scholarly journals Opening of a cryptic pocket in β-lactamase increases penicillinase activity

2021 ◽  
Vol 118 (47) ◽  
pp. e2106473118
Author(s):  
Catherine R. Knoverek ◽  
Upasana L. Mallimadugula ◽  
Sukrit Singh ◽  
Enrico Rennella ◽  
Thomas E. Frederick ◽  
...  
Keyword(s):  
Excited States ◽  
Protein Design ◽  
Protein Function ◽  
Drug Targets ◽  
Functional Role ◽  
Chemical Exchange ◽  
Thermal Fluctuations ◽  
Chemical Exchange Saturation Transfer ◽  
Open Population ◽  
Dynamics Simulations

Understanding the functional role of protein-excited states has important implications in protein design and drug discovery. However, because these states are difficult to find and study, it is still unclear if excited states simply result from thermal fluctuations and generally detract from function or if these states can actually enhance protein function. To investigate this question, we consider excited states in β-lactamases and particularly a subset of states containing a cryptic pocket which forms under the Ω-loop. Given the known importance of the Ω-loop and the presence of this pocket in at least two homologs, we hypothesized that these excited states enhance enzyme activity. Using thiol-labeling assays to probe Ω-loop pocket dynamics and kinetic assays to probe activity, we find that while this pocket is not completely conserved across β-lactamase homologs, those with the Ω-loop pocket have a higher activity against the substrate benzylpenicillin. We also find that this is true for TEM β-lactamase variants with greater open Ω-loop pocket populations. We further investigate the open population using a combination of NMR chemical exchange saturation transfer experiments and molecular dynamics simulations. To test our understanding of the Ω-loop pocket’s functional role, we designed mutations to enhance/suppress pocket opening and observed that benzylpenicillin activity is proportional to the probability of pocket opening in our designed variants. The work described here suggests that excited states containing cryptic pockets can be advantageous for function and may be favored by natural selection, increasing the potential utility of such cryptic pockets as drug targets.

scholarly journals Opening of a Cryptic Pocket in β-lactamase Increases Penicillinase Activity

2021 ◽  
Author(s):  
Catherine R Knoverek ◽  
Upasana L Mallimadugula ◽  
Sukrit Singh ◽  
Enrico Rennella ◽  
Thomas E Frederick ◽  
...  
Keyword(s):  
Excited States ◽  
Protein Design ◽  
Protein Function ◽  
Drug Targets ◽  
Functional Role ◽  
Thermal Fluctuations ◽  
Potential Utility ◽  
Open Population ◽  
Dynamics Simulations

AbstractUnderstanding the functional role of protein excited states has important implications in protein design and drug discovery. However, because these states are difficult to find and study, it is still unclear if excited states simply result from thermal fluctuations and generally detract from function or if these states can actually enhance protein function. To investigate this question, we consider excited states in β-lactamases and particularly a subset of states containing a cryptic pocket which forms under the Ω-loop. Given the known importance of the Ω-loop and the presence of this pocket in at least two homologs, we hypothesized that these excited states enhance enzyme activity. Using thiol labeling assays to probe Ω-loop pocket dynamics and kinetic assays to probe activity, we find that while this pocket is not completely conserved across β-lactamase homologs, those with the Ω-loop pocket have a higher activity against the substrate benzylpenicillin. We also find that this is true for TEM β-lactamase variants with greater open Ω-loop pocket populations. We further investigate the open population using a combination of NMR CEST experiments and molecular dynamics simulations. To test our understanding of the Ω-loop pocket’s functional role, we designed mutations to enhance/suppress pocket opening and observed that benzylpenicillin activity is proportional to the probability of pocket opening in our designed variants. The work described here suggests that excited states containing cryptic pockets can be advantageous for function and may be favored by natural selection, increasing the potential utility of such cryptic pockets as drug targets.

Determination of pseudocontact shifts of low-populated excited states by NMR chemical exchange saturation transfer

2016 ◽  
Vol 18 (20) ◽  
pp. 13794-13798 ◽  
Author(s):  
R. S. Ma ◽  
Q. F. Li ◽  
A. D. Wang ◽  
J. H. Zhang ◽  
Z. J. Liu ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
Excited States ◽  
Chemical Exchange ◽  
Chemical Exchange Saturation Transfer ◽  
Saturation Transfer ◽  
Pseudocontact Shifts ◽  
Distance Restraints

Angular and distance restraints for low populated excited conformations are studied using PCS–CEST NMR spectroscopy.

scholarly journals AlphaFold2 transmembrane protein structure prediction shines

2021 ◽  
Author(s):  
Tamas Hegedus ◽  
Markus Geisler ◽  
Gergely Lukacs ◽  
Bianka Farkas
Keyword(s):  
Prescription Drugs ◽  
Protein Function ◽  
Structure Prediction ◽  
Drug Targets ◽  
Transmembrane Protein ◽  
Structural Data ◽  
Data Bank ◽  
Rational Drug Design ◽  
Abc Protein ◽  
Dynamics Simulations

Transmembrane (TM) proteins are major drug targets, indicated by the high percentage of prescription drugs acting on them. For a rational drug design and an understanding of mutational effects on protein function, structural data at atomic resolution are required. However, hydrophobic TM proteins often resist experimental structure determination and in spite of the increasing number of cryo-EM structures, the available TM folds are still limited in the Protein Data Bank. Recently, the DeepMind's AlphaFold2 machine learning method greatly expanded the structural coverage of sequences, with high accuracy. Since the employed algorithm did not take specific properties of TM proteins into account, the validity of the generated TM structures should be assessed. Therefore, we investigated the quality of structures at genome scales, at the level of ABC protein superfamily folds, and also in specific individual cases. We tested template-free structure prediction also with a new TM fold, dimer modeling, and stability in molecular dynamics simulations. Our results strongly suggest that AlphaFold2 performs astoundingly well in the case of TM proteins and that its neural network is not overfitted. We conclude that a careful application of its structural models will advance TM protein associated studies at an unexpected level.

