Mateusz Chwastyk

Cotranslational folding of deeply knotted proteins.

Proper folding of deeply knotted proteins has a very low success rate even in structure-based models which favor formation of the native contacts but have no topological bias. By employing a structure-based model, we demonstrate that cotranslational folding on a model ribosome may enhance the odds to form trefoil knots for protein YibK without any need to introduce any non-native contacts. The ribosome is represented by a repulsive wall that keeps elongating the protein. On-ribosome folding proceeds through a a slipknot conformation. We elucidate the mechanics and energetics of its formation. We show that the knotting probability in on-ribosome folding is a function of temperature and that there is an optimal temperature for the process. Our model often leads to the establishment of the native contacts without formation of the knot.

Polysaccharide–Protein Complexes in a Coarse-Grained Model

We construct two variants of coarse-grained models of three hexaoses: one based on the centers of mass of the monomers and the other associated with the C4 atoms. The latter is found to be better defined and more suitable for studying interactions with proteins described within α-C based models. We determine the corresponding effective stiffness constants through all-atom simulations and two statistical methods. One method is the Boltzmann inversion (BI) and the other, named energy-based (EB), involves direct monitoring of energies as a function of the variables that define the stiffness potentials. The two methods are generally consistent in their account of the stiffness. We find that the elastic constants differ between the hexaoses and are noticeably different from those determined for the crystalline cellulose Iβ. The nonbonded couplings through hydrogen bonds between different sugar molecules are modeled by the Lennard-Jones potentials and are found to be stronger than the hydrogen bonds in proteins. We observe that the EB method agrees with other theoretical and experimental determinations of the nonbonded parameters much better than BI. We then consider the hexaose-Man5B catalytic complexes and determine the contact energies between their the C4-α-C atoms. These interactions are found to be stronger than the proteinic hydrogen bonds: about four times as strong for cellohexaose and two times for mannohexaose. The fluctuational dynamics of the coarse-grained complexes are found to be compatible with previous all-atom studies by Bernardi et al.

Synthesis of ZnAl2O4:(Er3+,Yb3+) spinel-type nanocrystalline upconverting luminescent marker in HeLa carcinoma cells, using a combustion aerosol method route

Efficiently upconverting, spherical ZnAl2O4 nanoparticles (NPs), doped with erbium and ytterbium, were synthesized by a combustion aerosol method (CAM) and transported to cytosol of carcinoma cell line (HeLa) for the first time. Spherical, 82–140 nm spinels were obtained at various concentrations of substrates. The nanoparticles were optimized to emit in the red luminescence range (Er3+, 661 nm, 4F9/2 → 4I15/2) when excited with near infrared light. Lower absorption and scattering by aqueous biological samples, compared to the green emission (Er3+, 550 nm, 2H11/2 → 4I15/2, 2S3/2 → 4I15/2), was responsible for the preferred upconversion. In addition, the application of the near infrared light significantly reduced the cellular autofluorescence and light scattering. X-ray diffraction, transmission electron microscopy, scanning electron microscopy, and photoluminescence spectroscopy were employed to characterize the synthesized samples. Energy dispersive X-ray microanalysis was used to confirm the composition and distribution of the nanoparticles through the spectrum and elemental mapping. The hydrophilic, spherical NPs, coated with PVP (polyvinylpyrrolidone) in the presence of a liposomal transfection factor, lipofectamine, were endocytosed into living HeLa cells and followed as luminescent markers by confocal laser scanning microscopy. We present the optimized protocols for the NPs synthesis and delivery of the spinels to cancer cells for bioimaging.

Structure-based analysis of thermodynamic and mechanical properties of cavity-containing proteins--case study of plant pathogenesis-related proteins of class 10.

We provide theoretical comparisons of the physical properties of eighteen proteins with the pathogenesis‐related proteins of class 10 (PR‐10) fold, which is characterized by a large hydrophobic cavity enclosed between a curved β‐sheet and a variable α‐helix. Our novel algorithm to calculate the volume of internal cavities within protein structures is used to demonstrate that, although the sizes of the cavities of the investigated PR‐10 proteins vary significantly, their other physical properties, such as thermodynamic and mechanical parameters or parameters related to folding, are very close. The largest variations (in the order of 20%) are predicted for the optimal folding times. We show that, on squeezing, the PR‐10 proteins behave differently from typical virus capsids.

Multiple folding pathways of proteins with shallow knots and co-translational folding

We study the folding process in the shallowly knotted protein MJ0366 within two variants of a structure-based model. We observe that the resulting topological pathways are much richer than identified in previous studies. In addition to the single knot-loop events, we find novel, and dominant, two-loop mechanisms. We demonstrate that folding takes place in a range of temperatures and the conditions of most successful folding are at temperatures which are higher than those required for the fastest folding. We also demonstrate that nascent conditions are more favorable to knotting than off-ribosome folding.

