Filip Persson

Can misfolded proteins be beneficial? The HAMLET case.

By changing the three-dimensional structure, a protein can attain new functions, distinct from those of the native protein. Amyloid-forming proteins are one example, in which conformational change may lead to fibril formation and, in many cases, neurodegenerative disease. We have proposed that partial unfolding provides a mechanism to generate new and useful functional variants from a given polypeptide chain. Here we present HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) as an example where partial unfolding and the incorporation of cofactor create a complex with new, beneficial properties. Native α-lactalbumin functions as a substrate specifier in lactose synthesis, but when partially unfolded the protein binds oleic acid and forms the tumoricidal HAMLET complex. When the properties of HAMLET were first described they were surprising, as protein folding intermediates and especially amyloid-forming protein intermediates had been regarded as toxic conformations, but since then structural studies have supported functional diversity arising from a change in fold. The properties of HAMLET suggest a mechanism of structure-function variation, which might help the limited number of human protein genes to generate sufficient structural diversity to meet the diverse functional demands of complex organisms.

Transient Access to the Protein Interior: Simulation versus NMR.

Many proteins rely on rare structural fluctuations for their function, whereby solvent and other small molecules gain transient access to internal cavities. In magnetic relaxation dispersion (MRD) experiments, water molecules buried in such cavities are used as intrinsic probes of the intermittent protein motions that govern their exchange with external solvent. While this has allowed a detailed characterization of exchange kinetics for several proteins, little is known about the exchange mechanism. Here, we use a millisecond all-atom MD trajectory produced by Shaw et al. (Science2010, 330, 341) to characterize water exchange from the four internal hydration sites in the protein bovine pancreatic trypsin inhibitor. Using a recently developed stochastic point process approach, we compute the survival correlation function probed by MRD experiments as well as other quantities designed to validate the exchange-mediated orientational randomization (EMOR) model used to interpret the MRD data. The EMOR model is found to be quantitatively accurate, and the simulation reproduces the experimental mean survival times for all four sites with activation energy discrepancies in the range 0-3 kBT. On the other hand, the simulated hydration sites are somewhat too flexible, and the water flip barrier is underestimated by up to 6 kBT. The simulation reveals that water molecules gain access to the internal sites by a transient aqueduct mechanism, migrating as single-file water chains through transient (<5 ns) tunnels or pores. The present study illustrates the power of state-of-the-art molecular dynamics simulations in validating and extending experimental results.

How amide hydrogens exchange in native proteins

Significance Proteins tend to be compactly folded but occasionally undergo conformational fluctuations that expose even the most deeply buried parts of the polypeptide chain to external solvent. As a consequence, the backbone amide hydrogens can exchange with water hydrogens. Monitoring such hydrogen exchange is a well-established method for characterizing protein flexibility, even though the nature of the ‟open” exchange-competent state is unknown. We have used an ultralong computer simulation to identify the elusive open state as transient (100 ps), locally distorted conformations where the N–H group coordinates two water molecules. Amide hydrogen exchange (HX) is widely used in protein biophysics even though our ignorance about the HX mechanism makes data interpretation imprecise. Notably, the open exchange-competent conformational state has not been identified. Based on analysis of an ultralong molecular dynamics trajectory of the protein BPTI, we propose that the open (O) states for amides that exchange by subglobal fluctuations are locally distorted conformations with two water molecules directly coordinated to the N–H group. The HX protection factors computed from the relative O-state populations agree well with experiment. The O states of different amides show little or no temporal correlation, even if adjacent residues unfold cooperatively. The mean residence time of the O state is ∼100 ps for all examined amides, so the large variation in measured HX rate must be attributed to the opening frequency. A few amides gain solvent access via tunnels or pores penetrated by water chains including native internal water molecules, but most amides access solvent by more local structural distortions. In either case, we argue that an overcoordinated N–H group is necessary for efficient proton transfer by Grotthuss-type structural diffusion.

