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Results for “protein domain motions”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Study of Protein Dynamics via Neutron Spin Echo Spectroscopy

Most human body proteins' activity and functionality are related to configurational changes of entire subdomains within the protein crystal structure. The crystal structures build the basis for any calculation that describes the structure or dynamics of a protein, most of the time with strong geometrical restrictions. However, these restrictions from the crystal structure are not present in the solution. The structure of the proteins in the solution may differ from the crystal due to rearrangements of loops or subdomains on the pico to nanosecond time scale (i.e., the internal protein dynamics time regime). This study describes how slow motions on timescales of several tens of nanoseconds can be accessed using neutron scattering. In particular, the dynamical characterization of two major human proteins, an intrinsically disordered protein that lacks a well-defined secondary structure and a classical antibody protein, is addressed by neutron spin echo spectroscopy (NSE) combined with a wide range of laboratory characterization methods. Further insights into protein domain dynamics were achieved using mathematical modeling to describe the experimental neutron data and determine the crossover between combined diffusive and internal protein motions. The extraction of the internal dynamic contribution to the intermediate scattering function obtained from NSE, including the timescale of the various movements, allows further vision into the mechanical properties of single proteins and the softness of proteins in their nearly natural environment in the crowded protein solution.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Intermediate scattering functions of a rigid body monoclonal antibody protein in solution studied by dissipative particle dynamic simulation

In the past decade, there was increased research interest in studying internal motions of flexible proteins in solution using Neutron Spin Echo (NSE) as NSE can simultaneously probe the dynamics at the length and time scales comparable to protein domain motions. However, the collective intermediate scattering function (ISF) measured by NSE has the contributions from translational, rotational, and internal motions, which are rather complicated to be separated. Widely used NSE theories to interpret experimental data usually assume that the translational and rotational motions of a rigid particle are decoupled and independent to each other. To evaluate the accuracy of this approximation for monoclonal antibody (mAb) proteins in solution, dissipative particle dynamic computer simulation is used here to simulate a rigid-body mAb for up to about 200 ns. The total ISF together with the ISFs due to only the translational and rotational motions as well as their corresponding effective diffusion coefficients is calculated. The aforementioned approximation introduces appreciable errors to the calculated effective diffusion coefficients and the ISFs. For the effective diffusion coefficient, the error introduced by this approximation can be as large as about 10% even though the overall agreement is considered reasonable. Thus, we need to be cautious when interpreting the data with a small signal change. In addition, the accuracy of the calculated ISFs due to the finite computer simulation time is also discussed.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗
DOE OSTI2022

Conformational motions and ligand-binding underlying gating and regulation in IP 3 R channel

Inositol-1,4,5-trisphosphate receptors (IP3Rs) are activated by IP 3 and Ca 2+ and their gating is regulated by various intracellular messengers that finely tune the channel activity. Here, using single particle cryo-EM analysis we determined 3D structures of the nanodisc-reconstituted IP 3 R1 channel in two ligand-bound states. These structures provide unprecedented details governing binding of IP 3 , Ca 2+ and ATP, revealing conformational changes that couple ligand-binding to channel opening. Using a deep-learning approach and 3D variability analysis we extracted molecular motions of the key protein domains from cryo-EM density data. We find that IP 3 binding relies upon intrinsic flexibility of the ARM2 domain in the tetrameric channel. Our results highlight a key role of dynamic side chains in regulating gating behavior of IP 3 R channels. This work represents a stepping-stone to developing mechanistic understanding of conformational pathways underlying ligand-binding, activation and regulation of the channel.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2003

