Carlos’s sci-art was just published as the front cover at JCIM journal. Great grand finale of cool project!

Mandal N, Surpeta B, Brezovsky J, 2024: Reinforcing Tunnel Network Exploration in Proteins using Gaussian Accelerated Molecular Dynamics. Journal of Chemical Information and Modeling, DOI: 10.1021/acs.jcim.4c00966. full text dataset dataset-trajectories

Tunnels are structural conduits in biomolecules responsible for transporting chemical compounds and solvent molecules from the active site. They have been shown to be present in a wide variety of enzymes across all functional and structural classes. However, the study of such pathways is experimentally challenging, because they are typically transient. Computational methods, such as molecular dynamics (MD) simulations, have been successfully proposed to explore tunnels. Conventional MD (cMD) provides structural details to characterize tunnels but suffers from sampling limitations to capture rare tunnel openings on longer time scales. Therefore, in this study, we explored the potential of Gaussian accelerated MD (GaMD) simulations to improve the exploration of complex tunnel networks in enzymes. We used the haloalkane dehalogenase LinB and its two variants with engineered transport pathways, which are not only well-known for their application potential but have also been extensively studied experimentally and computationally regarding their tunnel networks and their importance in multistep catalytic reactions. Our study demonstrates that GaMD efficiently improves tunnel sampling and allows the identification of all known tunnels for LinB and its two mutants. Furthermore, the improved sampling provided insight into a previously unknown transient side tunnel (ST). The extensive conformational landscape explored by GaMD simulations allowed us to investigate in detail the mechanism of ST opening. We determined variant-specific dynamic properties of ST opening, which were previously inaccessible due to limited sampling of cMD. Our comprehensive analysis supports multiple indicators of the functional relevance of the ST, emphasizing its potential significance beyond structural considerations. In conclusion, our research proves that the GaMD method can overcome the sampling limitations of cMD for the effective study of tunnels in enzymes, providing further means for identifying rare tunnels in enzymes with the potential for drug development, precision medicine, and rational protein engineering.

Sarkar DK, Surpeta B, Brezovsky J, 2024: Incorporating prior knowledge in the seeds of adaptive sampling molecular dynamics simulations of ligand transport in enzymes with buried active sites. Journal of Chemical Theory and Computation, DOI: 10.1021/acs.jctc.4c00452. full text dataset

Because most proteins have buried active sites, protein tunnels or channels play a crucial role in the transport of small molecules into buried cavities for enzymatic catalysis. Tunnels can critically modulate the biological process of protein–ligand recognition. Various molecular dynamics methods have been developed for exploring and exploiting the protein–ligand conformational space to extract high-resolution details of the binding processes, a recent example being energetically unbiased high-throughput adaptive sampling simulations. The current study systematically contrasted the role of integrating prior knowledge while generating useful initial protein–ligand configurations, called seeds, for these simulations. Using a nontrivial system of a haloalkane dehalogenase mutant with multiple transport tunnels leading to a deeply buried active site, simulations were employed to derive kinetic models describing the process of association and dissociation of the substrate molecule. The most knowledge-based seed generation enabled high-throughput simulations that could more consistently capture the entire transport process, explore the complex network of transport tunnels, and predict equilibrium dissociation constants, k_off/k_on, on the same order of magnitude as experimental measurements. Overall, the infusion of more knowledge into the initial seeds of adaptive sampling simulations could render analyses of transport mechanisms in enzymes more consistent even for very complex biomolecular systems, thereby promoting drug development efforts and the rational design of enzymes with buried active sites.

Sequeiros-Borja C, Bartlomiej Surpeta, Thirunavukarasu AS, Dongmo Foumthuim CJ, Igor Marchlewski, Brezovsky J, 2024: Water will find its way: transport through narrow tunnels in hydrolases. Journal of Chemical Information and Modeling, DOI: 10.1021/acs.jcim.4c00094. full text dataset-Hal dataset-Epx dataset-Lip dataset-hEpx dataset-E470G dataset-interactions dataset-Hal with different MD settings

An aqueous environment is vital for life as we know it, and water is essential for nearly all biochemical processes at the molecular level. Proteins utilize water molecules in various ways. Consequently, proteins must transport water molecules across their internal network of tunnels to reach the desired action sites, either within them or by functioning as molecular pipes to control cellular osmotic pressure. Despite water playing a crucial role in enzymatic activity and stability, its transport has been largely overlooked, with studies primarily focusing on water transport across membrane proteins. The transport of molecules through a protein’s tunnel network is challenging to study experimentally, making molecular dynamics simulations the most popular approach for investigating such events. In this study, we focused on the transport of water molecules across three different α/β-hydrolases: haloalkane dehalogenase, epoxide hydrolase, and lipase. Using a 5 μs adaptive simulation per system, we observed that only a few tunnels were responsible for the majority of water transport in dehalogenase, in contrast to a higher diversity of tunnels in other enzymes. Interestingly, water molecules could traverse narrow tunnels with subangstrom bottlenecks, which is surprising given the commonly accepted water molecule radius of 1.4 Å. Our analysis of the transport events in such narrow tunnels revealed a markedly increased number of hydrogen bonds formed between the water molecules and protein, likely compensating for the steric penalty of the process. Overall, these commonly disregarded narrow tunnels accounted for ∼20% of the total water transport observed, emphasizing the need to surpass the standard geometrical limits on the functional tunnels to properly account for the relevant transport processes. Finally, we demonstrated how the obtained insights could be applied to explain the differences in a mutant of the human soluble epoxide hydrolase associated with a higher incidence of ischemic stroke.

Bharadwaj P, Shet SM, Bisht M, Sarkar DK, Franklin G, Nataraj SK, Mondal D, 2023: Suitability of adenosine derivatives in improving the activity and stability of cytochrome c under stress: Insights into the effect of phosphate groups. The Journal of Physical Chemistry B doi: 10.1021/acs.jpcb.3c05996. full text

It is well known that adenosine and its phosphate derivatives play a crucial role in biological phenomena such as apoptosis and cell signaling and act as the energy currency of the cell. Although their interactions with various proteins and enzymes have been described, the focus of this work is to demonstrate the effect of the phosphate group on the activity and stability of the native heme metalloprotein cytochrome c (Cyt c), which is important from both biological and industrial aspects. In situ and in silico characterizations are used to correlate the relationship between the binding affinity of adenosine and its phosphate groups with unfolding behavior, corresponding peroxidase activities, and stability factors. Interaction of adenosine (ADN), adenosine monophosphate (AMP), adenosine 5′-diphosphate (ADP), and adenosine 5′-triphosphate (ATP) with Cyt c increases peroxidase-like activity by up to 1.8–6.5-fold compared to native Cyt c. This activity is significantly maintained even after multiple stress conditions such as oxidative stress and the presence of a chaotropic agent such as guanidine hydrochloride (GuHCl). With binding affinities on the order of ADN < AMP < ADP < ATP, adenosine derivatives were found to stabilize Cyt c by varying the secondary structural features of the protein. Thus, in addition to being a fundamental study, the current work also proposes a way of stabilizing protein systems to be used for real-time biocatalytic applications.