22 research outputs found
Physicochemical Properties of Ion Pairs of Biological Macromolecules
Ion pairs (also known as salt bridges) of electrostatically interacting cationic and anionic moieties are important for proteins and nucleic acids to perform their function. Although numerous three-dimensional structures show ion pairs at functionally important sites of biological macromolecules and their complexes, the physicochemical properties of the ion pairs are not well understood. Crystal structures typically show a single state for each ion pair. However, recent studies have revealed the dynamic nature of the ion pairs of the biological macromolecules. Biomolecular ion pairs undergo dynamic transitions between distinct states in which the charged moieties are either in direct contact or separated by water. This dynamic behavior is reasonable in light of the fundamental concepts that were established for small ions over the last century. In this review, we introduce the physicochemical concepts relevant to the ion pairs and provide an overview of the recent advancement in biophysical research on the ion pairs of biological macromolecules
Positive and negative impacts of nonspecific sites during target location by a sequence-specific DNA-binding protein: origin of the optimal search at physiological ionic strength
The inducible transcription factor Egr-1, which recognizes a 9-bp target DNA sequence via three zinc-finger domains, rapidly activates particular genes upon cellular stimuli such as neuronal signals and vascular stresses. Here, using the stopped-flow fluorescence method, we measured the target search kinetics of the Egr-1 zinc-finger protein at various ionic strengths between 40 and 400 mM KCl and found the most efficient search at 150 mM KCl. We further investigated the kinetics of intersegment transfer, dissociation, and sliding of this protein on DNA at distinct concentrations of KCl. Our data suggest that Egr-1's kinetic properties are well suited for efficient scanning of chromosomal DNA in vivo. Based on a newly developed theory, we analyzed the origin of the optimal search efficiency at physiological ionic strength. Target association is accelerated by nonspecific binding to nearby sites and subsequent sliding to the target as well as by intersegment transfer. Although these effects are stronger at lower ionic strengths, such conditions also favor trapping of the protein at distant nonspecific sites, decelerating the target association. Our data demonstrate that Egr-1 achieves the optimal search at physiological ionic strength through a compromise between the positive and negative impacts of nonspecific interactions with DNA
Deficient uracil base excision repair leads to persistent dUMP in HIV proviruses during infection of monocytes and macrophages.
Non-dividing cells of the myeloid lineage such as monocytes and macrophages are target cells of HIV that have low dNTP pool concentrations and elevated levels of dUTP, which leads to frequent incorporation of dUMP opposite to A during reverse transcription ("uracilation"). One factor determining the fate of dUMP in proviral DNA is the host cell uracil base excision repair (UBER) system. Here we explore the relative UBER capacity of monocytes (MC) and monocyte-derived macrophages (MDM) and the fate of integrated uracilated viruses in both cell types to understand the implications of viral dUMP on HIV diversification and infectivity. We find that the kinetics for MC infection is compatible with their lifetime in vivo and their near absence of hUNG2 activity is consistent with the retention of viral dUMP at high levels at least until differentiation into macrophages, where UBER becomes possible. Overexpression of human uracil DNA glycosylase in MDM prior to infection resulted in rapid removal of dUMP from HIV cDNA and near complete depletion of dUMP-containing viral copies. This finding establishes that the low hUNG2 expression level in these cells limits UBER but that hUNG2 is restrictive against uracilated viruses. In contrast, overexpression of hUNG2 after viral integration did not accelerate the excision of uracils, suggesting that they may poorly accessible in the context of chromatin. We found that viral DNA molecules with incorporated dUMP contained unique (+) strand transversion mutations that were not observed when dUMP was absent (G→T, T→A, T→G, A→C). These observations and other considerations suggest that dUMP introduces errors predominantly during (-) strand synthesis when the template is RNA. Overall, the likelihood of producing a functional virus from in vitro infection of MC is about 50-fold and 300-fold reduced as compared to MDM and activated T cells. The results implicate viral dUMP incorporation in MC and MDM as a potential viral diversification and restriction pathway during human HIV infection
AP-Endonuclease 1 Accelerates Turnover of Human 8‑Oxoguanine DNA Glycosylase by Preventing Retrograde Binding to the Abasic-Site Product
A major
product of oxidative DNA damage is 8-oxoguanine. In humans,
8-oxoguanine DNA glycosylase (hOGG1) facilitates removal of these
lesions, producing an abasic (AP) site in the DNA that is subsequently
incised by AP-endonuclease 1 (APE1). APE1 stimulates turnover of several
glycosylases by accelerating rate-limiting product release. However,
there have been conflicting accounts of whether hOGG1 follows a similar
mechanism. In pre-steady-state kinetic measurements, we found that
addition of APE1 had no effect on the rapid burst phase of 8-oxoguanine
excision by hOGG1 but accelerated steady-state turnover (<i>k</i><sub>cat</sub>) by ∼10-fold. The stimulation by APE1 required
divalent cations, could be detected under multiple-turnover conditions
using limiting concentrations of APE1, did not require flanking DNA
surrounding the hOGG1 lesion site, and occurred efficiently even when
the first 49 residues of APE1’s N-terminus had been deleted.
