An Olfactory Receptor Pseudogene whose Function emerged in Humans

Human olfactory receptor, hOR17-210, is identified as a pseudogene in the human genome. Experimental data has shown however, that the gene product of cloned hOR17-210 cDNA was able to bind an odorant-binding protein and is narrowly tuned for excitation by cyclic ketones. Supported by experimental results, we used the bioinformatics methods of sequence analysis, computational protein modeling and docking, to show that functionality in this receptor is retained due to sequence-structure features not previously observed in mammalian ORs. This receptor does not possess the first two transmembrane helical domains (of seven typically seen in GPCRs). It however, possesses an additional TM that has not been observed in other human olfactory receptors. By incorporating these novel structural features, we created two putative models for this receptor. We also docked odor ligands that were experimentally shown to bind hOR17-210 model. We show how and why structural modifications of OR17-210 do not hinder this receptor's functionality. Our studies reveal that novel gene rearrangement that result in sequence and structural diversity in has a bearing on OR and GPCR function and evolution

Folding and insertion thermodynamics of the transmembrane WALP peptide

The anchor of most integral membrane proteins consists of one or several helices spanning the lipid bilayer. The WALP peptide, GWW(LA)$_n$(L)WWA, is a common model helix to study the fundamentals of protein insertion and folding, as well as helix-helix association in the membrane. Its structural properties have been illuminated in a large number of experimental and simulation studies. In this combined coarse-grained and atomistic simulation study, we probe the thermodynamics of a single WALP peptide, focusing on both the insertion across the water-membrane interface, as well as folding in both water and a membrane. The potential of mean force characterizing the peptide's insertion into the membrane shows qualitatively similar behavior across peptides and three force fields. However, the Martini force field exhibits a pronounced secondary minimum for an adsorbed interfacial state, which may even become the global minimum---in contrast to both atomistic simulations and the alternative PLUM force field. Even though the two coarse-grained models reproduce the free energy of insertion of individual amino acids side chains, they both underestimate its corresponding value for the full peptide (as compared with atomistic simulations), hinting at cooperative physics beyond the residue level. Folding of WALP in the two environments indicates the helix as the most stable structure, though with different relative stabilities and chain-length dependence.Comment: 12 pages, 5 figure

Association of Protein Helices and Assembly of Foldamers: Stories in Membrane and Aqueous Environments

Solvents play an important role in association and assembly of molecules. Here we studied solvent effects on proteins and organic chemicals in different contexts. First, X-ray crystal structures show that helix dimers in membrane- and water-soluble proteins have distinct behaviors in packing and sequence selection. Transmembrane dimers are stabilized by compact packing and hydrogen bonding between small residues. Meanwhile, water-soluble dimers utilize hydrophobic residues for packing irrespective of the size of the interface and tight dimers are rare. Secondly, we apply the results learned above to a complex system in which a designed protein binds to single-walled carbon-nanotube in aqueous environments. Previous designs of the hexameric helical bundles utilized leucine and alanine residues to make two distinct helix-helix interfaces. Our molecular dynamics simulations showed that the alanine-comprising interface is much more labile than the leucine-comprising one. This result can be interpreted by the scarcity of tight soluble helix dimers as mentioned above. Thus more stable modular helix-helix interfaces have to be employed to design peptides binding to carbon-nanotubes with higher affinities. Lastly, we describe a serendipitous discovery of the crystalline framework structure by an amphiphilic triarylamide foldamer. Foldamers are peptide-like polymers of non-natural monomers arranged in defined sequence and chain length that are able to adopt protein-like secondary and tertiary structures. In contrast with traditional metal-organic and organic frameworks, which exploit strong directional coordination and hydrogen bonding for assembly in organic solvents, the crystal herein is built up from a combination of noncovalent hydrophobic, hydrogen-bonded, and electrostatic interactions in aqueous solution. The structure is in honeycomb geometry with each cubicle as a truncated octahedron. A new supramolecular synthon, in which hydrogen bonding and π-π stacking are encompassed, was discovered in the crystal structure. Through NMR experiments we probed the oligomeric states of the foldamer in the early stages prior to crystallization. The hierarchic crystal structure was discussed in terms of supramolecular synthons in crystal engineering

Computational studies on Membrane Proteins (bovine CNGA1 & mouse TSPO)

