Mark P. McPike

Engineering a Rigid Protein Tunnel for Biomolecular Detection

One intimidating challenge in protein nanopore-based technologies is designing robust protein scaffolds that remain functionally intact under a broad spectrum of detection conditions. Here, we show that an extensively engineered bacterial ferric hydroxamate uptake component A (FhuA), a β-barrel membrane protein, functions as a robust protein tunnel for the sampling of biomolecular events. The key implementation in this work was the coupling of direct genetic engineering with a refolding approach to produce an unusually stable protein nanopore. More importantly, this nanostructure maintained its stability under many experimental circumstances, some of which, including low ion concentration and highly acidic aqueous phase, are normally employed to gate, destabilize, or unfold β-barrel membrane proteins. To demonstrate these advantageous traits, we show that the engineered FhuA-based protein nanopore functioned as a sensing element for examining the proteolytic activity of an enzyme at highly acidic pH and for determining the kinetics of protein-DNA aptamer interactions at physiological salt concentration.

Acyclic Identification of Aptamers for Human alpha-Thrombin Using Over-Represented Libraries and Deep Sequencing

Background Aptamers are oligonucleotides that bind proteins and other targets with high affinity and selectivity. Twenty years ago elements of natural selection were adapted to in vitro selection in order to distinguish aptamers among randomized sequence libraries. The primary bottleneck in traditional aptamer discovery is multiple cycles of in vitro evolution. Methodology/Principal Findings We show that over-representation of sequences in aptamer libraries and deep sequencing enables acyclic identification of aptamers. We demonstrated this by isolating a known family of aptamers for human α-thrombin. Aptamers were found within a library containing an average of 56,000 copies of each possible randomized 15mer segment. The high affinity sequences were counted many times above the background in 2–6 million reads. Clustering analysis of sequences with more than 10 counts distinguished two sequence motifs with candidates at high abundance. Motif I contained the previously observed consensus 15mer, Thb1 (46,000 counts), and related variants with mostly G/T substitutions; secondary analysis showed that affinity for thrombin correlated with abundance (Kd = 12 nM for Thb1). The signal-to-noise ratio for this experiment was roughly 10,000∶1 for Thb1. Motif II was unrelated to Thb1 with the leading candidate (29,000 counts) being a novel aptamer against hexose sugars in the storage and elution buffers for Concanavilin A (Kd = 0.5 µM for α-methyl-mannoside); ConA was used to immobilize α-thrombin. Conclusions/Significance Over-representation together with deep sequencing can dramatically shorten the discovery process, distinguish aptamers having a wide range of affinity for the target, allow an exhaustive search of the sequence space within a simplified library, reduce the quantity of the target required, eliminate cycling artifacts, and should allow multiplexing of sequencing experiments and targets.

Footprinting, circular dichroism and UV melting studies on neomycin B binding to the packaging region of human immunodeficiency virus type-1 RNA

We have studied the binding of neomycin to a 171mer RNA (psi-RNA) from the packaging region of the LAI strain of human immunodeficiency virus type-1, HIV-1 (LAI). The RNase I footprinting studies reveal that the primary binding site for the drug is in stem-loop 1, which contains the dimer initiation site of HIV-1. Loading this site with neomycin causes a structural change in the RNA, allowing nucleotides in the neighboring stem-loop 2 to participate in the drug site. Drug binding to secondary sites induces structural changes in other stem-loops of the RNA. Footprinting plots, showing cutting at a site as a function of drug concentration, were analyzed using a two-state model to obtain relative site-specific binding constants. Circular dichroism measurements show that neomycin binding to psi-RNA changes the intensity of the strong negative CD band at 208 nm, confirming that neomycin induces structural changes. Melting studies of the RNA showed melting transitions in the absence of drug at 28.2, 37.2, 47.4, 55.5 and 60.8 degrees C. Only the first two were affected by drug binding, the reason for this being explained by our analysis.

Effects of the nature and concentration of salt on the interaction of the HIV-1 nucleocapsid protein with SL3 RNA.

