Martti Louhivuori

Release of content through mechano-sensitive gates in pressurized liposomes

Mechano-sensitive channels are ubiquitous membrane proteins that activate in response to increasing tension in the lipid membrane. They facilitate a sudden, nonselective release of solutes and water that safeguards the integrity of the cell in hypo- or hyper-osmotic shock conditions. We have simulated the rapid release of content from a pressurized liposome through a particular mechano-sensitive protein channel, MscL, embedded in the liposomal membrane. We show that a single channel is able to relax the liposome, stressed to the point of bursting, in a matter of microseconds. We map the full activation–deactivation cycle of MscL in near-atomic detail and are able to quantify the rapid decrease in liposomal stress as a result of channel activation. This provides a computational tool that opens the way to contribute to the rational design of functional nano-containers.

Protein Shape Change Has a Major Effect on the Gating Energy of a Mechanosensitive Channel

Increasing experimental evidence has shown that membrane protein functionality depends on molecular composition of cell membranes. However, the origin of this dependence is not fully understood. It is reasonable to assume that specific lipid-protein interactions are important, yet more generic effects due to mechanical properties of lipid bilayers likely play a significant role too. Previously it has been demonstrated using models for elastic properties of membranes and lateral pressure profiles of lipid bilayers that the mechanical properties of a lipid bilayer can contribute as much as ∼10 k(B)T to the free energy difference associated with a change in protein conformational state. Here, we extend those previous approaches to a more realistic model for a large mechanosensitive channel (MscL). We use molecular dynamics together with the MARTINI model to simulate the open and closed states of MscL embedded in a DOPC bilayer. We introduce a procedure to calculate the mechanical energy change in the channel gating using a three-dimensional pressure distribution inside a membrane, computed from the molecular dynamics simulations. We decompose the mechanical energy to terms associated with area dilation and shape contribution. Our results highlight that the lateral pressure profile of a lipid bilayer together with the shape change in gating can induce a contribution of ∼30 k(B)T on the gating energy of MscL. This contribution arises largely from the interfacial tension between hydrophobic and hydrophilic regions in a lipid bilayer.

Structural investigation of MscL gating using experimental data and coarse grained MD simulations.

The mechanosensitive channel of large conductance (MscL) has become a model system in which to understand mechanosensation, a process involved in osmoregulation and many other physiological functions. While a high resolution closed state structure is available, details of the open structure and the gating mechanism remain unknown. In this study we combine coarse grained simulations with restraints from EPR and FRET experiments to study the structural changes involved in gating with much greater level of conformational sampling than has previously been possible. We generated a set of plausible open pore structures that agree well with existing open pore structures and gating models. Most interestingly, we found that membrane thinning induces a kink in the upper part of TM1 that causes an outward motion of the periplasmic loop away from the pore centre. This previously unobserved structural change might present a new mechanism of tension sensing and might be related to a functional role in osmoregulation.

Structural, Dynamic Properties of Key Residues in Aβ Amyloidogenesis: Implications of an Important Role of Nanosecond Timescale Dynamics

The deposition of protein aggregates (amyloid) is associated with numerous debilitating human diseases, including Alzheimer’s and Parkinson’s diseases. A variety of polypeptides that form pathogenic amyloids have been identified. It has been demonstrated that the primary amino-acid sequence as well as tertiary structures of amyloidogenic proteins are highly diverse. In addition, polypeptides that are not related to amyloid diseases have been shown to form amyloids in vitro. These studies indicate that amyloid formation is a generic property of a polypeptide chain. Aggregation kinetics of polypeptides are, however, strongly dependent on the sequence. This suggests that there might exist critical regions that facilitate intermolecular interactions and subsequently promote aggregation. Identification of aggregation-prone sequences in amyloidogenic polypeptides has, therefore, been of great interest in structural biology. Recently, numerical algorithms have been developed to predict the aggregation-prone segment by considering physicochemical parameters (hydrophobicity, charge, and secondary-structure propensity) of individual amino-acid residues. Such relatively simple approaches have identified the amyloidogenic regions for various amyloid-forming polypeptides. For example, two hydrophobic regions, residues 17–21 and 31–42, of the AbACHTUNGTRENNUNG(1–42) peptide were predicted to have high aggregation propensities, which correlated well with the Ab fibril structure. However, residues 17–21, which were estimated to have a slightly lower aggregation score than the C terminus, have been shown to play a more critical role in nucleating Ab oligomerization. This indicates that additional properties of a polypeptide should be considered for a better understanding of the aggregation propensity. Amyloid formation involves intermolecular association of unfolded and/or partly folded monomeric amyloidogenic intermediates into b-structured insoluble amyloids; this renders the amyloid-forming process highly unfavorable in terms of ACHTUNGTRENNUNGentropy change. More ordered polypeptide segments with bsheet characteristics might, therefore, be more amenable to ACHTUNGTRENNUNGinitiating and promoting intermolecular associations that are necessary for amyloid formation. In this study, structural, dynamic properties of various forms of Ab peptides with different aggregation propensities were investigated in order to determine whether aggregation-prone regions posses distinct properties. Residual dipolar couplings (RDC) and N relaxation NMR experiments, which have provided invaluable structural information on disordered states of proteins, were employed to investigate the structural, dynamic features of Ab peptides. Residual local structures of the natively unfolded Ab ACHTUNGTRENNUNG(1–40) peptide were explored with RDC measurement in strained gels at the nonamyloidogenic temperature of 3 8C (Figure 1A). The experimental RDC profile (red) was compared to that calculated (blue) from a statistical coil model by using the program PALES (Figure 1A). A good agreement between the experimental and calculated RDC profiles was observed in the Nterminal region; this suggests that the local structural properties are intrinsic to the amino-acid sequence. RDC values of residues Tyr10, Phe19, and Phe20, however, notably deviated from the estimated values. The lower RDC values suggest that the aromatic residues might have a turn-like feature, which was observed in previous studies of disordered states of proteins. 34] It is also interesting to note that the calculated RDC values of the C-terminal residues 32–40 were averaged to zero; this indicates that the C terminus is randomly disordered in the statistical coil model. The experimental RDCs were, however, significantly higher than predicted. The higher RDC values of the C-terminal region clearly indicate that the C-terminal region adopts a more extended conformation than those in the statistical coil model. The locally ordered C terminus was not observed with chemical shift and NOE analyses; this suggests that RDCs are more sensitive to local ordering of a polypeptide segment. RDCs were also measured at a higher temperature (15 8C), that is, under more amyloidogenic conditions, to probe changes in the structural features (Figure 1B). Under the more amyloidogenic conditions, the overall pattern of the RDC profile did not change except for residues 19–20. The RDC values of the hydrophobic residues slightly increased at 15 8C; this suggests that these residues become slightly more extended at the higher temperature. Structural studies by using RDC measurements were carried out with the much more amyloidogenic AbACHTUNGTRENNUNG(1–42) peptide, which contains two more residues in the C terminus (Figure 1C). The overall RDC pattern of Ab ACHTUNGTRENNUNG(1–42) at the two temperatures tested was in close proximity to that of Ab ACHTUNGTRENNUNG(1–40). In addition, the experimental RDC values for residues 21–30 were very similar to the RDC pattern of a hairpin structure. Previ[a] Prof. Dr. K. H. Lim, G. L. Henderson Department of Chemistry, East Carolina University Greenville, NC 27858 (USA) Fax: (+1)252-328-6210 E-mail : limk@ecu.edu [b] A. Jha Biochemistry and Molecular Biology Institute for Biophysical Dynamics Department of Chemistry, University of Chicago Chicago, IL 60637 (USA) [c] M. Louhivuori Department of Physical Sciences Department of Chemistry, University of Helsinki P.O. Box 33 (Fabianinkatu 18), 00014 Helsinki (Finland) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

