coupling time-resolved fluorescence energy transfer (trFRET) to a continuous flow microfluidic mixer to obtain intramolecular distance distributions throughout the folding process. We have elucidated the folding mechanisms of two cytochromes---one that exhibits two-state folding (cytochrome cb₅₆₂) and one that has both a kinetic refolding intermediate ensemble and a distinct equilibrium unfolding intermediate (cytochrome c₅₅₂). Our data reveal that the distinct structural features of cytochrome c₅₅₂ contribute to its thermostability.

We have also investigated intrachain contact dynamics in unfolded cytochrome cb₅₆₂ by monitoring electron transfer, which occurs as the heme collides with a ruthenium photosensitizer, covalently bound to residues along the polypeptide. Intrachain diffusion for chemically denatured proteins proceeds on the microsecond timescale with an upper limit of 0.1 microseconds. The power-law dependence (slope = -1.5) of the rate constants on the number of peptide bonds between the heme and Ru complex indicate that cytochrome cb₅₆₂ is minimally frustrated.

In addition, we have explored the pathway dependence of electron tunneling rates between metal sites in proteins. Our research group has converted cytochrome b₅₆₂ to a c-type cytochrome with the porphyrin covalently bound to cysteine sidechains. We have investigated the effects of the changes to the protein structure (i.e., increased rigidity and potential new equatorial tunneling pathways) on the electron transfer rates, measured by transient absorption, in a series of ruthenium photosensitizer-modified proteins.

Identification of fucose-α(1-2)-galactose binding proteins in the mammalian brain

Fucose-α(1-2)-galactose (Fucα(1-2)Gal) carbohydrates have been implicated in cognitive functions. However, the underlying molecular mechanisms that govern these processes are not well understood. While significant progress has been made toward identifying glycoconjugates bearing this carbohydrate epitope, a major challenge remains the discovery of interactions mediated by these sugars. Here, we employ the use of multivalent glycopolymers to enable the proteomic identification of weak affinity, low abundant Fucα(1-2)Gal-binding proteins (i.e. lectins) from the brain. End-biotinylated glycopolymers containing photoactivatable crosslinkers were used to capture and enrich potential Fucα(1-2)Gal-specific lectins from rat brain lysates. Candidate lectins were tested for their ability to bind Fucα(1-2)Gal, and the functional significance of the interaction was investigated for one such candidate, SV2a, using a knock-out mouse system. Our results suggest an important role for this glycan-lectin interaction in facilitating synaptic changes necessary for neuronal communication. This study highlights the use of glycopolymer mimetics to discover novel lectins and identify functional interactions between fucosyl carbohydrates and lectins in the brain.

Engineering multi-step electron tunneling systems in proteins

Multi-step electron tunneling, or “hopping,” has become a fast-developing research field with studies ranging from theoretical modeling systems, inorganic complexes, to biological systems. In particular, the field is exploring hopping mechanisms in new proteins and protein complexes, as well as further understanding the classical biological hopping systems such as ribonuclease reductase, DNA photolyases, and photosystem II. Despite the plethora of natural systems, only a few biologically engineered systems exist. Engineered hopping systems can provide valuable information on key structural and electronic features, just like other kinds of biological model systems. Also, engineered systems can harness common biologic processes and utilize them for alternative reactions. In this thesis, two new hopping systems are engineered and characterized.

The protein Pseudomonas aeruginosa azurin is used as a building block to create the two new hopping systems. Besides being well studied and amenable to mutation, azurin already has been used to successfully engineer a hopping system. The two hopping systems presented in this thesis have a histidine-attached high potential rhenium 4,7-dimethyl-1,10-phenanthroline tricarbonyl [Re(dmp)(CO)3] + label which, when excited, acts as the initial electron acceptor. The metal donor is the type I copper of the azurin protein. The hopping intermediates are all tryptophan, an amino acid mutated into the azurin at select sites between the photoactive metal label and the protein metal site. One system exhibits an inter-molecular hopping through a protein dimer interface; the other system undergoes intra-molecular multi-hopping utilizing a tryptophan “wire.” The electron transfer reactions are triggered by excitation of the rhenium label and monitored by UV-Visible transient absorption, luminescence decays measurements, and time-resolved Infrared spectroscopy (TRIR). Both systems were structurally characterized by protein X-ray crystallography.

