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All retroviral proteases belong to the family of aspartic proteases. They are active as homodimers, each unit contributing one catalytic aspartate to the active site dyad. An important feature of all aspartic proteases is a conserved complex scaffold of hydrogen bonds supporting the active site, called the "fireman's grip," which involves the hydroxyl groups of two threonine (serine) residues in the active site Asp-Thr(Ser)-Gly triplets. It was shown previously that the fireman's grip is indispensable for the dimer stability of HIV protease. The retroviral proteases harboring Ser in their active site triplet are less active and, under natural conditions, are expressed in higher enzyme/substrate ratio than those having Asp-Thr-Gly triplet. To analyze whether this observation can be attributed to the different influence of Thr or Ser on dimerization, we prepared two pairs of the wild-type and mutant proteases from HIV and myeloblastosis-associated virus harboring either Ser or Thr in their Asp-Thr(Ser)-Gly triplet. The equilibrium dimerization constants differed by an order of magnitude within the relevant pairs. The proteases with Thr in their active site triplets were found to be ∼10 times more thermodynamically stable. The dimer association contributes to this difference more than does the dissociation. We propose that the fireman's grip might be important in the initial phases of dimer formation to help properly orientate the two subunits of a retroviral protease. The methyl group of threonine might contribute significantly to fixing such an intermediate conformation.Keywords: Retroviral protease; dimerization; HIV protease; fireman's grip; kinetic assay; fluorescence; pressure Retroviral proteases (PRs) belong to the family of retropepsins (Dunn 1998), which are dimeric aspartic proteases. The two monomers are interconnected by their C and N termini, which form an antiparallel ␤-sheet that is primarily responsible for the dimer stability (Wlodawer et al. 1989). The proteases are expressed as parts of the viral polyproteins Gag or Gag-Pol, from which they cleave themselves before cleaving the rest of the polyproteins to yield the structural proteins and replication enzymes. The Gag-Pol polyprotein is a product of ribosomal frame-shifting or readthrough suppression, which takes place with 5% to 10% frequency. In contrast, in the case of alpharetroviruses (e.g., myeloblastosis-associated virus [MAV]), the protease is a part of the Gag polyprotein and is thus expressed in equimolar ratio with its Gag substrate. In the case of betaretroviruses (Mason-Pfizer monkey virus [MPMV]) the protease also is expressed by frame-shifting event. It was observed previously that the activities of the alpharetroviral proteases are con-

G-Quadruplexes are four-stranded nucleic acid structures typically stabilized by GGGG tetrads. These structures are intrinsically fluorescent, which expands the known scope of nucleic acid function and raises the possibility that they could eventually be used as signaling components in label-free sensors constructed from DNA or RNA. In this study, we systematically investigated the effects of mutations in tetrads, loops, and overhanging nucleotides on the fluorescence intensity and maximum emission wavelength of >500 sequence variants of a reference DNA G-quadruplex. Some of these mutations modestly increased the fluorescence intensity of this G-quadruplex, while others shifted its maximum emission wavelength. Mutations that increased the fluorescence intensity were distinct from those that increased the maximum emission wavelength, suggesting a trade-off between these two biochemical properties. The fluorescence intensity and maximum emission wavelength were also correlated with multimeric state: the most fluorescent G-quadruplexes were monomers, while those with the highest maximum emission wavelengths typically formed dimeric structures. Oligonucleotides containing multiple G-quadruplexes were in some cases more fluorescent than those containing a single G-quadruplex, although this depended on the length and sequence of the spacer linking the G-quadruplexes. These experiments provide new insights into the properties of fluorescent G-quadruplexes and should aid in the development of improved label-free nucleic acid sensors.

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