The mature nucleocapsid protein of HIV-1, NCp7, and the NC domains in gag precursors are attractive targets for anti-AIDS drug discovery. The stability of the 1:1 complex of NCp7 with a 20mer mimic of stem-loop 3 RNA (SL3, also called psi-RNA, in the packaging domain of genomic RNA) is strongly affected by changes in ionic strength. NC domains recognize and specifically package genomic HIV-1 RNA, while electrostatic attractions and high concentrations of protein and RNA drive NCp7 to completely coat the RNA in the mature virion. The specific interactions of NCp7 binding to loop bases of SL3 produce 1:1 complexes in solutions that have a NaCl concentration of >or=0.2 M, while the electrostatic interactions can dominate at <or=0.15 M NaCl, leading to complexes that have a mainly 1:2 RNA:protein ratio. Persistent, nonequilibrium mixtures of 1:1 and protein-excess complexes can exist at these lower salt concentrations, where the distribution of complexes depends on the order of addition of RNA and protein. Adding salt causes rapid rearrangement of metastable multiprotein complexes to a 1:1 ratio. The stability of complexes is also affected by the nature of the added salt, with 0.018 M MgCl(2) and added 0.200 M NaCl producing the same K(d) (21 +/- 2 nM); acetate ion stabilizes the 1:1 complex by a factor of more than 2 compared to the same concentration of chloride ion. Maintaining a salt concentration of 0.2 M NaCl or 18 mM MgCl(2) is sufficient for experiments to distinguish drug candidates that disrupt the specific SL3-NCp7 interactions in the 1:1 complex.

Drug-RNA footprinting

RNase I and RNase T1 can be used to obtain high-quality footprinting information for paromomycin binding to a 176-mer RNA from the packaging region of HIV-1 (LAI). Controls and scanning procedures are necessary for quantitation of autoradiographic data, so that footprinting plots showing cutting behavior as a function of drug concentration can be used to identify binding sites and regions of altered structure on the 176-mer. From the RNase I footprinting results the primary paromomycin binding sites on the 176-mer are on the main stem and on the stem of SL1, but noncontiguous sequences may be involved in the same binding event. Strong enhancements in cleavage with added drug are also observed, indicating drug-induced structural changes. Drug binding may cause linker regions between stem-loops of the 176-mer to change structure, possibly providing a site or sites for additional drug binding. Because drug binding changes the structure of the packaging region, which may alter its function, paromomycin analogs with enhanced specificity for HIV psi RNA have potential as a new class of agent for treating AIDS.

Probing the RNA binding surface of the HIV-1 nucleocapsid protein by site-directed mutagenesis.

The highly conserved nucleocapsid protein domain in HIV-1 recognizes and binds SL3 in genomic RNA. In this work, we used the structure of the NCp7-SL3 RNA complex to guide the construction of 16 NCp7 mutants to probe the RNA binding surface of the protein [De Guzman, R. N., et al. (1998) Science 279, 384-388]. Thirteen residues with functional or structural significance were mutated individually to Ala (Asn(5), Phe(6), Val(13), Phe(16), Asn(17), Gly(19), Glu(21), Ile(24), Gln(45), Met(46), Gly(22), Pro(31), and Gly(40)), and three salt bridge switch mutants exchanged Lys and Glu (Lys(14)-Glu(21), Lys(33)-Glu(42), and Lys(38)-Glu(51)). Dissociation constants (Kd) determined by fluorescence titration and isothermal titration calorimetry were used to compare affinities of SL3 for the variant proteins to that for the wild type. The F16A (Phe(16) to Ala) variant showed a 25-fold reduction in affinity, consistent with a loss of organized structure in f1, the proteins first zinc finger. I24A, Q45A, and M46A reduced affinity by 2-5-fold; these residues occupy nearly equivalent positions in f1 and f2. E21A increased affinity by 3-fold, perhaps because of the mutants increased net positive charge. Among the salt bridge switch mutants, only K14E/E21K in f1 caused a substantial change in affinity (5-fold reduction), binding SL3 with a biphasic binding isotherm. Aside from these six variants, most of the mutations studied have relatively minor effects on the stability of the complex. We conclude that many side chain interactions in the wild-type complex contribute little to stability or can be compensated by new contacts in the mutants.