Isolation and characterization of proteins that bind to yeast origins of DNA replication

Yeast chromosomes contain sequences called ARSs which function as origins of replication in vitro and in vivo. We have carried out a systematic deletion analysis of ARS1, allowing us to define three functionally distinct domains, designated A, B, and C. Domain A is a sequence of 11 to 19bp, containing the core consensus element that is required for replication. The core consensus sequence, A/TTTTATPuTTTA/T, is conserved at all ARSs sequenced to date. A fragment containing only element A and 8 flanking nucleotides enables autonomous replication of centromeric plasmids. These plasmids replicate very inefficiently, suggesting that flanking sequences must be important for ARS function. Domain B also provides important sequences needed for efficient replication. Deletion of domain B drastically increases the doubling times of transformants and reduces plasmid stability. Domain B contains a potential consensus sequence conserved at some ARSs which overlaps a region of bent DNA. Mutational analysis suggests this bent DNA may be important for ARS function. Deletion of domain C has only a slight effect on replication of plasmids carrying those deletions.

We have identified a protein called ARS binding factor I (ABF-I) that binds to the HMR-E ARS and ARS1. We have purified this protein to homogeneity using conventional and oligonucleotide affinity chromatography. The protein has an apparent molecular weight of 135kDa and is present at about 700 molecules per diploid cell, based on the yield of purified protein and in situ antibody staining. DNaseI footprinting reveals that ABF-I binds sequence-specifically to an approximately 24bp sequence that overlaps element Bat ARSl. This same protein binds to and protects a similar size region at the HMR-E ARS.

We also find evidence for another ARS binding protein, ABF-III, based on DN asei footprint analysis and gel retardation assays. The protein protects approximately 22bp adjacent to the ABF-I site. There appears to be no interaction between ABF-I and ABF-III despite the proximity of their binding sites.

To address the function of ABF-I in DNA replication, we have cloned the ABF-I gene using rabbit polyclonal anti-sera and murine monoclonal antibodies against ABF-I to screen a λgt11 expression library. Four EcoRI restriction fragments were isolated which encoded proteins that were recognized by both polyclonal and monoclonal antibodies. A gene disruption can now be constructed to determine the in vivo function of ABF-I.

Revealing, illuminating, and modifying proteins in human diseases using noncanonical amino acids

To better understand human diseases, much recent work has focused on proteins to either identify disease targets through proteomics or produce therapeutics via protein engineering. Noncanonical amino acids (ncAAs) are tools for altering the chemical and physical properties of proteins, providing a facile strategy not only to label proteins but also to engineer proteins with novel properties. My thesis research has focused on the development and applications of noncanonical amino acids in identifying, imaging, and engineering proteins for studying human diseases. Chapter 1 introduces the concept of ncAAs and reveals insights to how I chose my thesis projects.

ncAAs have been incorporated to tag and enrich newly synthesized proteins for mass spectrometry through a method termed BONCAT, or bioorthogonal noncanonical amino acid tagging. Chapter 2 describes the investigation of the proteomic response of human breast cancer cells to induced expression of tumor suppressor microRNA miR-126 by combining BONCAT with another proteomic method, SILAC or stable isotope labeling by amino acids in cell culture. This proteomic analysis led to the discovery of a direct target of miR-126, shedding new light on its role in suppressing cancer metastasis.

In addition to mass spectrometry, ncAAs can also be utilized to fluorescently label proteins. Chapter 3 details the synthesis of a set of cell-permeant cyclooctyne probes and demonstration of selective labeling of newly synthesized proteins in live mammalian cells using azidohomoalanine. Similar to live cell imaging, the ability to selectively label a particular cell type within a mixed cell population is important to interrogating many biological systems, such as tumor microenvironments. By taking advantage of the metabolic differences between cancer and normal cells, Chapter 5 discusses efforts to develop selective labeling of cancer cells using a glutamine analogue.

Furthermore, Chapter 4 describes the first demonstration of global replacement at polar amino acid positions and its application in developing an alternative PEGylation strategy for therapeutic proteins. Polar amino acids typically occupy solvent-exposed positions on the protein surface, and incorporation of noncanonical amino acids at these positions should allow easier modification and cause less perturbation compared to replacements at the interior positions of proteins.

