10 research outputs found
All motors have to decide is what to do with the DNA that is given them
This is the published version. Copyright 2014 De Gruyter.DNA translocases are a diverse group of molecular motors responsible for a wide variety of cellular functions. The goal of this review is to identify common aspects in the mechanisms for how these enzymes couple the binding and hydrolysis of ATP to their movement along DNA. Not surprisingly, the shared structural components contained within the catalytic domains of several of these motors appear to give rise to common aspects of DNA translocation. Perhaps more interesting, however, are the differences between the families of translocases and the potential associated implications both for the functions of the members of these families and for the evolution of these families. However, as there are few translocases for which complete characterizations of the mechanisms of DNA binding, DNA translocation, and DNA-stimulated ATPase have been completed, it is difficult to form many inferences. We therefore hope that this review motivates the necessary further experimentation required for broader comparisons and conclusions
Disrupted Pathways: Generating Tunable Macromolecular Assembly Pathways
What follows is a pathway; a sequence of individual events, which together form a story. Yet it is still only a small part of what has come before. Biological structures also have individual stories; each composed of simple events in sequence. One story does not tell the whole, for that we must observe many stories, sample them if you will. Together, they bring understanding. Assembly is an emergent property of many individual binding events. Through this, all of the structures that make up life are created. Understanding the regime of possibilities provides insight into both the breadth and tendencies of the system. Cells contain numerous types of individual proteins many of which come together to form larger complexes. I will begin by introducing the elementary building blocks of those protein complexes. An introductory example will provide the first perspective, it will form common ground and allow the telling of the larger story with a shared perspective. Then a case study, a real biological complex and how understanding the progression of its pathways provided insight into the states which it reached. With the elementary operations described, I will move on to laying out the landscape of possible pathways; first for a specific case and then the structure of the assembly pathways themselves. Thus, providing a novel framework for the understanding of the stochastic space of protein complex assembly. Finally, I will provide an example of how making changes in the possible assembly pathways leads to non-intuitive changes in the conclusion of the protein complexes’ stories
Proteins are Not Recruited: A Plea for Better Diction
Nearly all biological processes proceed or are controlled by protein-protein or protein-ligand binding reactions. Using
anthropomorphic language to describe these interactions conveys an incorrect physical description of these processes
while simultaneously minimizing the importance of the thermodynamics underpinning the associated interactions.
Indeed, we should never say that proteins are recruited to binding partners or binding sites since this implies both a non-
existent level of communication within biological systems and a non-existent process by which proteins or binding sites
actively seek other proteins. Both of these fictions hinder our ability to determine quantitatively or qualitatively distinct
biophysical descriptions of the associated systems. Here we present examples of how interactions typically described as
protein recruitment can be more accurately and often more simply described as variations within binding equilibria. We
argue that this approach is better for describing protein-protein and protein-ligand binding, even when the objective is
only a qualitative description, especially for discussions with students in courses and research groups as it provides testable
models for these interaction
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Modeling reveals the strength of weak interactions in stacked-ring assembly.
