scholarly journals Controlling Protein Crystallization by Free Energy Guided Design of Interactions at Crystal Contacts

Crystals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 588
Author(s):  
Johannes Hermann ◽  
Daniel Bischoff ◽  
Phillip Grob ◽  
Robert Janowski ◽  
Dariusch Hekmat ◽  
...  
Keyword(s):  
Free Energy ◽  
Protein Interactions ◽  
Computational Study ◽  
Protein Crystallization ◽  
Lactobacillus Brevis ◽  
Protein Crystal ◽  
Crystal Formation ◽  
Contact Stability ◽  
Crystal Contacts ◽  
Energy Simulations

Protein crystallization can function as an effective method for protein purification or formulation. Such an application requires a comprehensive understanding of the intermolecular protein–protein interactions that drive and stabilize protein crystal formation to ensure a reproducible process. Using alcohol dehydrogenase from Lactobacillus brevis (LbADH) as a model system, we probed in our combined experimental and computational study the effect of residue substitutions at the protein crystal contacts on the crystallizability and the contact stability. Increased or decreased contact stability was calculated using molecular dynamics (MD) free energy simulations and showed excellent qualitative correlation with experimentally determined increased or decreased crystallizability. The MD simulations allowed us to trace back the changes to their physical origins at the atomic level. Engineered charge–charge interactions as well as engineered hydrophobic effects could be characterized and were found to improve crystallizability. For example, the simulations revealed a redesigning of a water mediated electrostatic interaction (“wet contact”) into a water depleted hydrophobic effect (“dry contact”) and the optimization of a weak hydrogen bonding contact towards a strong one. These findings explained the experimentally found improved crystallizability. Our study emphasizes that it is difficult to derive simple rules for engineering crystallizability but that free energy simulations could be a very useful tool for understanding the contribution of crystal contacts for stability and furthermore could help guide protein engineering strategies to enhance crystallization for technical purposes.

scholarly journals Protein crystal lattices are dynamic assemblies: the role of conformational entropy in the protein condensed phase

IUCrJ ◽  
2018 ◽  
Vol 5 (2) ◽  
pp. 130-140 ◽  
Author(s):  
Margarita Dimova ◽  
Yancho D. Devedjiev
Keyword(s):  
Condensed Phase ◽  
Protein Structures ◽  
Van Der Waals Interactions ◽  
Protein Crystal ◽  
Crystal Formation ◽  
Conformational Entropy ◽  
Crystal Lattices ◽  
Crystal Contacts ◽  
First Time

Until recently, the occurrence of conformational entropy in protein crystal contacts was considered to be a very unlikely event. A study based on the most accurately refined protein structures demonstrated that side-chain conformational entropy and static disorder might be common in protein crystal lattices. The present investigation uses structures refined using ensemble refinement to show that although paradoxical, conformational entropy is likely to be the major factor in the emergence and integrity of the protein condensed phase. This study reveals that the role of shape entropy and local entropic forces expands beyond the onset of crystallization. For the first time, the complete pattern of intermolecular interactions by protein atoms in crystal lattices is presented, which shows that van der Waals interactions dominate in crystal formation.

scholarly journals TELSAM polymers accelerate crystallization of fused target proteins by stabilizing minimal crystal contacts and in the absence of direct inter-TELSAM contacts

2021 ◽  
Author(s):  
Supeshala Dilrukshi Sarath Nawarathnage ◽  
Sara Soleimani ◽  
Moriah H Mathis ◽  
Braydan D Bezzant ◽  
Diana T Ramírez ◽  
...  
Keyword(s):  
Protein Crystallization ◽  
Target Protein ◽  
Protein Crystal ◽  
Protein Fusion ◽  
Definitive Evidence ◽  
Helical Polymer ◽  
Crystal Contacts ◽  
Genetic Fusion ◽  
Direct Inter ◽  
Future Work

We extend investigation into the usefulness of genetic fusion to TELSAM polymers as an effective protein crystallization strategy. We tested various numbers of the target protein fused per turn of the TELSAM helical polymer and various TELSAM–target connection strategies. We provide definitive evidence that: 1. A TELSAM–target protein fusion can crystallize more rapidly than the same target protein alone, 2. TELSAM–target protein fusions can form well-ordered, diffracting crystals using either flexible or rigid TELSAM–target linkers, 3. Well-ordered crystals can be obtained when either 2 or 6 copies of the target protein are presented per turn of the TELSAM helical polymer, 4. The TELSAM polymers themselves need not directly contact one another in the crystal lattice, and 5. Fusion to TELSAM polymer confers immense avidity to stabilize exquisitely weak inter-target protein crystal contacts. We report features of TELSAM-target protein crystals and outline future work needed to define the requirements for reliably obtaining optimal crystals of TELSAM–target protein fusions.

