341 research outputs found
Determination of barrier heights and prefactors from protein folding rate data
We determine both barrier heights and prefactors for protein folding by
applying constraints determined from experimental rate measurements to a
Kramers theory for folding rate. The theoretical values are required to match
the experimental values at two conditions of temperature and denaturant that
induce the same stability. Several expressions for the prefactor in the Kramers
rate equation are examined: a random energy approximation, a correlated energy
approximation, and an approximation using a single Arrhenius activation energy.
Barriers and prefactors are generally found to be large as a result of
implementing this recipe, i.e. the folding landscape is cooperative and smooth.
Interestingly, a prefactor with a single Arrhenius activation energy admits no
formal solution.Comment: 11 pages, 5 figures, 1 table, Accepted Biophys
Assortative mixing in Protein Contact Networks and protein folding kinetics
Starting from linear chains of amino acids, the spontaneous folding of
proteins into their elaborate three-dimensional structures is one of the
remarkable examples of biological self-organization. We investigated native
state structures of 30 single-domain, two-state proteins, from complex networks
perspective, to understand the role of topological parameters in proteins'
folding kinetics, at two length scales-- as ``Protein Contact Networks (PCNs)''
and their corresponding ``Long-range Interaction Networks (LINs)'' constructed
by ignoring the short-range interactions. Our results show that, both PCNs and
LINs exhibit the exceptional topological property of ``assortative mixing''
that is absent in all other biological and technological networks studied so
far. We show that the degree distribution of these contact networks is partly
responsible for the observed assortativity. The coefficient of assortativity
also shows a positive correlation with the rate of protein folding at both
short and long contact scale, whereas, the clustering coefficients of only the
LINs exhibit a negative correlation. The results indicate that the general
topological parameters of these naturally-evolved protein networks can
effectively represent the structural and functional properties required for
fast information transfer among the residues facilitating biochemical/kinetic
functions, such as, allostery, stability, and the rate of folding.Comment: Published in Bioinformatic
Cooperativity and the origins of rapid, single-exponential kinetics in protein folding
The folding of naturally occurring, single domain proteins is usually
well-described as a simple, single exponential process lacking significant
trapped states. Here we further explore the hypothesis that the smooth energy
landscape this implies, and the rapid kinetics it engenders, arises due to the
extraordinary thermodynamic cooperativity of protein folding. Studying
Miyazawa-Jernigan lattice polymers we find that, even under conditions where
the folding energy landscape is relatively optimized (designed sequences
folding at their temperature of maximum folding rate), the folding of
protein-like heteropolymers is accelerated when their thermodynamic
cooperativity enhanced by enhancing the non-additivity of their energy
potentials. At lower temperatures, where kinetic traps presumably play a more
significant role in defining folding rates, we observe still greater
cooperativity-induced acceleration. Consistent with these observations, we find
that the folding kinetics of our computational models more closely approximate
single-exponential behavior as their cooperativity approaches optimal levels.
These observations suggest that the rapid folding of naturally occurring
proteins is, at least in part, consequences of their remarkably cooperative
folding
Assessment of the optimization of affinity and specificity at protein–DNA interfaces
The biological functions of DNA-binding proteins often require that they interact with their targets with high affinity and/or high specificity. Here, we describe a computational method that estimates the extent of optimization for affinity and specificity of amino acids at a protein–DNA interface based on the crystal structure of the complex, by modeling the changes in binding-free energy associated with all individual amino acid and base substitutions at the interface. The extent to which residues are predicted to be optimal for specificity versus affinity varies within a given protein–DNA interface and between different complexes, and in many cases recapitulates previous experimental observations. The approach provides a complement to traditional methods of mutational analysis, and should be useful for rapidly formulating hypotheses about the roles of amino acid residues in protein–DNA interfaces
In Vivo Characterization of the Homing Endonuclease within the polB Gene in the Halophilic Archaeon Haloferax volcanii
