Biomedical subjects

Peter D Olmsted

Publications and source records attributed to Peter D Olmsted.

12 recordsLinked to original sources

PubMed2006

Free energy for protein folding from nonequilibrium simulations using the Jarzynski equality.

The equilibrium free energy difference between two long-lived molecular species or "conformational states" of a protein (or any other molecule) can in principle be estimated by measuring the work needed to shuttle the system between them, independent of the irreversibility of the process. This is the meaning of the Jarzynski equality (JE), which we test in this paper by performing simulations that unfold a protein by pulling two atoms apart. Pulling is performed fast relative to the relaxation time of the molecule and is thus far from equilibrium. Choosing a simple protein model for which we can independently compute its equilibrium properties, we show that the free energy can be exactly and effectively estimated from nonequilibrium simulations. To do so, one must carefully and correctly determine the ensemble of states that are pulled, which is more important the farther from equilibrium one performs simulations; this highlights a potential problem in using the JE to extract the free energy from forced unfolding experiments. The results presented here also demonstrate that the free energy difference between the native and denatured states of a protein measured in solution is not always equal to the free energy profile that can be estimated from forced unfolding simulations (or experiments) using the JE.

Algorithms↗

PubMed2006

Mechanical unfolding revisited through a simple but realistic model.

Single-molecule experiments and their application to probe the mechanical resistance and related properties of proteins provide a new dimension in our knowledge of these important and complex biological molecules. Single-molecule techniques may not have yet overridden solution experiments as a method of choice to characterize biophysical and biological properties of proteins, but have stimulated a debate and contributed considerably to bridge theory and experiment. Here we demonstrate this latter contribution by illustrating the reach of some theoretical findings using a solvable but nontrivial molecular model whose properties are analogous to those of the corresponding experimental systems. In particular, we show the relationship between the thermodynamic and the mechanical properties of a protein. The simulations presented here also illustrate how forced and spontaneous unfolding occur through different pathways and that folding and unfolding rates at equilibrium cannot in general be obtained from forced unfolding experiments or simulations. We also study the relationship between the energy surface and the mechanical resistance of a protein and show how a simple analysis of the native state can predict much of the mechanical properties of a protein.

Carrier Proteins↗

PubMed2006

Effect of hydrophobic mismatch on phase behavior of lipid membranes.

We investigate the competing effects of hydrophobic mismatch and chain stretching on the morphology and evolution of domains in lipid membranes via Monte Carlo techniques. We model the membrane as a binary mixture of particles that differ in their preferred lengths, with the shorter particles mimicking unsaturated nonraft lipids and the longer particles mimicking saturated raft lipids. We find that phase separation can be induced upon increasing either the ratio J/kappa of the hydrophobic surface tension J to the compressibility modulus kappa. J/kappa determines the decay length for thickness changes. When this decay length is larger than the system size the membrane remains mixed. Furthermore, increasing the thickness relaxation time can induce transient phase separation.

Computer Simulation↗

PubMed2005

Mechanical resistance of proteins explained using simple molecular models.

Recent experiments have demonstrated that proteins unfold when two atoms are mechanically pulled apart, and that this process is different to when heated or when a chemical denaturant is added to the solution. Experiments have also shown that the response of proteins to external forces is very diverse, some of them being "hard," and others "soft." Mechanical resistance originates from the presence of barriers on the energy landscape; together, experiment and simulation have demonstrated that unfolding occurs through alternative pathways when different pairs of atoms undergo mechanical extension. Here we use simulation to probe the mechanical resistance of six structurally diverse proteins when pulled in different directions. For this, we use two very different models: a detailed, transferable one, and a coarse-grained, structure-based one. The coarse-grained model gives results that are surprisingly similar to the detailed one and qualitatively agree with experiment; i.e., the mechanical resistance of different proteins or of a single protein pulled in different directions can be predicted by simulation. The results demonstrate the importance of pulling direction relative to the local topology in determining mechanical stability, and rationalize the effect of the location of importation/degradation tags on the rates of mitochondrial import or protein degradation in vivo.

