Link to publication Citation for published version (APA): Bille, A., Linse, B., Mohanty, S., & Irbäck, A. (2015). Equilibrium simulation of trp-cage in the presence of protein crowders. Journal of Chemical Physics, 143(17), [175102]. DOI: 10.1063/1.4934997General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.• Users may download and print one copy of any publication from the public portal for the purpose of private study or research.• You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. While steric crowders tend to stabilize globular proteins, it has been found that protein crowders can have an either stabilizing or destabilizing effect, where a destabilization may arise from nonspecific attractive interactions between the test protein and the crowders. Here, we use Monte Carlo replicaexchange methods to explore the equilibrium behavior of the miniprotein trp-cage in the presence of protein crowders. Our results suggest that the surrounding crowders prevent trp-cage from adopting its global native fold, while giving rise to a stabilization of its main secondary-structure element, an α-helix. With the crowding agent used (bovine pancreatic trypsin inhibitor), the trp-cage-crowder interactions are found to be specific, involving a few key residues, most of which are prolines. The effects of these crowders are contrasted with those of hard-sphere crowders. C 2015 AIP Publishing LLC. Equilibrium simulation of trp-cage in the presence of protein crowders[http://dx

Using Monte Carlo methods, we explore and compare the effects of two protein crowders, BPTI and GB1, on the folding thermodynamics of two peptides, the compact helical trp-cage and the β-hairpin-forming GB1m3. The thermally highly stable crowder proteins are modeled using a fixed backbone and rotatable side-chains, whereas the peptides are free to fold and unfold. In the simulations, the crowder proteins tend to distort the trp-cage fold, while having a stabilizing effect on GB1m3. The extent of the effects on a given peptide depends on the crowder type. Due to a sticky patch on its surface, BPTI causes larger changes than GB1 in the melting properties of the peptides. The observed effects on the peptides stem largely from attractive and specific interactions with the crowder surfaces, and differ from those seen in reference simulations with purely steric crowder particles.

Using NMR and Monte Carlo (MC) methods, we investigate the stability and dynamics of superoxide dismutase 1 (SOD1) in homogeneous crowding environments, where either bovine pancreatic trypsin inhibitor (BPTI) or the B1 domain of streptococcal protein G (PGB1) serves as a crowding agent. By NMR, we show that both crowders, and especially BPTI, cause a drastic loss in the overall stability of SOD1 in its apo monomeric form. Additionally, we determine chemical shift perturbations indicating that SOD1 interacts with the crowder proteins in a residue-specific manner that further depends on the identity of the crowding protein. Furthermore, the specificity of SOD1−crowder interactions is reciprocal: chemical shift perturbations on BPTI and PGB1 identify regions that interact preferentially with SOD1. By MC simulations, we investigate the local unfolding of SOD1 in the absence and presence of the crowders. We find that the crowders primarily interact with the long flexible loops of the folded SOD1 monomer. The basic mechanisms by which the SOD1 β-barrel core unfolds remain unchanged when adding the crowders. In particular, both with and without the crowders, the second β-sheet of the barrel is more dynamic and unfolding-prone than the first. Notably, the MC simulations (exploring the early stages of SOD1 unfolding) and the NMR experiments (under equilibrium conditions) identify largely the same set of PGB1 and BPTI residues as prone to form SOD1 contacts. Thus, contacts stabilizing the unfolded state of SOD1 in many cases appear to form early in the unfolding reaction.

Copper, zinc superoxide dismutase 1 (SOD1) is a ubiquitous homodimeric enzyme, whose misfolding and aggregation play a potentially key role in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). SOD1 aggregation is thought to be preceded by dimer dissociation and metal loss, but the mechanisms by which the metal-free monomer aggregates remain incompletely understood. Here we use implicit solvent all-atom Monte Carlo (MC) methods to investigate the local unfolding dynamics of the β-barrel-forming SOD1 monomer. Although event-to-event variations are large, on average, we find clear differences in dynamics among the eight strands forming the β-barrel. Most dynamic is the eighth strand, β8, which is located in the dimer interface of native SOD1. For the four strands in or near the dimer interface (β1, β2, β7, and β8), we perform aggregation simulations to assess the propensity of these chain segments to self-associate. We find that β1 and β2 readily self-associate to form intermolecular parallel β-sheets, whereas β8 shows a very low aggregation propensity.

Protein-based encapsulation systems have a wide spectrum of applications in targeted delivery of cargo molecules and for chemical transformations in confined spaces. By engineering affinity between cargo and container proteins it has been possible to enable the efficient and specific encapsulation of target molecules. Missing in current approaches is the ability to turn off the interaction after encapsulation to enable the cargo to freely diffuse in the lumen of the container. Separation between cargo and container is desirable in drug delivery applications and in the use of capsids as catalytic nanoparticles. We describe an encapsulation system based on the hepatitis B virus capsid in which an engineered high-affinity interaction between cargo and capsid proteins can be modulated by Ca . Cargo proteins are loaded into capsids in the presence of Ca , while ligand removal triggers unbinding inside the container. We observe that confinement leads to hindered rotation of cargo inside the capsid. Application of the designed container for catalysis was also demonstrated by encapsulation of an enzyme with β-glucosidase activity.

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Protein-based encapsulation systems have aw ide spectrum of applications in targeted delivery of cargo molecules and for chemical transformations in confined spaces.By engineering affinity between cargo and container proteins it has been possible to enable the efficient and specific encapsulation of target molecules.Missing in current approaches is the ability to turn off the interaction after encapsulation to enable the cargo to freely diffuse in the lumen of the container. Separation between cargo and container is desirable in drug delivery applications and in the use of capsids as catalytic nanoparticles.W ed escribe an encapsulation system based on the hepatitis Bv irus capsid in which an engineered highaffinity interaction between cargo and capsid proteins can be modulated by Ca 2+ .Cargo proteins are loaded into capsids in the presence of Ca 2+ ,while ligand removal triggers unbinding inside the container.W eo bserve that confinement leads to hindered rotation of cargo inside the capsid. Application of the designed container for catalysis was also demonstrated by encapsulation of an enzyme with b-glucosidase activity.Protein-based nanocontainers provide tantalizing opportunities for the encapsulation and targeted delivery of various drugs and imaging probes,t he construction of nanoreactors, and the development of biomaterials. [1] Virus-like particles (VLPs) have been widely used as encapsulation systems. [1] In natural container systems,l oading and release of cargo is typically exquisitely controlled. To facilitate an increased control of loading in engineered VLPs,s everal groups have mimicked natural container systems and introduced sites for noncovalent interactions between cargo and capsid proteins.Fore xample,V LPs have been modified to have highly negatively charged interiors to efficiently encapsulate cargo proteins tagged with positively charged sequence. [2] Specific protein-protein interactions have also been used to drive encapsulation selectivity by adding complementary coiledcoil sequences to capsid and guest proteins. [3] It has also been possible to use native RNA-capsid interactions to drive encapsulation of cargo into virus-derived VLPs. [4] An approach for metal-induced assembly and encapsulation has also been described. [5] An additional level of control might be achieved if the interaction between cargo and capsid proteins can be modulated post-encapsulation. In many applications,it would be highly beneficial to be able to turn off the noncovalent interaction between cargo and capsid after encapsulation. In protein delivery applications,t he cargo would ideally be separated from capsomers once capsids have disassembled. In nanoreactors,d etachment of enzymes attached to the container wall to move freely in the lumen of the capsid could enable more efficient catalysis and facilitate noncovalent interactions between proteins involved in multienzyme reaction cascades.Freely diffusing proteins inside the nanocontainer would also facilitate the study of protein crowding and protein-protein...