Theoretical tests of the mechanical protection strategy in protein nanomechanics

We provide theoretical tests of a novel experimental technique to determine mechanostability of proteins based on stretching a mechanically protected protein by single‐molecule force spectroscopy. This technique involves stretching a homogeneous or heterogeneous chain of reference proteins (single‐molecule markers) in which one of them acts as host to the guest protein under study. The guest protein is grafted into the host through genetic engineering. It is expected that unraveling of the host precedes the unraveling of the guest removing ambiguities in the reading of the force‐extension patterns of the guest protein. We study examples of such systems within a coarse‐grained structure‐based model. We consider systems with various ratios of mechanostability for the host and guest molecules and compare them to experimental results involving cohesin I as the guest molecule. For a comparison, we also study the force‐displacement patterns in proteins that are linked in a serial fashion. We find that the mechanostability of the guest is similar to that of the isolated or serially linked protein. We also demonstrate that the ideal configuration of this strategy would be one in which the host is much more mechanostable than the single‐molecule markers. We finally show that it is troublesome to use the highly stable cystine knot proteins as a host to graft a guest in stretching studies because this would involve a cleaving procedure. Proteins 2014; 82:717–726.

Topological transformations in proteins: effects of heating and proximity of an interface

Using a structure-based coarse-grained model of proteins, we study the mechanism of unfolding of knotted proteins through heating. We find that the dominant mechanisms of unfolding depend on the temperature applied and are generally distinct from those identified for folding at its optimal temperature. In particular, for shallowly knotted proteins, folding usually involves formation of two loops whereas unfolding through high-temperature heating is dominated by untying of single loops. Untying the knots is found to generally precede unfolding unless the protein is deeply knotted and the heating temperature exceeds a threshold value. We then use a phenomenological model of the air-water interface to show that such an interface can untie shallow knots, but it can also make knots in proteins that are natively unknotted.

Statistical radii associated with amino acids to determine the contact map: fixing the structure of a type I cohesin domain in the Clostridium thermocellum cellulosome

We propose to improve and simplify protein refinement procedures through consideration of which pairs of amino acid residues should form native contacts. We first consider 11 330 proteins from the CATH database to determine statistical distributions of contacts associated with a given type of amino acid. The distributions are set across the distances between the α-C atoms that are in contact. Based on this data, we determine typical radii of effective spheres that can be placed on the α-C atoms in order to reconstruct the distribution of the contact lengths. This is done by checking for overlaps with enlarged van der Waals spheres associated with heavy atoms on other amino acids.The resulting contacts can be used to identify non-native contacts that may arise during the time evolution of structure-based models. Here, the radii are used to guide reconstruction of nine missing side chains in a type I cohesin domain with the Protein Data Bank code 1AOH. We first identify the likely missing contacts and then sculpt the corresponding side chains by standard refinement tools to achieve consistency with the expected contact map. One ambiguity in refinement is resolved by determining all-atom conformational energies.

The volume of cavities in proteins and virus capsids.

An improved algorithm for the calculation of the volume of internal cavities within protein structures and virus capsids as well as the volumes occupied by single amino acid residues were presented. The geometrical approach was based on atomic van der Waals radii. The results obtained with two sets of the radii, those proposed by Pauling and those determined by Tsai et al were compared. The main improvement compared with our previous approach is a more elaborate treatment of the regions at the very boundary of the cavities, which yields a more accurate volume estimate. The cavity volume of a number of Plant Pathogenesis‐Related proteins of class 10 (PR‐10) were reevaluated and the volumes and other geometrical parameters for about 400 capsids of icosahedral viruses were reported. Using the same approach the volumes of amino acid residues in polypeptides as mean values averaged over multiple conformations of the side chain were also estimated. Proteins 2016; 84:1275–1286.

Structural entanglements in protein complexes

We consider multi-chain protein native structures and propose a criterion that determines whether two chains in the system are entangled or not. The criterion is based on the behavior observed by pulling at both termini of each chain simultaneously in the two chains. We have identified about 900 entangled systems in the Protein Data Bank and provided a more detailed analysis for several of them. We argue that entanglement enhances the thermodynamic stability of the system but it may have other functions: burying the hydrophobic residues at the interface and increasing the DNA or RNA binding area. We also study the folding and stretching properties of the knotted dimeric proteins MJ0366, YibK, and bacteriophytochrome. These proteins have been studied theoretically in their monomeric versions so far. The dimers are seen to separate on stretching through the tensile mechanism and the characteristic unraveling force depends on the pulling direction.