Biophysics↗

PubMed2005

Mechanically unfolding the small, topologically simple protein L.

beta-sheet proteins are generally more able to resist mechanical deformation than alpha-helical proteins. Experiments measuring the mechanical resistance of beta-sheet proteins extended by their termini led to the hypothesis that parallel, directly hydrogen-bonded terminal beta-strands provide the greatest mechanical strength. Here we test this hypothesis by measuring the mechanical properties of protein L, a domain with a topology predicted to be mechanically strong, but with no known mechanical function. A pentamer of this small, topologically simple protein is resistant to mechanical deformation over a wide range of extension rates. Molecular dynamics simulations show the energy landscape for protein L is highly restricted for mechanical unfolding and that this protein unfolds by the shearing apart of two structural units in a mechanism similar to that proposed for ubiquitin, which belongs to the same structural class as protein L, but unfolds at a significantly higher force. These data suggest that the mechanism of mechanical unfolding is conserved in proteins within the same fold family and demonstrate that although the topology and presence of a hydrogen-bonded clamp are of central importance in determining mechanical strength, hydrophobic interactions also play an important role in modulating the mechanical resistance of these similar proteins.

Bacterial Proteins↗

PubMed2005

The kinetics of phase separation in asymmetric membranes.

Phase separation in a model asymmetric membrane is studied using Monte Carlo techniques. The membrane comprises two species of particles, which mimic different lipids in lipid bilayers and separately possess either zero or non-zero spontaneous curvatures. We study the influence of phase separation on membrane shape and the influence of the coupling of composition and height dynamics on phase separation and domain growth, via both the degree of shape asymmetry and relative kinetic coefficients for height relaxation.

Biophysical Phenomena↗

PubMed2003

Pulling geometry defines the mechanical resistance of a beta-sheet protein.

Proteins show diverse responses when placed under mechanical stress. The molecular origins of their differing mechanical resistance are still unclear, although the orientation of secondary structural elements relative to the applied force vector is thought to have an important function. Here, by using a method of protein immobilization that allows force to be applied to the same all-beta protein, E2lip3, in two different directions, we show that the energy landscape for mechanical unfolding is markedly anisotropic. These results, in combination with molecular dynamics (MD) simulations, reveal that the unfolding pathway depends on the pulling geometry and is associated with unfolding forces that differ by an order of magnitude. Thus, the mechanical resistance of a protein is not dictated solely by amino acid sequence, topology or unfolding rate constant, but depends critically on the direction of the applied extension.

Anisotropy↗

PubMed2003

Unfolding dynamics of proteins under applied force.

Understanding the mechanisms of protein folding is a major challenge that is being addressed effectively by collaboration between researchers in the physical and life sciences. Recently, it has become possible to mechanically unfold proteins by pulling on their two termini using local force probes such as the atomic force microscope. Here, we present data from experiments in which synthetic protein polymers designed to mimic naturally occurring polyproteins have been mechanically unfolded. For many years protein folding dynamics have been studied using chemical denaturation, and we therefore firstly discuss our mechanical unfolding data in the context of such experiments and show that the two unfolding mechanisms are not the same, at least for the proteins studied here. We also report unexpected observations that indicate a history effect in the observed unfolding forces of polymeric proteins and explain this in terms of the changing number of domains remaining to unfold and the increasing compliance of the lengthening unstructured polypeptide chain produced each time a domain unfolds.

Hydrogen Bonding↗

PubMed2003

Are metastable, precrystallisation, density-fluctuations a universal phenomena?