Inteins are parasitic genetic elements, analogous to introns that excise themselves at the protein level by self-splicing, allowing the formation of functional non-disrupted proteins. Many inteins contain a homing endonuclease (HEN) gene, and rely on its activity for horizontal propagation. In the halophilic archaeon, Haloferax volcanii, the gene encoding DNA polymerase B (polB) contains an intein with an annotated but uncharacterized HEN. Here we examine the activity of the polB HEN in vivo, within its natural archaeal host. We show that this HEN is highly active, and able to insert the intein into both a chromosomal target and an extra-chromosomal plasmid target, by gene conversion. We also demonstrate that the frequency of its incorporation depends on the length of the flanking homologous sequences around the target site, reflecting its dependence on the homologous recombination machinery. Although several evolutionary models predict that the presence of an intein involves a change in the fitness of the host organism, our results show that a strain deleted for the intein sequence shows no significant changes in growth rate compared to the wild type
Phage T4 mobE promotes trans homing of the defunct homing endonuclease I-TevIII
Homing endonucleases are site-specific DNA endonucleases that typically function as mobile genetic elements by introducing a double-strand break (DSB) in genomes that lack the endonuclease, resulting in a unidirectional gene conversion event that mobilizes the homing endonuclease gene and flanking DNA. Here, we characterize phage T4-encoded mobE, a predicted free-standing HNH family homing endonuclease. We show that mobE is promoterless and dependent on upstream transcription for expression, and that an internal intrinsic terminator regulates mobE transcript levels. Crucially, in vivo mapping experiments revealed a MobE-dependent, strand-specific nick in the non-coding strand of the nrdB gene of phage T2. An internal deletion of the predicted HNH catalytic motif of MobE abolishes nicking, and reduces high-frequency inheritance of mobE. Sequence polymorphisms of progeny phage that inherit mobE are consistent with DSB repair pathways. Significantly, we found that mobility of the neighboring I-TevIII, a defunct homing endonuclease encoded within a group I intron interrupting the nrdB gene of phage T4, was dependent on an intact mobE gene. Thus, our data indicate that the stagnant nrdB intron and I-TevIII are mobilized in trans as a consequence of a MobE-dependent gene conversion event, facilitating persistence of genetic elements that have no inherent means of promoting their own mobility
Generation of single-chain LAGLIDADG homing endonucleases from native homodimeric precursor proteins
Homing endonucleases (HEs) cut long DNA target sites with high specificity to initiate and target the lateral transfer of mobile introns or inteins. This high site specificity of HEs makes them attractive reagents for gene targeting to promote DNA modification or repair. We have generated several hundred catalytically active, monomerized versions of the well-characterized homodimeric I-CreI and I-MsoI LAGLIDADG family homing endonuclease (LHE) proteins. Representative monomerized I-CreI and I-MsoI proteins (collectively termed mCreIs or mMsoIs) were characterized in detail by using a combination of biochemical, biophysical and structural approaches. We also demonstrated that both mCreI and mMsoI proteins can promote cleavage-dependent recombination in human cells. The use of single chain LHEs should simplify gene modification and targeting by requiring the expression of a single small protein in cells, rather than the coordinate expression of two separate protein coding genes as is required when using engineered heterodimeric zinc finger or homing endonuclease proteins
Expanding LAGLIDADG endonuclease scaffold diversity by rapidly surveying evolutionary sequence space
LAGLIDADG homing endonucleases (LHEs) are a family of highly specific DNA endonucleases capable of recognizing target sequences ∼20 bp in length, thus drawing intense interest for their potential academic, biotechnological and clinical applications. Methods for rational design of LHEs to cleave desired target sites are presently limited by a small number of high-quality native LHEs to serve as scaffolds for protein engineering—many are unsatisfactory for gene targeting applications. One strategy to address such limitations is to identify close homologs of existing LHEs possessing superior biophysical or catalytic properties. To test this concept, we searched public sequence databases to identify putative LHE open reading frames homologous to the LHE I-AniI and used a DNA binding and cleavage assay using yeast surface display to rapidly survey a subset of the predicted proteins. These proteins exhibited a range of capacities for surface expression and also displayed locally altered binding and cleavage specificities with a range of in vivo cleavage activities. Of these enzymes, I-HjeMI demonstrated the greatest activity in vivo and was readily crystallizable, allowing a comparative structural analysis. Taken together, our results suggest that even highly homologous LHEs offer a readily accessible resource of related scaffolds that display diverse biochemical properties for biotechnological applications
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