Biomedical subjects

Stephen G Brohawn

Publications and source records attributed to Stephen G Brohawn.

5 recordsLinked to original sources

PubMed2026

A noncontiguous code for RNA-guided DNA recognition at the origin of CRISPR-Cas.

CRISPR-Cas provides RNA-mediated adaptive immunity, but how its first RNA-guided effector arose is unclear. In this study, we report the discovery of Viral Interference Programmable Repeat (VIPR) systems consisting of a Vipr protein ancestral to the earliest CRISPR-Cas effectors and VIPR RNAs (vrRNAs) comprising alternating GGY/NN motifs. Unlike canonical guide RNAs that pair with target nucleic acids through contiguous complementarity, vrRNAs recognize double-stranded DNA through a noncontiguous code in which the variable NN dinucleotides collectively specify a gapped target sequence. Natural vrRNA targets suggest that VIPR systems act against competing phages, and we demonstrate programmable phage defense by redirecting the complex for transcriptional repression. These results suggest that adaptive immunity originated from ancient warfare between viruses, revealing a previously unidentified logic for encoding information in sequence.

CRISPR-Cas Systems↗

PubMed2026

VIPR RNA-guided DNA recognition by noncontiguous geometric triplex formation.

Viral interference programmable repeat (VIPR) systems use a noncontiguous code for RNA-guided transcriptional silencing. How the Vipr protein and a VIPR RNA (vrRNA) comprising alternating GGY and NN segments achieve precise DNA targeting is unknown. Here, we present 21 cryo-electron microscopy structures that help explain the mechanism of target engagement. Vipr protomers oligomerize along the vrRNA to form a right-handed helical filament, sequestering each GGY motif and positioning the adjacent NN bases for target base pairing. DNA binding, in which every third nucleotide is skipped, results in a gapped vrRNA-DNA hybrid helix that encircles the nontarget DNA strand to form a geometric triplex. These findings suggest that triplex-mediated target-strand handoff could enable noncontiguous and programmable RNA-guided DNA recognition in VIPR systems.

DNA↗

PubMed2006

Homodimerization of the G protein SRbeta in the nucleotide-free state involves proline cis/trans isomerization in the switch II region.

Protein translocation across and insertion into membranes is essential to all life forms. Signal peptide-bearing nascent polypeptide chains emerging from the ribosome are first sampled by the signal-recognition particle (SRP), then targeted to the membrane via the SRP receptor (SR), and, finally, transferred to the protein-conducting channel. In eukaryotes, this process is tightly controlled by the concerted action of three G proteins, the 54-kD subunit of SRP and the alpha- and beta-subunits of SR. We have determined the 2.2-A crystal structure of the nucleotide-free SRbeta domain. Unexpectedly, the structure is a homodimer with a highly intertwined interface made up of residues from the switch regions of the G domain. The remodeling of the switch regions does not resemble any of the known G protein switch mechanisms. Biochemical analysis confirms homodimerization in vitro, which is incompatible with SRalpha binding. The switch mechanism involves cis/trans isomerization of a strictly conserved proline, potentially implying a new layer of regulation of cotranslational transport.

Amino Acid Sequence↗

PubMed2004

New water-soluble phosphines as reductants of peptide and protein disulfide bonds: reactivity and membrane permeability.

Tris(2-carboxyethyl)phosphine (TCEP) is a widely used substitute for dithiothreitol (DTT) in the reduction of disulfide bonds in biochemical systems. Although TCEP has been recently shown to be a substrate of the flavin-dependent sulfhydryl oxidases, there is little quantitative information concerning the rate by which TCEP reduces other peptidic disulfide bonds. In this study, mono-, di-, and trimethyl ester analogues of TCEP were synthesized to evaluate the role of carboxylate anions in the reduction mechanism, and to expand the range of phosphine reductants. The effectiveness of all four phosphines relative to DTT has been determined using model disulfides, including a fluorescent disulfide-containing peptide (H(3)N(+)-VTWCGACKM-NH(2)), and with protein disulfide bonds in thioredoxin and sulfhydryl oxidase. Mono-, di-, and trimethyl esters exhibit phosphorus pK values of 6.8, 5.8, and 4.7, respectively, extending their reactivity with the model peptide to correspondingly lower pH values relative to that of TCEP (pK = 7.6). At pH 5.0, the order of reactivity is as follows: trimethyl- > dimethyl- > monomethyl- > TCEP >> DTT; tmTCEP is 35-fold more reactive than TCEP, and DTT is essentially unreactive. Esterification also increases lipophilicity, allowing tmTCEP to penetrate phospholipid bilayers rapidly (>30-fold faster than DTT), whereas the parent TCEP is impermeant. Although more reactive than DTT toward small-molecule disulfides at pH 7.5, all phosphines are markedly less reactive toward protein disulfides at this pH. Molecular modeling suggests that the nucleophilic phosphorus of TCEP is more sterically crowded than the thiolate of DTT, contributing to the lower reactivity of the phosphine with protein disulfides. In sum, these data suggest that there is considerable scope for the synthesis of phosphine analogues tailored for specific applications in biological systems.

Disulfides↗

PubMed2003

Avian sulfhydryl oxidase is not a metalloenzyme: adventitious binding of divalent metal ions to the enzyme.

Metal- and flavin-dependent sulfhydryl oxidases catalyze the generation of disulfide bonds with reduction of oxygen to hydrogen peroxide. The mammalian skin enzyme has been reported to be copper-dependent, but a recent protein sequence shows it belongs to the Quiescin/sulfhydryl oxidase (QSOX) flavoprotein family. This work demonstrates that avian QSOX is not a metalloenzyme, and that copper and zinc ions inhibit the oxidation of reduced pancreatic ribonuclease by the enzyme. Studies with Zn(2+), as a redox inactive surrogate for copper, show that one Zn(2+) binds to four-electron-reduced QSOX by diverting electrons away from the flavin and into two of the three redox active disulfide bridges in the enzyme. The resulting zinc complex is modestly air-stable, reverting to a spectrum of the native protein with a t(1/2) of 40 min, whereas the four-electron-reduced native QSOX is reoxidized in less than a second under comparable conditions. Using tris(2-carboxyethyl)phosphine hydrochloride (TCEP), an alternate substrate of QSOX that binds Zn(2+) relatively weakly (unlike dithiothreitol), allows rapid inhibition of oxidase activity to be demonstrated at low micromolar metal levels. Zinc binding was followed by rapid-scanning spectrophotometry. Copper also binds the four-electron-reduced form of QSOX with a visible spectrum suggestive of active site occupancy. In addition to interactions with the reduced enzyme, dialysis experiments show that multiple copper and zinc ions can bind to the oxidized enzyme without the perturbation of the flavin spectrum seen earlier. These data suggest that a reinvestigation of the metal content of skin sulfhydryl oxidases is warranted. The redox-modulated binding of zinc to QSOX is considered in light of evidence for a role of zinc-thiolate interactions in redox signaling and zinc mobilization.

Amino Acid Sequence↗