How Molecular Crowding Differs from Macromolecular Crowding: A Femtosecond Mid-Infrared Pump–Probe Study

Verma, Pramod Kumar; Kundu, Achintya; Cho, Minhaeng

doi:10.1021/acs.jpclett.8b03153

Cited by 19 publications

(9 citation statements)

References 57 publications

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“…[29][30][31][32][33][34][35][36][37][38] Protein crowding is typically simulated experimentally by the addition of co-solvents such as polyethylene glycol, carbohydrates, or other macromolecules. 31,[39][40][41][42] Some limitations to these approaches are the use of cosolvents that are electrostatically neutral or have low solubility. Other approaches that mimic crowding have included encapsulating proteins in sol-gels 29 and other nano-scale confinements.…”

Section: Introductionmentioning

confidence: 99%

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Measuring how two proteins affect each other's net charge in a crowded environment

et al. 2021

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Theory predicts that the net charge (Z) of a protein can be altered by the net charge of a neighboring protein as the two approach one another below the Debye length. This type of charge regulation suggests that a protein's charge and perhaps function might be affected by neighboring proteins without direct binding. Charge regulation during protein crowding has never been directly measured due to analytical challenges. Here, we show that lysine specific protein crosslinkers (NHS ester-Staudinger pairs) can be used to mimic crowding by linking two non-interacting proteins at a maximal distance of $7.9 Å. The net charge of the regioisomeric dimers and preceding monomers can then be determined with lysine-acyl "protein charge ladders" and capillary electrophoresis. As a proof of concept, we covalently linked myoglobin (Z monomer = À0.43 ± 0.01) and α-lactalbumin (Z monomer = À4.63 ± 0.05). Amide hydrogen/deuterium exchange and circular dichroism spectroscopy demonstrated that crosslinking did not significantly alter the structure of either protein or result in direct binding (thus mimicking crowding). Ultimately, capillary electrophoretic analysis of the dimeric charge ladder detected a change in charge of ΔZ = À0.04 ± 0.09 upon crowding by this pair (Z dimer = À5.10 ± 0.07). These small values of ΔZ are not necessarily general to protein crowding (qualitatively or quantitatively) but will vary per protein size, charge, and solvent conditions.

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Section: Introductionmentioning

confidence: 99%

“…Protein crowding is typically simulated experimentally by the addition of co‐solvents such as polyethylene glycol, carbohydrates, or other macromolecules 31,39–42 . Some limitations to these approaches are the use of co‐solvents that are electrostatically neutral or have low solubility.…”

Section: Introductionmentioning

confidence: 99%

Measuring how two proteins affect each other's net charge in a crowded environment

et al. 2021

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“…Two-dimensional infrared (2DIR) spectroscopy has proven to be a powerful tool to probe ultrafast equilibrium dynamics of enzymes as well as models of their active sites on femtosecond to picosecond timescales. − ,− Polarization-dependent mid-IR pump–probe spectroscopy has also been widely used to extract the vibrational population and orientational relaxation dynamics of various systems. − In this manuscript, we apply both polarization-dependent mid-IR pump–probe spectroscopy and 2DIR spectroscopy to a series of copper coordination complexes to determine how the structural variation in the secondary coordination sphere impacts the ultrafast dynamics at a small-molecule binding site where a series of trigonal pyramidal Cu I complexes bind O 2 .…”

Section: Introductionmentioning

confidence: 99%

Probing Ligand Effects on the Ultrafast Dynamics of Copper Complexes via Midinfrared Pump–Probe and 2DIR Spectroscopies

et al. 2021

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The effects of ligand structural variation on the ultrafast dynamics of a series of copper coordination complexes were investigated using polarization-dependent mid-IR pump− probe spectroscopy and two-dimensional infrared (2DIR) spectroscopy. The series consists of three copper complexes [( R 3 P 3 tren)Cu I I N 3 ]BAr 4 F ( 1 P R 3 , R 3 P 3 tren = tris[2-( p h o s p h i n i m i n a t o ) e t h y l ] a m i n e , B A r 4 F = tetrakis-(pentafluorophenyl)borate) where the number of methyl and phenyl groups in the PR 3 ligand are systematically varied across the series (PR 3 = PMe 3 , PMe 2 Ph, PMePh 2 ). The asymmetric stretching mode of azide in the 1 PR3 series is used as a vibrational probe of the small-molecule binding site. The results of the pump−probe measurements indicate that the vibrational energy of azide dissipates through intramolecular pathways and that the bulkier phenyl groups lead to an increase in the spatial restriction of the diffusive reorientation of bound azide. From 2DIR experiments, we characterize the spectral diffusion of the azide group and find that an increase in the number of phenyl groups maps to a broader inhomogeneous frequency distribution (Δ 2 ). This indicates that an increase in the steric bulk of the secondary coordination sphere acts to create more distinct configurations in the local environment that are accessible to the azide group. This work demonstrates how ligand structural variation affects the ultrafast dynamics of a small molecular group bound to the metal center, which could provide insight into the structure−function relationship of the copper coordination complexes and transition-metal coordination complexes in general.

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“…3,7−9 Recent studies have explored this hypothesis, providing evidence of structural interruptions, domain formation, and diffusive motion as a consequence of membrane protein encounters approaching biological concentrations. 1,3,7,8,10,11 Key biological processes take place in interfacial environments, and thus, it is crucial to characterize the effect of crowding on the local environment at the interface between the hydrophobic and hydrophilic regions of the membrane from an atomistic perspective, including H-bond populations and dynamics. 12 Two-dimensional infrared (2D IR) spectroscopy is an ideal tool for probing ultrafast dynamics at the lipid−water interface.…”

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confidence: 99%

Ultrafast Spectroscopy of Lipid–Water Interfaces: Transmembrane Crowding Drives H-Bond Dynamics

Flanagan

Cárdenas

Baiz

2020

J. Phys. Chem. Lett.

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Biology takes place in crowded, heterogeneous environments, and it is therefore essential to account for crowding effects in our understanding of biophysical processes at the molecular level. Comparable to the cytosol, proteins occupy approximately 30% of the plasma membrane surface; thus, crowding should have an effect on the local structure and dynamics at the lipid−water interface. Using a combination of ultrafast two-dimensional infrared spectroscopy and molecular dynamics simulations, we quantify the effects of membrane peptide concentration on the picosecond interfacial H-bond dynamics. The measurements reveal a nonmonotonic dependence of water orientation and dynamics as a function of transmembrane peptide:lipid ratio. We observe three dynamical regimes: a "pure lipid-like" regime at low peptide concentrations, a bulk-like region at intermediate peptide concentrations where dynamics are faster by ∼20% compared to those of the pure lipid bilayer, and a crowded regime where high peptide concentrations slow dynamics by ∼50%.

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How Molecular Crowding Differs from Macromolecular Crowding: A Femtosecond Mid-Infrared Pump–Probe Study

Cited by 19 publications

References 57 publications

Measuring how two proteins affect each other's net charge in a crowded environment

Measuring how two proteins affect each other's net charge in a crowded environment

Probing Ligand Effects on the Ultrafast Dynamics of Copper Complexes via Midinfrared Pump–Probe and 2DIR Spectroscopies

Ultrafast Spectroscopy of Lipid–Water Interfaces: Transmembrane Crowding Drives H-Bond Dynamics

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