Electrochemical and electron transfer investigations of copper proteins

A study of the pH and temperature dependence of the redox potentials of azurins from five species of bacteria has been performed. The variations in the potentials with pH have been interpreted in terms of electrostatic interactions between the copper site and titrating histidine residues, including the effects of substitutions in the amino acid sequences of the proteins on the electrostatic interactions. A comparison of the observed pH dependences with predictions based on histidine pK_a values known for Pseudomonas aeruginosa (Pae), Alcaligenes denitrificans (Ade), and Alcaligenes faecalis (Afa) azurins indicates that the Pae and Ade redox potentials exhibit pH dependences in line with electrostatic arguments, while Afa azurin exhibits more complex behavior. Redox enthalpies and entropies for four of the azurins at low and high pH values have also been obtained. Based on these results in conjuction with the variable pH experiments, it appears that Bordetella bronchiseptica azurin may undergo a more substantial conformational change with pH than has been observed for other species of azurin.

The temperature dependence of the redox potential of bovine erythrocyte superoxide dismutase (SOD) has been determined at pH 7.0, with potassium ferricyanide as the mediator. The following thermodynamic parameters have been obtained (T = 25°C): E°' = 403±5 mV vs. NHE, ΔG°' = -9.31 kcal/mol, ΔH°' = -21.4 kcal/mol, ΔS°' = -40.7 eu, ΔS°'_(rc) = -25.1 eu. It is apparent from these results that ΔH°', rather than ΔS°', is the dominant factor in establishing the high redox potential of SOD. The large negative enthalpy of reduction may also reflect the factors which give SOD its high specificity toward reduction and oxidation by superoxide.

Structural and mechanistic motifs in membrane proteins: the three-dimensional modelling of rhodopsin, band 3, and the nicotinic acetylcholine receptor

Because so little is known about the structure of membrane proteins, an attempt has been made in this work to develop techniques by which to model them in three dimensions. The procedures devised rely heavily upon the availability of several sequences of a given protein. The modelling procedure is composed of two parts. The first identifies transmembrane regions within the protein sequence on the basis of hydrophobicity, β-turn potential, and the presence of certain amino acid types, specifically, proline and basic residues. The second part of the procedure arranges these transmembrane helices within the bilayer based upon the evolutionary conservation of their residues. Conserved residues are oriented toward other helices and variable residues are positioned to face the surrounding lipids. Available structural information concerning the protein's helical arrangement, including the lengths of interhelical loops, is also taken into account. Rhodopsin, band 3, and the nicotinic acetylcholine receptor have all been modelled using this methodology, and mechanisms of action could be proposed based upon the resulting structures.

Specific residues in the rhodopsin and iodopsin sequences were identified, which may regulate the proteins' wavelength selectivities. A hinge-like motion of helices M3, M4, and M5 with respect to the rest of the protein was proposed to result in the activation of transducin, the G-protein associated with rhodopsin. A similar mechanism is also proposed for signal transduction by the muscarinic acetylcholine and β-adrenergic receptors.

The nicotinic acetylcholine receptor was modelled with four trans-membrane helices per subunit and with the five homologous M2 helices forming the cation channel. Putative channel-lining residues were identified and a mechanism of channel-opening based upon the concerted, tangential rotation of the M2 helices was proposed.

Band 3, the anion exchange protein found in the erythrocyte membrane, was modelled with 14 transmembrane helices. In general the pathway of anion transport can be viewed as a channel composed of six helices that contains a single hydrophobic restriction. This hydrophobic region will not allow the passage of charged species, unless they are part of an ion-pair. An arginine residue located near this restriction is proposed to be responsible for anion transport. When ion-paired with a transportable anion it rotates across the barrier and releases the anion on the other side of the membrane. A similar process returns it to its original position. This proposed mechanism, based on the three-dimensional model, can account for the passive, electroneutral, anion exchange observed for band 3. Dianions can be transported through a similar mechanism with the additional participation of a histidine residue. Both residues are located on M10.