Structural Basis for "Flip-Flop" Action of Human Pyruvate Dehydrogenase

The derivative of vitamin B1, thiamin pyrophosphate is a cofactor of pyruvate dehydrogenase, a component enzyme of the mitochondrial pyruvate dehydrogenase multienzyme complex that plays a major role in directing energy metabolism in the cell. This cofactor is used to cleave the C(sup alpha)-C(=O) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase. In alpha(sub 2)beta(sub 2)-tetrameric human pyruvate dehydrogenase, there are two cofactor binding sites, each of them being a center of independently conducted, although highly coordinated enzymatic reactions. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites can now be understood based on the recently determined crystal structure of the holo-form of human pyruvate dehydrogenase at 1.95A resolution. The structure of pyruvate dehydrogenase was determined using a combination of MAD phasing and molecular replacement followed by rounds of torsion-angles molecular-dynamics simulated-annealing refinement. The final pyruvate dehydrogenase structure included coordinates for all protein amino acids two cofactor molecules, two magnesium and two potassium ions, and 742 water molecules. The structure was refined to R = 0.202 and R(sub free) = 0.244. Our structural analysis of the enzyme folding and domain assembly identified a simple mechanism of this protein motion required for the conduct of catalytic action.

Ciszak, Ewa↗
NASA NTRS2010

Interactions of Ku70/80 with Double-Strand DNA: Energetic, Dynamics, and Functional Implications

Space radiation is a proficient inducer of DNA damage leading to mutation, aberrant cell signaling, and cancer formation. Ku is among the first responding proteins in nucleus to recognize and bind the DNA double strand breaks (DSBs) whenever they are introduced. Once loaded Ku works as a scaffold to recruit other repair factors of non-homologous end joining and facilitates the following repair processes. The crystallographic study of the Ku70/80 heterodimer indicate the core structure of this protein shows virtually no conformational change after binding with DNA. To investigate the dynamical features as well as the energetic characteristics of Ku-DNA binding, we conduct multi-nanosecond molecular dynamics simulations of a modeled Ku70/80 structure and several complexes with two 24-bp DNA duplexes. Free energy calculations show significant energy differences between the complexes with Ku bound at DSBs and those with Ku associated at an internal site of a chromosome. The results also reveal detailed interactions between different nucleotides and the amino acids along the DNA-binding cradle of Ku, indicating subtle binding preference of Ku at specific DNA sequences. The covariance matrix analyses along the trajectories demonstrate the protein is stimulated to undergo correlated motions of different domains once bound to DNA ends. Additionally, principle component analyses identify these low frequency collective motions suitable for binding with and translocation along duplex DNA. It is proposed that the modification of dynamical properties of Ku upon binding with DSBs may provide a signal for the further recruitment of other repair factors such as DNA-PKcs, XLF, and XRCC4.

Hu, Shaowen↗
DOE OSTI2022

Conformational spread drives the evolution of the calcium–calmodulin protein kinase II

The calcium calmodulin (Ca 2+ /CaM) dependent protein kinase II (CaMKII) decodes Ca 2+ frequency oscillations. The CaMKIIα isoform is predominantly expressed in the brain and has a central role in learning. I matched residue and organismal evolution with collective motions deduced from the atomic structure of the human CaMKIIα holoenzyme to learn how its ring architecture abets function. Protein dynamic simulations showed its peripheral kinase domains (KDs) are conformationally coupled via lateral spread along the central hub. The underlying β-sheet motions in the hub or association domain (AD) were deconvolved into dynamic couplings based on mutual information. They mapped onto a coevolved residue network to partition the AD into two distinct sectors. A second, energetically stressed sector was added to ancient bacterial enzyme dimers for assembly of the ringed hub. The continued evolution of the holoenzyme after AD–KD fusion targeted the sector’s ring contacts coupled to the KD. Among isoforms, the α isoform emerged last and, it alone, mutated rapidly after the poikilotherm–homeotherm jump to match the evolution of memory. The correlation between dynamics and evolution of the CaMKII AD argues single residue substitutions fine-tune hub conformational spread. The fine-tuning could increase CaMKIIα Ca 2+ frequency response range for complex learning functions.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2022