Stimulation by APE1 does not involve relief from product inhibition
because thymine DNA glycosylase, an enzyme that binds more tightly
to AP sites than hOGG1 does, could not effectively substitute for
APE1. A stimulation mechanism involving stable protein–protein
interactions between free APE1 and hOGG1, or the DNA-bound forms,
was excluded using protein cross-linking assays. The combined results
indicate a mechanism whereby dynamic excursions of hOGG1 from the
AP site allow APE1 to invade the site and rapidly incise the phosphate
backbone. This mechanism, which allows APE1 to access the AP site
without forming specific interactions with the glycosylase, is a simple
and elegant solution to passing along unstable intermediates in base
excision repair
NMR Scalar Couplings across Intermolecular Hydrogen Bonds between Zinc-Finger Histidine Side Chains and DNA Phosphate Groups
Signal Transmission in Escherichia coli Cyclic AMP Receptor Protein for Survival in Extreme Acidic Conditions
El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado.During the life cycle of enteric bacterium Escherichia coli, it encounters a wide spectrum of pH changes. The asymmetric dimer of the cAMP receptor protein, CRP, plays a key role in regulating the expressions of genes and the survival of E. coli. To elucidate the pH effects on the mechanism of signal transmission, we present a combination of results derived from ITC, crystallography, and computation. CRP responds to a pH change by inducing a differential effect on the affinity for the binding events to the two cAMP molecules, ensuing in a reversible conversion between positive and negative cooperativity at high and low pH, respectively. The structures of four crystals at pH ranging from 7.8 to 6.5 show that CRP responds by inducing a differential effect on the structures of the two subunits, particularly in the DNA binding domain. Employing the COREX/BEST algorithm, computational analysis shows the change in the stability of residues at each pH. The change in residue stability alters the connectivity between residues including those in cAMP and DNA binding sites. Consequently, the differential impact on the topology of the connectivity surface among residues in adjacent subunits is the main reason for differential change in affinity; that is, the pH-induced differential change in residue stability is the biothermodynamic basis for the change in allosteric behavior. Furthermore, the structural asymmetry of this homodimer amplifies the differential impact of any perturbations. Hence, these results demonstrate that the combination of these approaches can provide insights into the underlying mechanism of an apparent complex allostery signal and transmission in CRP.National Institutes of HealthRevisión por pare
Influence of Quasi-Specific Sites on Kinetics of Target DNA Search by a Sequence-Specific DNA-Binding Protein
Dynamic Equilibria of Short-Range Electrostatic Interactions at Molecular Interfaces of Protein–DNA Complexes
Intermolecular ion pairs (salt bridges)
are crucial for protein–DNA
association. For two protein–DNA complexes, we demonstrate
that the ion pairs of protein side-chain NH<sub>3</sub><sup>+</sup> and DNA phosphate groups undergo dynamic transitions between distinct
states in which the charged moieties are either in direct contact
or separated by water. While the crystal structures of the complexes
show only the solvent-separated ion pair (SIP) state for some interfacial
lysine side chains, our NMR hydrogen-bond scalar coupling data clearly
indicate the presence of the contact ion pair (CIP) state for the
same residues. The 0.6-μs molecular dynamics (MD) simulations
confirm dynamic transitions between the CIP and SIP states. This behavior
is consistent with our NMR order parameters and scalar coupling data
for the lysine side chains. Using the MD trajectories, we also analyze
the free energies of the CIP–SIP equilibria. This work illustrates
the dynamic nature of short-range electrostatic interactions in DNA
recognition by proteins