In-situ observations of crystallisation in minerals and organic polymers have been made by simultaneous, time-resolved small angle X-ray scattering (SAXS) and wide angle X-ray scattering (WAXS) techniques. In isotactic polypropylene slow quiescent crystallisation shows the onset of large scale ordering prior to crystal growth. Rapid crystallisations studied by melt extrusion indicate the development of well resolved oriented SAXS patterns associated with long range order before the development of crystalline peaks in the WAXS region. Block copolymers self-assemble into mesophases in polymer melts above a critical chain length (or above a critical temperature) and this self-assembly process is shown to be susceptible to an incipient crystallisation. Mesophase formation is observed at anomalously high temperatures in ethylene-oxide containing block copolymers below the normal melting point of the polyoxy ethylene chains. Formation of calcium carbonate from aqueous solutions of sodium carbonate and calcium nitrate is observed to be a two-stage process and precipitation proceeds by the production of an amorphous metastable phase. This phase grows until it is volume filling and leads to the formation of the two polymorphs Calcite and Vaterite. These three sets of results suggest pre-nucleation density fluctuations, leading to a metastable phase, play an integral role in all three classes of crystallisation. In due course, this phase undergoes transformation to "normal" crystals.

Journal Article↗

PubMed2002

Dynamical coarse-graining of highly fluctuating membranes under shear flow.

The effect of strong shear flow on highly fluctuating lamellar systems stabilized by intermembrane collisions via the Helfrich interaction is studied. Advection enters the microscopic equation of motion for a single membrane via a nonlinear coupling. Upon coarse-graining the theory for a single bilayer up to the length scale of the collision length, at which a hydrodynamic description applies, an additional dynamical coupling is generated which is of the form of a wave-vector-dependent tension that is nonlinear in the applied shear rate. This new term has consequences for the effects of strong flow on the stability and dynamics of lamellar surfactant phases.

Journal Article↗

PubMed2002

The effect of core destabilization on the mechanical resistance of I27.

It is still unclear whether mechanical unfolding probes the same pathways as chemical denaturation. To address this point, we have constructed a concatamer of five mutant I27 domains (denoted (I27)(5)*) and used it for mechanical unfolding studies. This protein consists of four copies of the mutant C47S, C63S I27 and a single copy of C63S I27. These mutations severely destabilize I27 (DeltaDeltaG(UN) = 8.7 and 17.9 kJ mol(-1) for C63S I27 and C47S, C63S I27, respectively). Both mutations maintain the hydrogen bond network between the A' and G strands postulated to be the major region of mechanical resistance for I27. Measuring the speed dependence of the force required to unfold (I27)(5)* in triplicate using the atomic force microscope allowed a reliable assessment of the intrinsic unfolding rate constant of the protein to be obtained (2.0 x 10(-3) s(-1)). The rate constant of unfolding measured by chemical denaturation is over fivefold faster (1.1 x 10(-2) s(-1)), suggesting that these techniques probe different unfolding pathways. Also, by comparing the parameters obtained from the mechanical unfolding of a wild-type I27 concatamer with that of (I27)(5)*, we show that although the observed forces are considerably lower, core destabilization has little effect on determining the mechanical sensitivity of this domain.

Amino Acid Sequence↗

PubMed2002

Mechanically unfolding proteins: the effect of unfolding history and the supramolecular scaffold.

The mechanical resistance of a folded domain in a polyprotein of five mutant I27 domains (C47S, C63S I27)(5)is shown to depend on the unfolding history of the protein. This observation can be understood on the basis of competition between two effects, that of the changing number of domains attempting to unfold, and the progressive increase in the compliance of the polyprotein as domains unfold. We present Monte Carlo simulations that show the effect and experimental data that verify these observations. The results are confirmed using an analytical model based on transition state theory. The model and simulations also predict that the mechanical resistance of a domain depends on the stiffness of the surrounding scaffold that holds the domain in vivo, and on the length of the unfolded domain. Together, these additional factors that influence the mechanical resistance of proteins have important consequences for our understanding of natural proteins that have evolved to withstand force.

Computer Simulation↗