Quantifying Structural Relationships of Metal-Binding Sites Suggests Origins of Biological Electron Transfer

Biological redox reactions drive planetary biogeochemical cycles. Using a novel, structure-guided sequence analysis of proteins, we explored the patterns of evolution of enzymes responsible for these reactions. Our analysis reveals that the folds that bind transition metal–containing ligands have similar structural geometry and amino acid sequences across the full diversity of proteins. Similarity across folds reflects the availability of key transition metals over geological time and strongly suggests that transition metal–ligand binding had a small number of common peptide origins. We observe that structures central to our similarity network come primarily from oxidoreductases, suggesting that ancestral peptides may have also facilitated electron transfer reactions. Last, our results reveal that the earliest biologically functional peptides were likely available before the assembly of fully functional protein domains over 3.8 billion years ago. Thus, life is a special, very complex form of motion of matter, but this form did not always exist, and it is not separated from inorganic nature by an impassable abyss; rather, it arose from inorganic nature as a new property in the process of evolution of the world. We must study the history of this evolution if we want to solve the problem of the origin of life.

Yana Bromberg↗
NASA NTRS2003

Structural Basis for Flip-Flop Action of Thiamin Pyrophosphate-Dependent Enzymes Revealed by Human Pyruvate Dehydrogenase

Thiamin pyrophosphate (TPP), the biologically active form of vitamin BI, is a cofactor of enzymes catalyzing reactions involving the cleavage of a carbon-carbon bond adjacent to an oxo group. TPP-dependent enzymes show a common mechanism of TPP activation by: (1) forming the ionic N-H...O(sup -) hydrogen bonding between the N1' atom of the aminopirymidine ring of the coenzyme and intrinsic gamma-carboxylate group of glutamate and (2) imposing an "active" V-conformation that brings the N4' atom of the aminopirymidine to the distance required for the intramolecular C-H.. .N hydrogen bonding with the thiazolium C2 atom. Within these two hydrogen bonds that rapidly exchange protons, protonation of the N1' atom is strictly coordinated with the deprotonation of the 4' -amino group and eventually abstraction of the proton from C2. The human pyruvate dehydrogenase Elp, component of human pyruvate dehydrogenase complex, catalyzes the irreversible decarboxylation of the pyruvate followed by the reductive acetylation of the lipoyl group of dihydrolipoyl acyltransferase. Elp is alpha(sub 2)beta(sub2)-heterotetrameric with a molecular mass of I54 kDa, which has two catalytic sites, each providing TPP and magnesium ion as cofactors and each formed on the interface between the PP and PYR domains. The dynamic nonequivalence of two otherwise chemically equivalent catalytic sites has been observed and the flip-flop mechanism was suggested, according to which two active sites affect each other and in which different steps of the catalytic reaction are performed in each of the sites at any given moment. Based on specific futures of human pyruvate dehydrogenase including rigid and flexible connections between domains that bind the cofactor we propose a mechanistic model for the flip-flop action of this enzyme. We postulate that the dynamic protein environment drives the exchange of tautomers in the 4' -aminopyrimidine ring of the cofactor through a concerted shuttl-like motion of tightly connected domains. The dynamic exchange of those tautomers, in turns, is required during the reactions of pyruvate decarboxylation and reductive acetylation of lipoamide. Thus the shuttle-like motion of the domains is coordinated with the reactions of decarboxylation and acetylation, which are carried out in each of the cofactor sites resulting in a flip-flop action of the enzyme. The structure-derived mechanism of action of human pyruvate dehydrogenase may be likely common for other TPP-dependent enzymes.

