Engineering novel allostery into existing proteins is a challenging endeavor to obtain novel sensors, therapeutic proteins, or modulate metabolic and cellular processes. The RG13 protein achieves such allostery by inserting a circularly permuted TEM-1 β-lactamase gene into the maltose binding protein (MBP). RG13 is positively regulated by maltose yet is, serendipitously, inhibited by Zn(2+) at ...

The HIV-1 nucleocapsid protein (NC) is a small basic protein containing two zinc fingers (ZF) separated by a short linker. It is involved in several steps of the replication cycle and acts as a nucleic acid chaperone protein in facilitating nucleic acid strand transfers occurring during reverse transcription. Recent analysis of three-dimensional structures of NC-nucleic acids complexes establis...

The membrane proximal region (MPR, residues 649-683) and transmembrane domain (TMD, residues 684-705) of the gp41 subunit of HIV-1's envelope protein are highly conserved and are important in viral mucosal transmission, virus attachment and membrane fusion with target cells. Several structures of the trimeric membrane proximal external region (residues 662-683) of MPR have been reported at the ...

We report herein the synthesis and biological testing of several glycosylated derivatives of some fluoroquinolone antibiotics. In particular, we have prepared several glycosylated derivatives of ciprofloxacin (2) in which the carbohydrate units are linked to the free secondary amine of the piperazine unit by: (a) no linker (e.g., a glycosylamine), (b) a beta-oxyethyl linker, and (c) a gamma-oxy...

The tandem C2-domains of synaptotagmin 1 (syt) function as Ca(2+)-binding modules that trigger exocytosis; in the absence of Ca(2+), syt inhibits spontaneous release. Here, we used proline linkers to constrain and alter the relative orientation of these C2-domains. Short poly-proline helices have a period of three, so large changes in the relative disposition of the C2-domains result from chang...

Nucleoid-associated proteins (NAPs) facilitate chromosome organization in bacteria, but the precise mechanism remains elusive. H-NS is a NAP that also plays a major role in silencing pathogen genes. We used genetics, single-particle tracking in live cells, superresolution microscopy, atomic force microscopy, and molecular dynamics simulations to examine H-NS/DNA interactions in single cells. We...

The double-stranded Kpn I linker, CGGTACCG, is readily crosslinked by 4’-hydroxymethyl4.5’,8-trimethylpsoralen (HMT). Under identical conditions, the corresponding Bam HI linker, CGGATCCG, is resistant to modification. The differential reactivity of HMT towards the two sequences is also observed with SV40 DNA where the Kpn I restriction site is ten-fold more susceptible to HMT modification than...

This paper reports dissociation constants and "effective molarities" (M(eff)) for the intramolecular binding of a ligand covalently attached to the surface of a protein by oligo(ethylene glycol) (EG(n)) linkers of different lengths (n = 0, 2, 5, 10, and 20) and compares these experimental values with theoretical estimates from polymer theory. As expected, the value of M(eff) is lowest when the ...

Cytosine methylation regulates essential genome functions across eukaryotes, but the fundamental question of whether nucleosomal or naked DNA is the preferred substrate of plant and animal methyltransferases remains unresolved. Here, we show that genetic inactivation of a single DDM1/Lsh family nucleosome remodeler biases methylation toward inter-nucleosomal linker DNA in Arabidopsis thaliana a...

cAMP mediates autonomic regulation of heart rate by means of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which underlie the pacemaker current If. cAMP binding to the C-terminal cyclic nucleotide binding domain enhances HCN open probability through a conformational change that reaches the pore via the C-linker. Using structural and functional analysis, we identified a bin...