A genetic and biochemical analysis of pre-mRNA splicing in Saccharomyces cerevisiae

Pre-mRNA splicing requires interaction of cis- acting intron sequences with trans -acting factors: proteins and small nuclear ribonucleoproteins (snRNPs). The assembly of these factors into a large complex, the spliceosome, is essential for the subsequent two step splicing reaction. First, the 5' splice site is cleaved and free exon 1 and a lariat intermediate (intron- exon2) form. In the second reaction the 3' splice site is cleaved the exons ligated and lariat intron released. A combination of genetic and biochemical techniques have been used here to study pre-mRNA splicing in yeast.

Yeast introns have three highly conserved elements. We made point mutations within these elements and found that most of them affect splicing efficiency in vivo and in vitro, usually by inhibiting spliceosome assembly.

To study trans -acting splicing factors we generated and screened a bank of temperature- sensitive (ts) mutants. Eleven new complementation groups (prp17 to prp27) were isolated. The four phenotypic classes obtained affect different steps in splicing and accumulate either: 1) pre-mRNA, 2) lariat intermediate, 3) excised intron or 4) both pre-mRNA and intron. The latter three classes represent novel phenotypes. The excised intron observed in one mutant: prp26 is stabilized due to protection in a snRNP containing particle. Extracts from another mutant: prpl8 are heat labile and accumulate lariat intermediate and exon 1. This is especially interesting as it allows analysis of the second splicing reaction. In vitro complementation of inactivated prp18 extracts does not require intact snRNPs. These studies have also shown the mutation to be in a previously unknown splicing protein. A specific requirement for A TP is also observed for the second step of splicing. The PRP 18 gene has been cloned and its polyadenylated transcript identified.

Electron flow through cytochrome P450

The cytochromes P450 (P450s) are a remarkable class of heme enzymes that catalyze the metabolism of xenobiotics and the biosynthesis of signaling molecules. Controlled electron flow into the thiolate-ligated heme active site allows P450s to activate molecular oxygen and hydroxylate aliphatic C–H bonds via the formation of high-valent metal-oxo intermediates (compounds I and II). Due to the reactive nature and short lifetimes of these intermediates, many of the fundamental steps in catalysis have not been observed directly. The Gray group and others have developed photochemical methods, known as “flash-quench,” for triggering electron transfer (ET) and generating redox intermediates in proteins in the absence of native ET partners. Photo-triggering affords a high degree of temporal precision for the gating of an ET event; the initial ET and subsequent reactions can be monitored on the nanosecond-to-second timescale using transient absorption (TA) spectroscopies. Chapter 1 catalogues critical aspects of P450 structure and mechanism, including the native pathway for formation of compound I, and outlines the development of photochemical processes that can be used to artificially trigger ET in proteins. Chapters 2 and 3 describe the development of these photochemical methods to establish electronic communication between a photosensitizer and the buried P450 heme. Chapter 2 describes the design and characterization of a Ru-P450-BM3 conjugate containing a ruthenium photosensitizer covalently tethered to the P450 surface, and nanosecond-to-second kinetics of the photo-triggered ET event are presented. By analyzing data at multiple wavelengths, we have identified the formation of multiple ET intermediates, including the catalytically relevant compound II; this intermediate is generated by oxidation of a bound water molecule in the ferric resting state enzyme. The work in Chapter 3 probes the role of a tryptophan residue situated between the photosensitizer and heme in the aforementioned Ru-P450 BM3 conjugate. Replacement of this tryptophan with histidine does not perturb the P450 structure, yet it completely eliminates the ET reactivity described in Chapter 2. The presence of an analogous tryptophan in Ru-P450 CYP119 conjugates also is necessary for observing oxidative ET, but the yield of heme oxidation is lower. Chapter 4 offers a basic description of the theoretical underpinnings required to analyze ET. Single-step ET theory is first presented, followed by extensions to multistep ET: electron “hopping.” The generation of “hopping maps” and use of a hopping map program to analyze the rate advantage of hopping over single-step ET is described, beginning with an established rhenium-tryptophan-azurin hopping system. This ET analysis is then applied to the Ru-tryptophan-P450 systems described in Chapter 2; this strongly supports the presence of hopping in Ru-P450 conjugates. Chapter 5 explores the implementation of flash-quench and other phototriggered methods to examine the native reductive ET and gas binding events that activate molecular oxygen. In particular, TA kinetics that demonstrate heme reduction on the microsecond timescale for four Ru-P450 conjugates are presented. In addition, we implement laser flash-photolysis of P450 ferrous–CO to study the rates of CO rebinding in the thermophilic P450 CYP119 at variable temperature. Chapter 6 describes the development and implementation of air-sensitive potentiometric redox titrations to determine the solution reduction potentials of a series of P450 BM3 mutants, which were designed for non-native cyclopropanation of styrene in vivo. An important conclusion from this work is that substitution of the axial cysteine for serine shifts the wild type reduction potential positive by 130 mV, facilitating reduction by biological redox cofactors in the presence of poorly-bound substrates. While this mutation abolishes oxygenation activity, these mutants are capable of catalyzing the cyclopropanation of styrene, even within the confines of an E. coli cell. Four appendices are also provided, including photochemical heme oxidation in ruthenium-modified nitric oxide synthase (Appendix A), general protocols (Appendix B), Chapter-specific notes (Appendix C) and Matlab scripts used for data analysis (Appendix D).

