From Bacteriorhodopsin to Spike Protein S

Membrane-bound proteins that change protonation during function use specific protein groups to bind and transfer protons. Knowledge of the identity of the proton-binding groups is of paramount importance to decipher the reaction mechanism of the protein, and protonation states of prominent are studied extensively using experimental and computational approaches. Analyses of model transporters and receptors from different organisms, and with widely different biological functions, indicate common structure-sequence motifs at internal proton-binding sites. Proton-binding dynamic hydrogen-bond networks that are exposed to the bulk might provide alternative proton-binding sites and proton-binding pathways. In this perspective article I discuss protonation coupling and proton binding at internal and external carboxylate sites of proteins that use proton transfer for function. An inter-helical carboxylate-hydroxyl hydrogen-bond motif is present at functionally important sites of membrane proteins from archaea to the brain. External carboxylate-containing H-bond clusters are observed at putative proton-binding sites of protonation-coupled model proteins, raising the question of similar functionality in spike protein S

Computational Molecular Biophysics of Membrane Reactions

Proteins are nanoscale molecules that perform functions essential for biological life. Membranes surrounding cells, for example, contain receptor proteins that mediate communication between the cell and the external milieu, membrane transporters that transport ions and larger compounds across the membranes, and enzymes that catalyze chemical reactions. Likewise, soluble proteins found in interior of the cell include motor proteins that move other proteins around, enzymes that bind to and repair breaks in the DNA, and proteins that help control the cellular clock. Mutations in genes that encode proteins can cause disease, as is the case of cystic fibrosis, a disease that associates with mutation of a chloride channel called the cystic fibrosis transmembrane conductance regulator.1 The essential functions they perform in the cell makes proteins essential drug targets for modern bio-medical applications. An important example here is the programmed death ligand-1 (PD-L1), which is a valuable target for modern immunotherapy.2-4 Predicting how a protein responds to a drug molecule, or using the protein as inspiration for biotechnological applications, require knowledge of how that protein works. As proteins are dynamic entities and protein dynamics are essential for function,5-8 describing the mechanism of action of a protein requires knowledge about the protein motions in fluid environments. Theoretical biophysics provides valuable tools to characterize protein reaction mechanisms and protein motions at the atomic level of detail. This Habilitation Thesis presents research on using theoretical biophysics approaches to decipher how proteins work. The focus of the research is on membrane proteins and reactions that occur at lipid membrane interfaces. The central question I address is the role of dynamic hydrogen (H) bonds in protein function and membrane interactions. The methods used include quantum mechanical (QM) computations of small molecules, combined quantum mechanics/molecular mechanics (QM/MM) of chemical reactions in protein environments, classical mechanical computations of large protein and membrane systems, and bridging numerical simulations to bioinformatics. In my research group we developed algorithms to identify H-bond networks in proteins and membrane environments, and to characterize the dynamics of these networks. To extend the applicability of numerical computations to bio-systems that bind drug-like compounds, we derive parameters for a potential energy function widely used in the field. The main research topics and specific questions addressed are summarized below together with a discussion of the computational approaches used

Phosphatidylglyerol Lipid Binding at the Active Site of an Intramembrane Protease

Transmembrane substrate cleavage by the small Escherichia coli rhomboid protease GlpG informs on mechanisms by which lipid interactions shape reaction coordinates of membrane-embedded enzymes. Here, I review and discuss new work on the molecular picture of protein-lipid interactions that might govern the formation of the substrate-enzyme complex in fluid lipid membranes. Negatively charged PG-type lipids are of particular interest, because they are a major component of bacterial membranes. Atomistic computer simulations indicate POPG and DOPG lipids bridge remote parts of GlpG and might pre-occupy the substrate-docking site. Inhibition of catalytic activity by PG lipids could arise from ligand-like lipid binding at the active site, which could delay or prevent substrate docking. Dynamic protein-lipid H-bond networks, water access to the active site, and fluctuations in the orientation of GlpG suggest that GlpG has lipid-coupled dynamics that could shape the energy landscape of transmembrane substrate docking

Extended protein/water H-bond networks in photosynthetic water oxidation

Oxidation of water molecules in the photosystem II (PSII) protein complex proceeds at the manganese–calcium complex, which is buried deeply in the lumenal part of PSII. Understanding the PSII function requires knowledge of the intricate coupling between the water-oxidation chemistry and the dynamic proton management by the PSII protein matrix. Here we assess the structural basis for long-distance proton transfer in the interior of PSII and for proton management at its surface. Using the recent high-resolution crystal structure of PSII, we investigate prominent hydrogen-bonded networks of the lumenal side of PSII. This analysis leads to the identification of clusters of polar groups and hydrogen-bonded networks consisting of amino acid residues and water molecules. We suggest that long-distance proton transfer and conformational coupling is facilitated by hydrogen-bonded networks that often involve more than one protein subunit. Proton-storing Asp/Glu dyads, such as the D1-E65/D2-E312 dyad connected to a complex water-wire network, may be particularly important for coupling protonation states to the protein conformation. Clusters of carboxylic amino acids could participate in proton management at the lumenal surface of PSII. We propose that rather than having a classical hydrophobic protein interior, the lumenal side of PSII resembles a complex polyelectrolyte with evolutionary optimized hydrogen-bonding networks. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial

Hydrogen bond dynamics in membrane protein function

Changes in inter-helical hydrogen bonding are associated with the conformational dynamics of membrane proteins. The function of the protein depends on the surrounding lipid membrane. Here we review through specific examples how dynamical hydrogen bonds can ensure an elegant and efficient mechanism of long-distance intra-protein and protein–lipid coupling, contributing to the stability of discrete protein conformational substates and to rapid propagation of structural perturbations. This article is part of a Special Issue entitled: Protein Folding in Membranes

Dynamic water bridging and proton transfer at a surface carboxylate cluster of photosystem II

Proton-transfer proteins are often exposed to the bulk clusters of carboxylate groups that might bind protons transiently. This raises important questions as to how the carboxylate groups of a protonated cluster interact with each other and with water, and how charged protein groups and hydrogen-bonded waters could have an impact on proton transfers at the cluster. We address these questions by combining classical mechanical and quantum mechanical computations with the analysis of cyanobacterial photosystem II crystal structures from Thermosynechococcus elongatus. The model system we use consists of an interface between PsbO and PsbU, which are two extrinsic proteins of photosystem II. We find that a protonated carboxylate pair of PsbO is part of a dynamic network of protein–water hydrogen bonds which extends across the protein interface. Hydrogen-bonded waters and a conserved lysine sidechain largely shape the energetics of proton transfer at the carboxylate cluster

Graph-Based Analyses of Dynamic Water-Mediated Hydrogen-Bond Networks in Phosphatidylserine: Cholesterol Membranes

Phosphatidylserine lipids are anionic molecules present in eukaryotic plasma membranes, where they have essential physiological roles. The altered distribution of phosphatidylserine in cells such as apoptotic cancer cells, which, unlike healthy cells, expose phosphatidylserine, is of direct interest for the development of biomarkers. We present here applications of a recently implemented Depth-First-Search graph algorithm to dissect the dynamics of transient water-mediated lipid clusters at the interface of a model bilayer composed of 1-palmytoyl-2-oleoyl-sn-glycero-2-phosphatidylserine (POPS) and cholesterol. Relative to a reference POPS bilayer without cholesterol, in the POPS:cholesterol bilayer there is a somewhat less frequent sampling of relatively complex and extended water-mediated hydrogen-bond networks of POPS headgroups. The analysis protocol used here is more generally applicable to other lipid:cholesterol bilayers

Rhomboid Protease Dynamics and Lipid Interactions

Intramembrane proteases, which cleave transmembrane (TM) helices, participate in numerous biological processes encompassing all branches of life. Several crystallographic structures of Escherichia coli GlpG rhomboid protease have been determined. In order to understand GlpG dynamics and lipid interactions in a native-like environment, we have examined the molecular dynamics of wild-type and mutant GlpG in different membrane environments. The irregular shape and small hydrophobic thickness of the protein cause significant bilayer deformations that may be important for substrate entry into the active site. Hydrogen-bond interactions with lipids are paramount in protein orientation and dynamics. Mutations in the unusual L1 loop cause changes in protein dynamics and protein orientation that are relayed to the His-Ser catalytic dyad. Similarly,mutations in TM5 change the dynamics and structure of the L1 loop. These results imply that the L1 loop has an important regulatory role in proteolysis.National Institute of General Medical Sciences (GM-74637

Voltage Sensing in Bacterial Protein Translocation

The bacterial channel SecYEG efficiently translocates both hydrophobic and hydrophilic proteins across the plasma membrane. Translocating polypeptide chains may dislodge the plug, a half helix that blocks the permeation of small molecules, from its position in the middle of the aqueous translocation channel. Instead of the plug, six isoleucines in the middle of the membrane supposedly seal the channel, by forming a gasket around the translocating polypeptide. However, this hypothesis does not explain how the tightness of the gasket may depend on membrane potential. Here, we demonstrate voltage-dependent closings of the purified and reconstituted channel in the presence of ligands, suggesting that voltage sensitivity may be conferred by motor protein SecA, ribosomes, signal peptides, and/or translocating peptides. Yet, the presence of a voltage sensor intrinsic to SecYEG was indicated by voltage driven closure of pores that were forced-open either by crosslinking the plug to SecE or by plug deletion. We tested the involvement of SecY’s half-helix 2b (TM2b) in voltage sensing, since clearly identifiable gating charges are missing. The mutation L80D accelerated voltage driven closings by reversing TM2b’s dipolar orientation. In contrast, the L80K mutation decelerated voltage induced closings by increasing TM2b’s dipole moment. The observations suggest that TM2b is part of a larger voltage sensor. By partly aligning the combined dipole of this sensor with the orientation of the membrane-spanning electric field, voltage may drive channel closure