Detection of Chemical Exchange in Methyl Groups of Macromolecules

2019 ◽  
Author(s):  
Michelle Gill ◽  
Andrew Hsu ◽  
Arthur G. Palmer, III
Keyword(s):  
Relaxation Rate ◽  
Chemical Exchange ◽  
Double Quantum ◽  
Methyl Groups ◽  
Multiple Quantum ◽  
Relaxation Data ◽  
E Coli ◽  
Methyl Trosy ◽  
Dynamics Simulations ◽  
Exchange Broadening

<div> <div> <div> <p>The zero- and double-quantum methyl TROSY Hahn-echo and the methyl <sup>1</sup>H-<sup>1</sup>H dipole- dipole cross-correlation nuclear magnetic resonance experiments enable estimation of multiple quantum chemical exchange broadening in methyl groups in proteins. The two relaxation rate constants are established to be linearly dependent using molecular dynamics simulations and empirical analysis of experimental data. This relationship allows chemical exchange broadening to be recognized as an increase in the Hahn-echo relaxation rate constant. The approach is illustrated by analyzing relaxation data collected at three temperatures for <i>E. coli </i>ribonuclease HI and by analyzing relaxation data collected for different cofactor and substrate complexes of <i>E. coli </i>AlkB. </p> </div> </div> </div>

Disturbing Gtp-Binding Protein Function Through Microinjection Into The Visual Cell Of Limulus

1992 ◽  
Vol 47 (11-12) ◽  
pp. 915-921 ◽  
Author(s):  
Henmg Stieve ◽  
Barbara Niemeyer ◽  
Klaus Aktories ◽  
Heidi E. Hamm
Keyword(s):  
Monoclonal Antibody ◽  
G Protein ◽  
Protein Function ◽  
Clostridium Botulinum ◽  
Functional Role ◽  
Electrical Response ◽  
Visual Cell ◽  
Gtp Binding ◽  
Dim Light ◽  
Ventral Nerve Photoreceptor

We have tested the action of three agents microinjected into the ventral nerve photoreceptor of Limulus on the electrical response to dim light. 1. A monoclonal antibody (mAb 4 A) against the Gɑ subunit of frog transducin reduces the size of the receptor current to 60%, suggesting an interaction with Gɑ in the Limulus photoreceptor. 2. Injection of Clostridium botulinum ADPribosyltransferase C 3 reduces the size to 46%; latency is not affected. The results imply that small GTP-binding proteins play a functional role in photoreception of invertebrates. 3. Injection of GD P-β-S reduces dose-dependently the size of the receptor current to 15% and prolongs the latency to 200%, presumably by reducing number and rate of G-protein activations

Amide proton transfer imaging for differentiation of tuberculomas from high-grade gliomas: Preliminary experience

2021 ◽  
pp. 197140092110027
Author(s):  
Karthik Kulanthaivelu ◽  
Shumyla Jabeen ◽  
Jitender Saini ◽  
Sanita Raju ◽  
Atchayaram Nalini ◽  
...  
Keyword(s):  
Proton Transfer ◽  
Chemical Exchange ◽  
Spin Echo ◽  
Chemical Exchange Saturation Transfer ◽  
Amide Proton ◽  
High Grade ◽  
Saturation Pulse ◽  
Amide Proton Transfer ◽  
High Grade Gliomas ◽  
Amide Protons

Purpose Tuberculomas can occasionally masquerade as high-grade gliomas (HGG). Evidence from magnetisation transfer (MT) imaging suggests that there is lower protein content in the tuberculoma microenvironment. Building on the principles of chemical exchange saturation transfer and MT, amide proton transfer (APT) imaging generates tissue contrast as a function of the mobile amide protons in tissue’s native peptides and intracellular proteins. This study aimed to further the understanding of tuberculomas using APT and to compare it with HGG. Method Twenty-two patients ( n = 8 tuberculoma; n = 14 HGG) were included in the study. APT was a 3D turbo spin-echo Dixon sequence with inbuilt B0 correction. A two-second, 2 μT saturation pulse alternating over transmit channels was applied at ±3.5 ppm around water resonance. The APT-weighted image (APTw) was computed as the MT ratio asymmetry (MTRasym) at 3.5 ppm. Mean MTRasym values in regions of interest (areas = 9 mm2; positioned in component with homogeneous enhancement/least apparent diffusion coefficient) were used for the analysis. Results MTRasym values of tuberculomas ( n = 14; 8 cases) ranged from 1.34% to 3.11% ( M = 2.32 ± 0.50). HGG ( n = 17;14 cases) showed MTRasym ranging from 2.40% to 5.70% ( M = 4.32 ± 0.84). The inter-group difference in MTRasym was statistically significant ( p < 0.001). APTw images in tuberculomas were notable for high MTRasym values in the perilesional oedematous-appearing parenchyma (compared to contralateral white matter; p < 0.001). Conclusion Tuberculomas demonstrate lower MTRasym ratios compared to HGG, reflective of a relative paucity of mobile amide protons in the ambient microenvironment. Elevated MTRasym values in perilesional parenchyma in tuberculomas are a unique observation that may be a clue to the inflammatory milieu.

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