Vibrational Spectra of a Mechanosensitive Channel

We report the simulated vibrational spectra of a mechanosensitive membrane channel in different gating states. Our results show that while linear absorption is insensitive to structural differences, linear dichroism and sum-frequency generation spectroscopies are sensitive to the orientation of the transmembrane helices, which is changing during the opening process. Linear dichroism cannot distinguish an intermediate structure from the closed structure, but sum-frequency generation can. In addition, we find that two-dimensional infrared spectroscopy can be used to distinguish all three investigated gating states of the mechanosensitive membrane channel.

Evidence of molecular alignment fluctuations in aqueous dilute liquid crystalline media

Protein dynamics can be studied by NMR measurements of aqueous dilute liquid crystalline samples. However, the measured residual dipolar couplings are sensitive not only to internal fluctuations but to all changes in internuclear vectors relative to the laboratory frame. We show that side-chain fluctuations and bond librations in the ps–ns time scale perturb the molecular shape and charge distribution of a small globular protein sufficiently to cause a noticeable variation in the molecular alignment. The alignment variation disperses the bond vectors of a conformational ensemble even further from the dispersion already caused by internal fluctuations of a protein. Consequently RDC-probed order parameters are lower than those obtained by laboratory frame relaxation measurements.

Sensitivity of Coarse-Grained Mechano-Sensation

Mechano-sensitive channels are ubiquitous membrane proteins that activate in response to increasing tension in the lipid membrane. They facilitate a sudden, non-selective release of solutes and water that safe-guards the integrity of the cell in hypo- or hyper-osmotic shock conditions. Mechano-sensitive channels react to a sudden increase in membrane tension by forming trans-membrane pores that counteract the pressure gradient build-up by balancing the osmotic conditions on either side of the cell membrane. They have a crucial role in diverse biological functions from sensory feedback to the prevention of cell death by membrane rupture.A varity of different mechano-sensitive channels exist with names such as MscL, MscS, and MscM describing their experimentally observed gating properties. Recent crystal structures of MscL (2OAR) and MscS (2OAU, 2VV5) have opened the way for computer simulation studies of the function, dynamics, and molecular mechanisms of these exciting channels. The coarse-grained MARTINI force-field, which has been build to reproduce accurately macroscopic and thermodynamic properties of biological systems, is ideally suited for the task. We have looked at the sensitivity of the coarse-grained channels to internal and external changes and their impact on the channel function.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Mixing Martinis: Hybrid Atomistic/Coarse-Grained Models for Protein Molecular Dynamics

In recent years, the development and deployment of coarse grained models for simulations of proteins has taken an enormous flight. The main reason for this is that such models provide significant alleviation of the time scale limits that otherwise restrict the use of molecular simulations for biological processes. Coarse graining allows assessment of processes that occur on the scale of microseconds and micrometers, rather than nanoseconds and nanometers, albeit with the obvious consequence that detail is lost. This loss of detail has proven acceptable in many cases, but poses problems for the assessment of mechanical features of proteins, especially where local dynamics is intimately linked with overall conformational changes.To bring back the detail, yet only where it is needed, we have developed an integrative approach, coupling a Martini Coarse Grained model to an atomistic description of part of the system. This method involves a novel treatment of the interaction of the all-atom parts with the surrounding coarse grained particles, using virtual sites, rather than specific cross interactions. The potential applications of the method are manifold and include high-throughput protein-ligand binding studies, adsorption and protein folding.