The Get3 ATPase drives unidirectional targeting of tail-anchored membrane proteins

Efficient and accurate localization of membrane proteins is essential to all cells and requires a complex cascade of interactions between protein machineries. This is exemplified in the recently discovered Guided Entry of Tail-anchored protein pathway, in which the central targeting factor Get3 must sequentially interact with three distinct binding partners (Get4, Get1 and Get2) to ensure the targeted delivery of Tail-anchored proteins to the endoplasmic reticulum membrane. To understand the molecular and energetic principles that provide the vectorial driving force of these interactions, we used a quantitative fluorescence approach combined with mechanistic enzymology to monitor the effector interactions of Get3 at each stage of Tail-anchored protein targeting. We show that nucleotide and membrane protein substrate generate a gradient of interaction energies that drive the cyclic and ordered transit of Get3 from Get4 to Get2 and lastly to Get1. These data also define how the Get3/Tail-anchored complex is captured, handed over, and disassembled by the Get1/2 receptor at the membrane, and reveal a novel role for Get4/5 in recycling Get3 from the endoplasmic reticulum membrane at the end of the targeting reaction. These results provide general insights into how complex cascades of protein interactions are coordinated and coupled to energy inputs in biological systems.

Biological electron transfer in copper proteins

Nature has used a variety of protein systems to mediate electron transfer. In this thesis I examine aspects of the control of biological electron transfer by two copper proteins that act as natural electron carriers.

In the first study, I have made a mutation to one of the ligand residues in the azurin blue copper center, methionine 121 changed to a glutamic acid. Studies of intramolecular electron transfer rates from that mutated center to covalently attached ruthenium complexes indicate that the weak axial methionine ligand is important not only for tuning the reduction potential of the blue copper site but also for maintaining the low reorganization energy that is important for fast electron transfer at long distances.

In the second study, I begin to examine the reorganization energy of the purple copper center in the CuA domain of subunit II of cytochrome c oxidase. In this copper center, the unpaired electron is delocalized over the entire binuclear site. Because long-range electron transfer into and out of this center occurs over long distances with very small driving forces, the reorganization energy of the CuA center has been predicted to be extremely low. I describe a strategy for measuring this reorganization energy starting with the construction of a series of mutations introducing surface histidines. These histidines can then be labeled with a series of ruthenium compounds that differ primarily in their reduction potentials. The electron transfer rates to these ruthenium compounds can then be used to determine the reorganization energy of the CuA site.

Computational design of self-assembling proteins and protein-DNA nanowires

Computational protein design (CPD) is a burgeoning field that uses a physical-chemical or knowledge-based scoring function to create protein variants with new or improved properties. This exciting approach has recently been used to generate proteins with entirely new functions, ones that are not observed in naturally occurring proteins. For example, several enzymes were designed to catalyze reactions that are not in the repertoire of any known natural enzyme. In these designs, novel catalytic activity was built de novo (from scratch) into a previously inert protein scaffold. In addition to de novo enzyme design, the computational design of protein-protein interactions can also be used to create novel functionality, such as neutralization of influenza. Our goal here was to design a protein that can self-assemble with DNA into nanowires. We used computational tools to homodimerize a transcription factor that binds a specific sequence of double-stranded DNA. We arranged the protein-protein and protein-DNA binding sites so that the self-assembly could occur in a linear fashion to generate nanowires. Upon mixing our designed protein homodimer with the double-stranded DNA, the molecules immediately self-assembled into nanowires. This nanowire topology was confirmed using atomic force microscopy. Co-crystal structure showed that the nanowire is assembled via the desired interactions. To the best of our knowledge, this is the first example of a protein-DNA self-assembly that does not rely on covalent interactions. We anticipate that this new material will stimulate further interest in the development of advanced biomaterials.