Enhancement of nucleation of protein crystals on nano-wrinkled surfaces

2016 ◽  
Vol 186 ◽  
pp. 187-197 ◽  
Author(s):  
Praveen K. Bommineni ◽  
Sudeep N. Punnathanam
Keyword(s):  
Free Energy ◽  
Protein Concentration ◽  
Protein Crystallization ◽  
Crystal Nucleation ◽  
Protein Crystal ◽  
Protein Crystals ◽  
Free Energy Barrier ◽  
High Quality ◽  
High Quality Protein ◽  
Sinusoidal Surface

The synthesis of high quality protein crystals is essential for determining their structure. Hence the development of strategies to facilitate the nucleation of protein crystals is of prime importance. Recently, Ghatak and Ghatak [Langmuir 2013, 29, 4373] reported heterogeneous nucleation of protein crystals on nano-wrinkled surfaces. Through a series of experiments on different proteins, they were able to obtain high quality protein crystals even at low protein concentrations and sometimes without the addition of a precipitant. In this study, the mechanism of protein crystal nucleation on nano-wrinkled surfaces is studied through Monte Carlo simulations. The wrinkled surface is modeled by a sinusoidal surface. Free-energy barriers for heterogeneous crystal nucleation on flat and wrinkled surfaces are computed and compared. The study reveals that the enhancement of nucleation is closely related to the two step nucleation process seen during protein crystallization. There is an enhancement of protein concentration near the trough of the sinusoidal surface which aids in nucleation. However, the high curvature at the trough acts as a deterrent to crystal nucleus formation. Hence, significant lowering of the free-energy barrier is seen only if the increase in the protein concentration at the trough is very high.

scholarly journals Transfer of a Rational Crystal Contact Engineering Strategy between Diverse Alcohol Dehydrogenases

Crystals ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 975
Author(s):  
Brigitte Walla ◽  
Daniel Bischoff ◽  
Robert Janowski ◽  
Nikolas von den Eichen ◽  
Dierk Niessing ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Crystal Packing ◽  
Protein Crystallization ◽  
Lactobacillus Brevis ◽  
Protein Crystal ◽  
Wild Type ◽  
Alcohol Dehydrogenases ◽  
Crystallization Conditions ◽  
Contact Engineering ◽  
Crystal Contact

Protein crystallization can serve as a purification step in biotechnological processes but is often limited by the non-crystallizability of proteins. Enabling or improving crystallization is mostly achieved by high-throughput screening of crystallization conditions and, more recently, by rational crystal contact engineering. Two selected rational crystal contact mutations, Q126K and T102E, were transferred from the alcohol dehydrogenases of Lactobacillus brevis (LbADH) to Lactobacillus kefir (LkADH). Proteins were expressed in E. coli and batch protein crystallization was performed in stirred crystallizers. Highly similar crystal packing of LkADH wild type compared to LbADH, which is necessary for the transfer of crystal contact engineering strategies, was achieved by aligning purification tag and crystallization conditions, as shown by X-ray diffraction. After comparing the crystal sizes after crystallization of LkADH mutants with the wild type, the mean protein crystal size of LkADH mutants was reduced by 40–70% in length with a concomitant increase in the total amount of crystals (higher number of nucleation events). Applying this measure to the LkADH variants studied results in an order of crystallizability T102E > Q126K > LkADH wild type, which corresponds to the results with LbADH mutants and shows, for the first time, the successful transfer of crystal contact engineering strategies.

Prediction of Alkanolamine pKa Values by Combined Molecular Dynamics Free Energy Simulations and ab initio Calculations

2019 ◽  
Author(s):  
Javad Noroozi ◽  
William Smith
Keyword(s):  
Molecular Dynamics ◽  
Free Energy ◽  
Ab Initio Calculations ◽  
Gas Phase ◽  
Quantum Chemical ◽  
Phase Reaction ◽  
Pka Values ◽  
Gas Phase Reaction ◽  
Reaction Free Energy ◽  
Energy Simulations

We use molecular dynamics free energy simulations in conjunction with quantum chemical calculations of gas phase reaction free energy to predict alkanolamines pka values. <br>

Forces mediating protein–protein interactions: a computational study of p53 “approaching” MDM2

2009 ◽  
Vol 125 (3-6) ◽  
pp. 621-635 ◽  
Author(s):  
Shubhra Ghosh Dastidar ◽  
Arumugam Madhumalar ◽  
Gloria Fuentes ◽  
David P. Lane ◽  
Chandra S. Verma
Keyword(s):  
Protein Interactions ◽  
Computational Study ◽  
Protein Protein Interactions
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