Around thirty percent of total proteins are present in the membrane and play an important role to communicate intracellular and extracellular region. Their presence in the membrane is one of the limiting steps to determine protein structure and to understand their mechanisms. Hence bioinformatics techniques and computational tools play an important role to overcome these issues in characterizing the structural/functional mechanism of membrane proteins. In this thesis, I have developed and used state of the art computational techniques applied to two different pharmaceutically relevant membrane proteins, Cyclic nucleotide-gated channels (CNG) and translocator membrane protein (TSPO). CNG ion channels are embedded into the neuronal membrane. Till date, the structure and their gating mechanism are subject to interest. Different approaches like electrophysiology, single molecule force spectroscopy, biophysics, etc. have been employed to study these channels. Here I studied the gating mechanism of the CNGA1 ion channel by use of homology modeling and coarse-grained molecular dynamics. TSPO is a key biomarker for the diagnostics of inflammation in the brain. Limited Structural and functional information available on mammalian TSPOs homodimers. Computational studies suggested that the NMR-solved structure is not prone to dimer formation and is not stable in a membrane environment and has been an object of vivid criticism. To address this issue we use homology modeling technique and molecular dynamics approach. Principle results are: 1. I have successfully created homology models for CNGA1 homotetramer and performed coarse-grained simulation in the presence and absence of cGMP molecule and developed the coarse-grained force-field parameters for cGMP. 2. I have proposed a new model of the functionally relevant dimeric form of mTSPO. The model is fully consistent with solid-state NMR spectral data. Our predictions provide for the first time structural insights on this pharmaceutically important target fully consistent with experimental data. 3. During these studies, and in order to optimize the preparation of the systems it was necessary to develop an automated tool for creating the input files for doing coarse-grained simulations. These tools are shared with the community through a publically available online web-server that simplifies the task of generating input files which help in performing simulation and retrieving the result data for small simulations. The web-server, MERMAID is available at MERMAID (http://molsim.sci.univr.it/mangesh/). The application of novel computational approaches in this thesis allowed me to characterize extensively both systems by offering a rational to a huge amount of experimental data on biological relevant systems

Modeling and Dynamics of the Inward-Facing State of a Na+/Cl− Dependent Neurotransmitter Transporter Homologue

The leucine transporter (LeuT) has recently commanded exceptional attention due mainly to two distinctions; it provides the only crystal structures available for a protein homologous to the pharmacologically relevant neurotransmitter: sodium symporters (NSS), and, it exhibits a hallmark 5-TM inverted repeat (“LeuT-fold”), a fold recently discovered to also exist in several secondary transporter families, underscoring its general role in transporter function. Constructing the transport cycle of “LeuT-fold” transporters requires detailed structural and dynamic descriptions of the outward-facing (OF) and inward-facing (IF) states, as well as the intermediate states. To this end, we have modeled the structurally unknown IF state of LeuT, based on the known crystal structures of the OF state of LeuT and the IF state of vSGLT, a “LeuT-fold” transporter. The detailed methodology developed for the study combines structure-based alignment, threading, targeted MD and equilibrium MD, and can be applied to other proteins. The resulting IF-state models maintain the secondary structural features of LeuT. Water penetration and solvent accessibility calculations show that TM1, TM3, TM6 and TM8 line the substrate binding/unbinding pathway with TM10 and its pseudosymmetric partner, TM5, participating in the extracellular and intracellular halves of the lumen, respectively. We report conformational hotspots where notable changes in interactions occur between the IF and OF states. We observe Na2 exiting the LeuT-substrate- complex in the IF state, mainly due to TM1 bending. Inducing a transition in only one of the two pseudosymmetric domains, while allowing the second to respond dynamically, is found to be sufficient to induce the formation of the IF state. We also propose that TM2 and TM7 may be facilitators of TM1 and TM6 motion. Thus, this study not only presents a novel modeling methodology applied to obtain the IF state of LeuT, but also describes structural elements involved in a possibly general transport mechanism in transporters adopting the “LeuT-fold”

Computational methods to design biophysical experiments for the study of protein dynamics

In recent years, new software and automated instruments have enabled us to imagine autonomous or "self-driving" laboratories of the future. However, ways to design new scientific studies remain unexplored due to challenges such as minimizing associated time, labor, and expense of sample preparation and data acquisition. In the field of protein biophysics, computational simulations such as molecular dynamics and spectroscopy-based experiments such as double electron-electron resonance and Fluorescence resonance energy transfer techniques have emerged as critical experimental tools to capture protein dynamic behavior, a change in protein structure as a function of time which is important for their cellular functions. These techniques can lead to the characterization of key protein conformations and can capture protein motions over a diverse range of timescales. This work addresses the problem of the choice of probe positions in a protein, which residue-pairs should experimentalists choose for spectroscopy experiments. For this purpose, molecular dynamics simulations and Markov state models of protein conformational dynamics are utilized to rank sets of labeled residue-pairs in terms of their ability to capture the conformational dynamics of the protein. The applications of our experimental study design methodology called OptimalProbes on different types of proteins and experimental techniques are examined. In order to utilize this method for a previously uncharacterized protein, atomistic molecular dynamics simulations are performed to study a bacterial di/tri-peptide transporter a typical representative of the Major Facilitator Superfamily of membrane proteins. This was followed by ideal double electron-electron resonance experimental choice predictions based on the simulation data. The predicted choices are superior to the residue-pair choices made by experimentalists which failed to capture the slowest dynamical processes in the conformational ensemble obtained from our long timescale simulations. For molecular dynamics simulations based design of experimental studies to succeed both ensembles need to be comparable. Since this has not been the case for double electron-electron resonance distance distributions and molecular simulations, we explore possible reasons that can lead to mismatches between experiments and simulations in order to reconcile simulated ensembles with experimentally obtained distance traces. This work is one of the first studies towards integrating spectroscopy experiment design into a computational method systematically based on molecular simulations