Cells employ many large macromolecular machines for the execution and regulation of processes that are vital for cell and organismal viability. Interestingly, cells cannot synthesize these machines as functioning units. Instead, cells synthesize the molecular parts that must then assemble into the functional complex. Many important machines, including chaperones such as GroEL and proteases such as the proteasome, comprise protein rings that are stacked on top of one another. While there is some experimental data regarding how stacked-ring complexes such as the proteasome self-assemble, a comprehensive understanding of the dynamics of stacked-ring assembly is currently lacking. Here, we developed a mathematical model of stacked-trimer assembly and performed an analysis of the assembly of the stacked homomeric trimer, which is the simplest stacked-ring architecture. We found that stacked rings are particularly susceptible to a form of kinetic trapping that we term deadlock, in which the system gets stuck in a state where there are many large intermediates that are not the fully assembled structure but that cannot productively react. When interaction affinities are uniformly strong, deadlock severely limits assembly yield. We thus predicted that stacked rings would avoid situations where all interfaces in the structure have high affinity. Analysis of available crystal structures indicated that indeed the majority-if not all-of stacked trimers do not contain uniformly strong interactions. Finally, to better understand the origins of deadlock, we developed a formal pathway analysis and showed that, when all the binding affinities are strong, many of the possible pathways are utilized. In contrast, optimal assembly strategies utilize only a small number of pathways. Our work suggests that deadlock is a critical factor influencing the evolution of macromolecular machines and provides general principles for understanding the self-assembly efficiency of existing machines
Effects of nucleosome stability on remodeler-catalyzed repositioning
Chromatin remodelers are molecular motors that play essential roles in the regulation of nucleosome positioning and chromatin accessibility. These machines couple the energy obtained from the binding and hydrolysis of ATP to the mechanical work of manipulating chromatin structure through processes that are not completely understood. Here we present a quantitative analysis of nucleosome repositioning by the imitation switch (ISWI) chromatin remodeler and demonstrate that nucleosome stability significantly impacts the observed activity. We show how DNA damage induced changes in the affinity of DNA wrapping within the nucleosome can affect ISWI repositioning activity and demonstrate how assay-dependent limitations can bias studies of nucleosome repositioning. Together, these results also suggest that some of the diversity seen in chromatin remodeler activity can be attributed to the variations in the thermodynamics of interactions between the remodeler, the histones, and the DNA, rather than reflect inherent properties of the remodeler itself
ISWI Remodels Nucleosomes through a Random Walk
The chromatin remodeler ISWI is capable
of repositioning clusters
of nucleosomes to create well-ordered arrays or moving single nucleosomes
from the center of DNA fragments toward the ends without disrupting
their integrity. Using standard electrophoresis assays, we have monitored
the ISWI-catalyzed repositioning of different nucleosome samples each
containing a different length of DNA symmetrically flanking the initially
centrally positioned histone octamer. We find that ISWI moves the
histone octamer between distinct and thermodynamically stable positions
on the DNA according to a random walk mechanism. Through the application
of a spectrophotometric assay for nucleosome repositioning, we further
characterized the repositioning activity of ISWI using short nucleosome
substrates and were able to determine the macroscopic rate of nucleosome
repositioning by ISWI. Additionally, quantitative analysis of repositioning
experiments performed at various ISWI concentrations revealed that
a monomeric ISWI is sufficient to obtain the observed repositioning
activity as the presence of a second ISWI bound had no effect on the
rate of nucleosome repositioning. We also found that ATP hydrolysis
is poorly coupled to nucleosome repositioning, suggesting that DNA
translocation by ISWI is not energetically rate-limiting for the repositioning
reaction. This is the first calculation of a microscopic ATPase coupling
efficiency for nucleosome repositioning and also further supports
our conclusion that a second bound ISWI does not contribute to the
repositioning reaction
Quantitative Determination of Binding of ISWI to Nucleosomes and DNA Shows Allosteric Regulation of DNA Binding by Nucleotides
The
regulation of chromatin structure is controlled by a family
of molecular motors called chromatin remodelers. The ability of these
enzymes to remodel chromatin structure is dependent on their ability
to couple ATP binding and hydrolysis into the mechanical work that
drives nucleosome repositioning. The necessary first step in determining
how these essential enzymes perform this function is to characterize
both how they bind nucleosomes and how this interaction is regulated
by ATP binding and hydrolysis. With this goal in mind, we monitored
the interaction of the chromatin remodeler ISWI with fluorophore-labeled
nucleosomes and DNA through associated changes in fluorescence anisotropy
of the fluorophore upon binding of ISWI to these substrates. We determined
that one ISWI molecule binds to a 20 bp double-stranded DNA substrate
with an affinity of 18 ± 2 nM. In contrast, two ISWI molecules
can bind to the core nucleosome with short linker DNA with stoichiometric
macroscopic equilibrium constants: 1/β<sub>1</sub> = 1.3 ±
0.6 nM, and 1/β<sub>2</sub> = 13 ± 7 nM<sup>2</sup>. Furthermore,
to improve our understanding of the mechanism of DNA translocation
by ISWI, and hence nucleosome repositioning, we determined the effect
of nucleotide analogues on substrate binding by ISWI. While the affinity
of ISWI for the nucleosome substrate with short lengths of flanking
DNA was not affected by the presence of nucleotides, the affinity
of ISWI for the DNA substrate is weakened in the presence of nonhydrolyzable
ATP analogues but not by ADP