Proteolytic processing of the nonstructural proteins of dengue 2 virus

The genomes of many positive stranded RNA viruses and of all retroviruses are translated as large polyproteins which are proteolytically processed by cellular and viral proteases. Viral proteases are structurally related to two families of cellular proteases, the pepsin-like and trypsin-like proteases. This thesis describes the proteolytic processing of several nonstructural proteins of dengue 2 virus, a representative member of the Flaviviridae, and describes methods for transcribing full-length genomic RNA of dengue 2 virus. Chapter 1 describes the in vitro processing of the nonstructural proteins NS2A, NS2B and NS3. Chapter 2 describes a system that allows identification of residues within the protease that are directly or indirectly involved with substrate recognition. Chapter 3 describes methods to produce genome length dengue 2 RNA from cDNA templates.

The nonstructural protein NS3 is structurally related to viral trypsinlike proteases from the alpha-, picorna-, poty-, and pestiviruses. The hypothesis that the flavivirus nonstructural protein NS3 is a viral proteinase that generates the termini of several nonstructural proteins was tested using an efficient in vitro expression system and antisera specific for the nonstructural proteins NS2B and NS3. A series of cDNA constructs was transcribed using T7 RNA polymerase and the RNA translated in reticulocyte lysates. Proteolytic processing occurred in vitro to generate NS2B and NS3. The amino termini of NS2B and NS3 produced in vitro were found to be the same as the termini of NS2B and NS3 isolated from infected cells. Deletion analysis of cDNA constructs localized the protease domain necessary and sufficient for correct cleavage to the first 184 amino acids of NS3. Kinetic analysis of processing events in vitro and experiments to examine the sensitivity of processing to dilution suggested that an intramolecular cleavage between NS2A and NS2B preceded an intramolecular cleavage between NS2B and NS3. The data from these expression experiments confirm that NS3 is the viral proteinase responsible for cleavage events generating the amino termini of NS2B and NS3 and presumably for cleavages generating the termini of NS4A and NS5 as well.

Biochemical and genetic experiments using viral proteinases have defined the sequence requirements for cleavage site recognition, but have not identified residues within proteinases that interact with substrates. A biochemical assay was developed that could identify residues which were important for substrate recognition. Chimeric proteases between yellow fever and dengue 2 were constructed that allowed mapping of regions involved in substrate recognition, and site directed mutagenesis was used to modulate processing efficiency.

Expression in vitro revealed that the dengue protease domain efficiently processes the yellow fever polyprotein between NS2A and NS2B and between NS2B and NS3, but that the reciprocal construct is inactive. The dengue protease processes yellow fever cleavage sites more efficiently than dengue cleavage sites, suggesting that suboptimal cleavage efficiency may be used to increase levels of processing intermediates in vivo. By mutagenizing the putative substrate binding pocket it was possible to change the substrate specificity of the yellow fever protease; changing a minimum of three amino acids in the yellow fever protease enabled it to recognize dengue cleavage sites. This system allows identification of residues which are directly or indirectly involved with enzyme-substrate interaction, does not require a crystal structure, and can define the substrate preferences of individual members of a viral proteinase family.

Full-length cDNA clones, from which infectious RNA can be transcribed, have been developed for a number of positive strand RNA viruses, including the flavivirus type virus, yellow fever. The technology necessary to transcribe genomic RNA of dengue 2 virus was developed in order to better understand the molecular biology of the dengue subgroup. A 5' structural region clone was engineered to transcribe authentic dengue RNA that contains an additional 1 or 2 residues at the 5' end. A 3' nonstructural region clone was engineered to allow production of run off transcripts, and to allow directional ligation with the 5' structural region clone. In vitro ligation and transcription produces full-length genomic RNA which is noninfectious when transfected into mammalian tissue culture cells. Alternative methods for constructing cDNA clones and recovering live dengue virus are discussed.