Session F08: Physics of Proteins II: Structure & Dynamics of Proteins

Kinetics of pHILIP Insertion into a Membrane with Varying Model Parameters

One of the features of pH Low Insertion Peptide (pHILIP) is to detect the level of pH in the extracellular environment and insert across the membrane as a helix when it senses low pH in the environment. By exploiting this feature, pHLIP can open new avenues in cancer diagnosis and treatment. To bring pHLIP technology to a reality, it is very important to know how long does a pHLIP molecule take to insert across the cell membrane. Two different time scales that account for the transformation of a pHILIP molecule from the coil to interfacial helix and the insertion of the helix into the membrane with transmembrane orientation are very important. This talk will mainly focus on kinetics of pHILIP insertion into a membrane with varying model parameters that control level of pH and value of pKa.   

Intramolecular Structural Vibrations of Triose Phosphate Isomerase

It has long been contended that protein conformational access is through thermally populated intramolecular structural vibrations (ISV). The possible optimization of the ISV is particularly intriguing for the ubiquitous “perfect enzyme” triose phosphate isomerase (TIM). TIM is found in the two domains of life: archaea and bacteria . Beyond this, TIM’s structural motif, named the TIM-barrel fold, is present in ~10% of known protein catalyst structures. Raising the question, do the structural dynamics of the TIM-barrel fold provide biochemical advantages? In particular the possible long range dynamical coupling between the dimer interface and the substrate access gating by loop 6. the structural dynamics can be examined by Stationary Sample Anisotropic Terahertz Microspectroscopy (SSATM). TIM SSATM measurements were done on large crystals made using a batch microseeding method. This uses smaller crystals harvested, crushed, and diluted into a crystal slurry. Then a whisker tool picks up microcrystals from the slurry to drop some in the wells to stimulate growth of larger crystals. Crystals are used so the orientation of the molecules is known from the unit cell by having a known crystal facet on the aperture. The unit cell and crystal facets are characterized using X-ray crystallography and face indexing. Modeling can then be used to assign collective displacements to the ISV SSATM spectra. Our early measurements have shown reproducibility of an anisotropic band around 26cm^-1 that may be loop 6 motions.   

Effects of Salt and Lipids on Conformational Dynamics of the Aβ42 Protein

It is well established that amyloid β-protein (Aβ) self-assembly is involved in triggering of Alzheimer's disease. However, evidence of physiological function of Aβ has only begun to emerge. Details of Aβ-lipid interactions, which may underlie physiological and pathological activities of Aβ, are not well understood. Here, the effects of salt and 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipids on conformational dynamics of Aβ42 monomer in water are examined by all-atom molecular dynamics (MD). Ten 250-ns long trajectories were acquired in six sets of conditions: 0, 27, and 109 mM of DMPC lipids and pure water or 150 mM salt. We show that salt facilitates long-range tertiary contacts in Aβ42 and more compact conformations. Adding lipids results in lipid-concentration dependent Aβ42 unfolding. At the high lipid concentration, salt enables the N-terminal region of Aβ42 to form long-range tertiary contacts and interact with lipids, resulting in the formation of a parallel β-strand. Stable lipid-protein complexes whereby the protein is adhered to the lipid cluster, rather than embedded into it, are observed. Aβ residing on the cluster surface may be important for facilitating repair of leaks in the blood-brain barrier without penetrating and damaging cellular membranes.   

Triggered functional dynamics of AsLOV2 by time-resolved electron paramagnetic resonance at high magnetic fields

Proteins are fundamental building blocks of life; understanding their function is key to understanding biological processes. Recent EPR structural analyses of proteins, including Gd-DEER and nitroxide rapid freeze-quench techniques, provide static distance distributions at various times after activation [1]. However, an in-depth functional understanding of proteins requires a technique for tracking their inter-residue movement in real time. Such techniques exist, and include time-resolved (tr) X-ray spectroscopy, tr IR spectroscopy, tr NMR, and Förster resonance energy transfer, though each presents challenges when working with light-activated proteins in vitro. To mitigate these challenges, we present rapidscan-enabled, high-field, 240 GHz Gd-Gd tr EPR (TiGGER) and demonstrate it on AsLOV2, a light-activated protein found in oats. It had been established that upon 450 nm illumination in solution, AsLOV2’s J?-helix unfolds and becomes disordered, though there were not direct measurements of this motion, until now. The mechanical relaxation time constants recorded by TiGGER and UV-Vis spectroscopy are similar, confirming a relationship between photoswitching of the chromophore and mechanical action [2].

[1] Potapov and Goldfarb, Appl Magn Reson, Jan. 2010.

[2] Maity*, Price*, et al., bioRxiv, Oct. 2022.   

Molecular dynamics (MD) study of the structural and dynamical properties of an intrinsically disordered protein (IDP)

Mechanical gradients in the hard tissues of biological materials are essential for dissipation of stress and large forces that lead to damage. Despite the lack of mineralization, the Nereis virens worm jaw has mechanical properties comparable to human dentin and superior to synthetically engineered polymers. These remarkable properties arise from an extensive coordination of Zn²⁺ with a histidine rich IDP, Nvjp-1, imparting the jaw with mechanical and chemical gradients that greatly enhance its functionality. In a recent study [1], we used all atom MD simulations to investigate how Zn²⁺ binding to Nvjp-1 mediates sclerotization of the Nereis virens jaw. It was shown that zinc capture occurs at low pH mainly by polar residues with at most two residues interacting with a given zinc ion. A pH shift to alkaline conditions leads to additional zinc-residue coordination resulting in a reduction of the radius of gyration that correlates with sclerotization. Here we present results on structure factor, intermediate scattering function, and backbone N-H vector correlations for pristine and metal-bound Nvjp -1. These results may be combined with small angle x-ray scattering (SAXS) and nuclear magnetic resonance (NMR) data to understand the role of metal centers on the structural and dynamical properties of biological materials.

