Computational Studies of Protein Structure, Dynamics, and Function in Native-like Environments

Proteins are among the four unique organic constituents of cells. They are responsible for a variety of important cell functions ranging from providing structural support to catalyzing biological reactions. They vary in shape, dynamic behavior, and localization. All of these together determine the specificity in their functions, but the question is how. The ultimate goal of the research conducted in this thesis is to answer this question. Two types of proteins are of particular interest. They include transmembrane proteins and protein assemblies. Using computer simulations with available experimental data to validate the simulation results, the research described here aims to reveal the structure and dynamics of proteins in their native-like environment and the indication on the mechanism of their functions. The first part of the thesis focuses on studying the structure and functions of transmembrane proteins. These proteins are consisted of transmembrane α-helices or β-strands, and each of the secondary structure elements adopts a unique orientation in the membrane following its local interactions. The structure of the entire protein is a collection of the orientations of these elements and their relative positions with respect to one another. These two basic aspects of membrane protein structure are studied in Chapter II and III. In Chapter II, efforts are given to determine the favorable orientation of a β-hairpin peptide, protegrin-1, in different lipid bilayers. The orientational preference results from the interplay between the protein and the surrounding lipid molecules. Chapter III is centered on revealing the structure and dynamics of caveolin-1 in DMPC bilayers. Caveolin-1 forms a re-entrant helix-turn-helix structure with two α-helices embedded in the membrane bilayer. The study shows that caveolin-1 monomer is rather dynamic and maintains its inserted conformation via both specific and non-specific protein-lipid interactions. To investigate the structural and dynamic impact on the function of a membrane protein, molecular dynamics simulations of the voltage-dependent anion channel are performed and the results are presented in Chapter IV. It is found in this chapter that the electrostatic interactions between charged residues on the channel wall facing the lumen are responsible for retarding the cation current, therefore giving the channel its anion selectivity. The second category of protein that is of interest in this thesis is the assembled protein complex, especially the ones that are highly symmetric. Actually, many membrane proteins belong to this category as well, but the study presented here in Chapter V involves simulations performed on a soluble protein complex, bacterioferritin B from Pseudomonas Aeruginosa. It is revealed by the simulations that the dynamic behavior of the protein is magnified by the symmetry and is tightly associated to its function

Membrane Tension, Lipid Adaptation, Conformational Changes, and Energetics in MscL Gating

This is the publisher's version. Copyright 2011 by Elsevier.This study aims to explore gating mechanisms of mechanosensitive channels in terms of membrane tension, membrane adaptation, protein conformation, and energetics. The large conductance mechanosensitive channel from Mycobacterium tuberculosis (Tb-MscL) is used as a model system; Tb-MscL acts as a safety valve by releasing small osmolytes through the channel opening under extreme hypoosmotic conditions. Based on the assumption that the channel gating involves tilting of the transmembrane (TM) helices, we have performed free energy simulations of Tb-MscL as a function of TM helix tilt angle in a dimyristoylphosphatidylcholine bilayer. Based on the change in system dimensions, TM helix tilting is shown to be essentially equivalent to applying an excess surface tension to the membrane, causing channel expansion, lipid adaptation, and membrane thinning. Such equivalence is further corroborated by the observation that the free energy cost of Tb-MscL channel expansion is comparable to the work done by the excess surface tension. Tb-MscL TM helix tilting results in an expanded water-conducting channel of an outer dimension similar to the proposed fully open MscL structure. The free energy decomposition indicates a possible expansion mechanism in which tilting and expanding of TM2 facilitates the iris-like motion of TM1, producing an expanded Tb-MscL

Comparative Molecular Dynamics Simulation Studies of Protegrin-1 Monomer and Dimer in Two Different Lipid Bilayers

This is the publisher's version. Copyright 2009 by Elsevier.Antimicrobial peptides interact specifically with the membrane of a pathogen and kill the pathogen by releasing its cellular contents. Protegrin-1 (PG-1), a β-hairpin antimicrobial peptide, is known to exist as a transmembrane monomer in a 1,2-dilauroylphosphatidylcholine (DLPC) bilayer and shows concentration-dependent oligomerization in a 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayer. To examine its structure, dynamics, orientation, and interaction in membranes, we performed comparative molecular dynamics simulations of PG-1 monomer and dimer in DLPC and POPC bilayers for a total of 840 ns. The PG-1 monomer exhibits larger tilting in DLPC than in POPC due to a hydrophobic mismatch. PG-1 tilting is dependent on its rotation angle. The specific orientation of PG-1 in membranes is governed by the interactions of its aromatic residues with lipid headgroups. The calculated 15N and 13CO chemical shifts of Val16 in DLPC reveal that there are different sets of tilt and rotation angles that satisfy the experimental values reasonably, suggesting that more experiments are needed to determine its orientation. The dimer simulations show that the dimer interface is better preserved in POPC than in DLPC because POPC's greater hydrophobic thickness causes reduced flexibility of the C-terminal strands. Both monomer and dimer simulations show membrane thinning around PG-1, largely due to arginine-lipid interactions