Nano-aperture fabrication for single quantum dot spectroscopy

We present a simple and controllable method for fabricating nano-apertures in a metal film using polystyrene nano-spheres as masks during the metal evaporation. We show how the processing conditions used during deposition of the spheres such as spin velocity, nano-sphere concentration and a reduction of the surface tension interplay and control the distribution of spheres. The fabrication method is ideal for luminescence studies by isolating individual nanometre-sized objects, which is exemplified by photoluminescence spectroscopy of single self-assembled Stranski-Krastanow quantum dots.

Analysis of Protein Dynamics Simulations by a Stochastic Point Process Approach

MD simulations can now explore the complex dynamics of proteins and their associated solvent in atomic detail on a millisecond time scale. Among the phenomena that thereby become amenable to detailed study are intermittent conformational transitions where the protein accesses transient high-energy states that often play key roles in biology. Here, we present a coherent theoretical framework, based on the stochastic theory of stationary point processes, that allows the essential dynamical characteristics of such processes to be efficiently extracted from the MD trajectory without assuming Poisson statistics. Since the complete information content of a point process is contained in the sequence of residence or interevent times, the experimentally relevant survival correlation function can be computed several orders of magnitude more efficiently than with the conventional approach, involving averaging over initial times. We also present a detailed analysis of the statistical and binning errors, of particular importance when MD results are compared with experiment. As an illustration of the general theoretical framework, we use a 1 ms MD trajectory of the protein BPTI to analyze the exchange kinetics of an internal water molecule and the dynamics of the rare conformational fluctuations that govern the rate of this exchange process.

The spatial range of protein hydration

Proteins interact with their aqueous surroundings, thereby modifying the physical properties of the solvent. The extent of this perturbation has been investigated by numerous methods in the past half-century, but a consensus has still not emerged regarding the spatial range of the perturbation. To a large extent, the disparate views found in the current literature can be traced to the lack of a rigorous definition of the perturbation range. Stating that a particular solvent property differs from its bulk value at a certain distance from the protein is not particularly helpful since such findings depend on the sensitivity and precision of the technique used to probe the system. What is needed is a well-defined decay length, an intrinsic property of the protein in a dilute aqueous solution, that specifies the length scale on which a given physical property approaches its bulk-water value. Based on molecular dynamics simulations of four small globular proteins, we present such an analysis of the structural and dynamic properties of the hydrogen-bonded solvent network. The results demonstrate unequivocally that the solvent perturbation is short-ranged, with all investigated properties having exponential decay lengths of less than one hydration shell. The short range of the perturbation is a consequence of the high energy density of bulk water, rendering this solvent highly resistant to structural perturbations. The electric field from the protein, which under certain conditions can be long-ranged, induces a weak alignment of water dipoles, which, however, is merely the linear dielectric response of bulk water and, therefore, should not be thought of as a structural perturbation. By decomposing the first hydration shell into polarity-based subsets, we find that the hydration structure of the nonpolar parts of the protein surface is similar to that of small nonpolar solutes. For all four examined proteins, the mean number of water-water hydrogen bonds in the nonpolar subset is within 1% of the value in bulk water, suggesting that the fragmentation and topography of the nonpolar protein-water interface has evolved to minimize the propensity for protein aggregation by reducing the unfavorable free energy of hydrophobic hydration.

Nanoimprint - a tool for realizing nano-bio research

In this paper, we present a status report on how implementation of nanoimprint lithography has advanced our research. Contact guidance nerve growth experiments have so far primarily been done on micrometer-structured surfaces. We have made a stamp with 17 areas of different, submicron, line width and spacing covering a total 2.6 mm/spl times/0.45 mm. This has been imprinted, in PMMA, and consequently used in experiments to investigate how axonal outgrowth is affected by the nanopatterns. Protein interactions with nanostructured surfaces are also studied in a system exploring and controlling biomolecular motors, i.e., the muscle motor proteins actin and myosin.