1. J. Phys. Chem. B 2022, 126, 6614.

    

Integrating Machine Learning and Molecular Modelling for Predicting Protein-Ligand Interactions

Computational approaches have increasingly become an indispensable part in predicting protein-ligand interactions and attracted much attention. In this talk, I will present our most recent progress along this direction by integrating machine learning and molecular modeling.   

Robustness of Min protein system In vivo pattern formation on [MinE] vs. [MinD] phase space

The oscillation of the Min proteins represents one of the most striking dynamic pattern formations in biology. Despite its long history, some of the most fundamental aspects of the Min system remain unanswered. They are, (1) how many Min proteins do cells need to start oscillations? (2) Does the cell produce just enough Min proteins for oscillations or in excess? (3) How do the answers change under different growth conditions? In this work, we answer these questions by constructing a phase diagram in the MinD vs. MinE space, two key Min proteins required for the reaction-diffusion system. To this end, we engineered a E. coli strain to independently produce MinD and MinE using two inducible promoters, covering about two orders of magnitude dynamic range in concentrations. We find that the concentrations of Min proteins in the wildtype cells are close to the minimum concentrations, below which the system cannot sustain its oscillatory behavior. As we increased the concentration of MinD or MinE, we discovered various regimes such as standing wave, traveling wave, and their phase coexistence, in vivo. Our work, for the first time, shows how the biophysical properties of the Min proteins and the cellular physiology are intimately linked.   

Interfacial charge transfer of amyloid-β proteins with graphene examined using Raman spectroscopy

The abnormal aggregation of amyloid-β (Aβ) protein is associated with many human diseases, including Alzheimer’s disease. Therefore, for early diagnosis and treatment, methods to identify the Aβ aggregation stages – monomer, oligomer, fibril – has been rigorously investigated. Previously, Aβ states were determined based on their structural properties, namely their morphologies and vibrational modes. However, there is little known about the electronic properties of Aβ proteins.

In this work, we investigate the electronic states of Aβ proteins at different fibrillization stages. We achieve this by monitoring the interfacial charge transfer between Aβ proteins and monolayer graphene using Raman spectroscopy. Graphene, an atomically thin semimetal, reacts sensitively to charge-transfer-induced doping and the shifts in the carrier concentrations and in the Fermi levels can be distinguished from the characteristic Raman bands in graphene (G and 2D). In particular, we found that graphene was p-doped by monomers while n-doped by fibrils, indicating that the electronic energy levels of Aβs shift as they aggregate. This charge-transfer polarity between the monomer and the fibril states provides critical insights into the electronic properties of Aβs that are essential to identifying the onset of neurotoxic Aβ fibril formation and developing a rapid, label-free diagnosis protocol based on two-dimensional materials.

    

Hard X-ray spectroscopy: a versatile tool for biophysicists

Metals play a key role in the variety of processes in nature. Metal activity is usually associated with changes in the oxidation state, spin state, and metal-ligand covalency of corresponding metallocofactors. Thus, the local electronic structure of the transition metal is essential for elucidating mechanisms underlying the dynamic processes involving metal ions. The inner-shell X-ray spectroscopy is the way to directly probe the electronic structure of metal which makes spectroscopic methods crucial for studying these processes. I am going to overview the x-ray spectroscopy techniques used in our laboratory for studying a variety of objects ranging from enzymes such as photosystem II, which catalyzes the fundamental biosphere-sustaining process of light-induced water splitting, to the application of X-ray spectroscopy for situ studies of battery cells. To highlight the sensitivity and powerfulness of the technique I will also present the results of our recent study conducted on the heme-dependent enzyme indoleamine (IDO). IDO is a crucial component to the metabolism of the amino acids suppressing the immune system which makes it a key for disease treatment, in particular cancer. To date the mechanisms of the underlying process are not completely understood, specifically, the spin states of iron and the mechanics of its formation are ambiguous. For determining the spin states of the associated metalloenzyme reaction we collected the Fe Kβ main line emission from crystals of the IDO using a specially designed von Hamos spectrometer.

    

Graphene-Based Field Effect Transistors for Use in Biodetection of Hemoglobin

Biosensors which utilize both well-known properties of optoelectronic devices and the vibrational fingerprints of biomolecules have been a promising avenue for detection and characterization of biomaterials. Graphene with outstanding properties has been used as an inorganic component and biomolecules of interest will excite resonantly in the graphene channel at particular wavelengths will generate an electrical signal. Hemoglobin as a measurable particle is of particular interest, as it has an absorbance spectrum in the visible range, meaning that it will readily create a large electrical signal when combined with graphene and illuminated with visible light. We have fabricated graphene field-effect transistor devices and use them to detect the concentration of hemoglobin in aqueous solutions. In these measurements, we have found that the electrical signal depends strongly on both the incident wavelength as well as the concentration of hemoglobin in solutions.