Protein Dynamics and Ion Traffic in Bacterioferritin

Bacterioferritin (Bfr) is a spherical protein composed of 24 subunits and 12 heme molecules. Bfrs contribute to regulate iron homeostasis in bacteria by capturing soluble but potentially toxic Fe2+ and by compartmentalizing it in the form of a bioavailable ferric mineral inside the protein’s hollow cavity. When iron is needed, Fe3+ is reduced and mobilized into the cytosol as Fe2+. Hence, key to the function of Bfr is its ability to permeate iron ions in and out of its interior cavity, which is likely imparted by a flexible protein shell. To examine the conformational flexibility of Bfrs in a native-like environment and the way in which the protein shell interacts with monovalent cations, we have performed molecular dynamics (MD) simulations of BfrB from Pseudomonas aeruginosa (Pa BfrB) in K2HPO4 solutions at different ionic strengths. The results indicate the presence of coupled thermal fluctuations (dynamics) in the 4-fold and B-pores of the protein, which is key to enable passage of monovalent cations through the protein shell using B-pores as conduits. The MD simulations also show that Pa BfrB ferroxidase centers are highly dynamic and permanently populated by transient cations exchanging with other cations in the interior cavity, as well as the solution bathing the protein. Taken together, the findings suggest that Fe2+ traffic across the Pa BfrB shell via B-pores and that the ferroxidase pores enable capture and oxidation of Fe2+, followed by translocation of Fe3+ to the interior cavity, aided by the conformationally active H130

Comparative Molecular Dynamics Simulation Studies of Protegrin-1 Monomer and Dimer in Two Different Lipid Bilayers

This is the publisher's version. Copyright 2009 by Elsevier.Antimicrobial peptides interact specifically with the membrane of a pathogen and kill the pathogen by releasing its cellular contents. Protegrin-1 (PG-1), a β-hairpin antimicrobial peptide, is known to exist as a transmembrane monomer in a 1,2-dilauroylphosphatidylcholine (DLPC) bilayer and shows concentration-dependent oligomerization in a 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayer. To examine its structure, dynamics, orientation, and interaction in membranes, we performed comparative molecular dynamics simulations of PG-1 monomer and dimer in DLPC and POPC bilayers for a total of 840 ns. The PG-1 monomer exhibits larger tilting in DLPC than in POPC due to a hydrophobic mismatch. PG-1 tilting is dependent on its rotation angle. The specific orientation of PG-1 in membranes is governed by the interactions of its aromatic residues with lipid headgroups. The calculated 15N and 13CO chemical shifts of Val16 in DLPC reveal that there are different sets of tilt and rotation angles that satisfy the experimental values reasonably, suggesting that more experiments are needed to determine its orientation. The dimer simulations show that the dimer interface is better preserved in POPC than in DLPC because POPC's greater hydrophobic thickness causes reduced flexibility of the C-terminal strands. Both monomer and dimer simulations show membrane thinning around PG-1, largely due to arginine-lipid interactions

Evaluating child engagement in digital story stems using facial data

Engagement is a key factor in understanding people’s psychology and behaviours and is an understudied topic in children. The area of focus in this thesis is child engagement in the story-stems used in child Attachment evaluations such as the Manchester Child Attachment Task (MCAST). Due to the high cost and time required for conducting Attachment assessments, automated assessments are being developed. These present story-stems in a cost-effective way on a laptop screen to digitalise the interaction between the child and the story, without disrupting the storytelling. However, providing such tests via computer relies on the child being engaged in the digital story-stem. If they are not engaged, then the tests will not be successful and the collected data will be of poor-quality, which will not allow for successful detection of Attachment status. Therefore, the aim of this research is to investigate a range of aspects of child engagement to understand how to engage children in story-stems, and how to measure their engagement levels. This thesis focuses on measuring the levels of child engagement in digital story-stems and specifically on understanding the effect of multimedia digital story-stems on children’s engagement levels to create a better and more engaging digital story-stem. Data sources used in this thesis include the observation of each child’s facial behaviours and a questionnaire with Smiley-o-meter scale. Measurement tools are developed and validated through analyses of facial data from children when watching digital story-stems with different presentation and voice types. Results showed that facial data analysis, using eye-tracking measures and facial action units (AUs) recognition, can be used to measure children’s engagement levels in the context of viewing digital story-stems. Using eye-tracking measures, engaged children have longer fixation durations in both mean and sum of fixation durations, which reflect that a child was deeply engaged in the story-stems. Facial AU recognition had better performance in a binary classification for discriminating engaged or disengaged children than eye-tracking measurements. The most frequently occurring facial action units taken from the engaged classes show that children’s facial action units indicated signs of fear, which suggest that children felt anxiety and distress while watching the story-stems. These feeling of anxiety and distress show that children have a strong emotional engagement and can locate themselves in the story-stems, showing that they were strong engaged. A further contribution in this thesis was to investigate the best way of creating an engaging story-stem. Results showed that an animated video narrated by a female expressive voice was most engaging. Compared to the live-action MCAST video, data showed that children were more engaged in the animated videos. Voice gender and voice expressiveness were two factors of the quality of storytelling voice that were evaluated and both affected children’s engagement levels. The distribution of child engagement across different voice types was compared to find the best storytelling voice type for story-stem design. A female expressive voice had a better performance for displaying the ‘distress’ in the story-stem than other voice types and engaged children more in the story-stems. The quality of the storytelling voice used to narrate story-stems and animated videos both significantly affected children’s levels of engagement. Such digital story-stems make children more engaged in the digital MCAST test