Dominiak, Paulina↗
DOE OSTI2019

A bacterial surface layer protein exploits multistep crystallization for rapid self-assembly

Surface layers (S-layers) are crystalline protein coats surrounding microbial cells. S-layer proteins (SLPs) regulate their extracellular self-assembly by crystallizing when exposed to an environmental trigger. However, molecular mechanisms governing rapid protein crystallization in vivo or in vitro are largely unknown. Here, we demonstrate that the Caulobacter crescentus SLP readily crystallizes into sheets in vitro via a calcium-triggered multistep assembly pathway. This pathway involves 2 domains serving distinct functions in assembly. The C-terminal crystallization domain forms the physiological 2-dimensional (2D) crystal lattice, but full-length protein crystallizes multiple orders of magnitude faster due to the N-terminal nucleation domain. Observing crystallization using a time course of electron cryo-microscopy (Cryo-EM) imaging reveals a crystalline intermediate wherein N-terminal nucleation domains exhibit motional dynamics with respect to rigid lattice-forming crystallization domains. Dynamic flexibility between the 2 domains rationalizes efficient S-layer crystal nucleation on the curved cellular surface. Rate enhancement of protein crystallization by a discrete nucleation domain may enable engineering of kinetically controllable self-assembling 2D macromolecular nanomaterials.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structures of honeybee-infecting Lake Sinai virus reveal domain functions and capsid assembly with dynamic motions

Understanding the structural diversity of honeybee-infecting viruses is critical to maintain pollinator health and manage the spread of diseases in ecology and agriculture. We determine cryo-EM structures of $T$ = 4 and $T$ = 3 capsids of virus-like particles (VLPs) of Lake Sinai virus (LSV) 2 and delta-N48 LSV1, belonging to tetraviruses, at resolutions of 2.3–2.6 Å in various pH environments. Structural analysis shows that the LSV2 capsid protein (CP) structural features, particularly the protruding domain and C-arm, differ from those of other tetraviruses. The anchor loop on the central β-barrel domain interacts with the neighboring subunit to stabilize homo-trimeric capsomeres during assembly. Delta-N48 LSV1 CP interacts with ssRNA via the rigid helix α1’, α1’–α1 loop, β-barrel domain, and C-arm. Cryo-EM reconstructions, combined with X-ray crystallographic and small-angle scattering analyses, indicate that pH affects capsid conformations by regulating reversible dynamic particle motions and sizes of LSV2 VLPs. C-arms exist in all LSV2 and delta-N48 LSV1 VLPs across varied pH conditions, indicating that autoproteolysis cleavage is not required for LSV maturation. The observed linear domino-scaffold structures of various lengths, made up of trapezoid-shape capsomeres, provide a basis for icosahedral $T$ = 4 and $T$ = 3 architecture assemblies. These findings advance understanding of honeybee-infecting viruses that can cause Colony Collapse Disorder.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Viscoelastic analysis of mussel threads reveals energy dissipative mechanisms

Mussels use byssal threads to secure themselves to rocks and as shock absorbers during cyclic loading from wave motion. Byssal threads combine high strength and toughness with extensibility of nearly 200%. Researchers attribute tensile properties of byssal threads to their elaborate multi-domain collagenous protein cores. Because the elastic properties have been previously scrutinized, we instead examined byssal thread viscoelastic behaviour, which is crucial for withstanding cyclic loading. By targeting protein domains in the collagenous core via chemical treatments, stress relaxation experiments provided insights on domain contributions and were coupled with in situ small-angle X-ray scattering to investigate relaxation-specific molecular reorganizations. Results show that when silk-like domains in the core were disrupted, the stress relaxation of the threads decreased by nearly 50% and lateral molecular spacing also decreased, suggesting that these domains are essential for energy dissipation and assume a compressed molecular rearrangement when disrupted. A generalized Maxwell model was developed to describe the stress relaxation response. The model predicts that maximal damping (energy dissipation) occurs at around 0.1 Hz which closely resembles the wave frequency along the California coast and implies that these materials may be well adapted to the cyclic loading of the ambient conditions.

36 MATERIALS SCIENCE↗
DOE OSTI2025

Photoactivation Transition State and Dynamical Response of the Orange Carotenoid Protein

The orange carotenoid protein (OCP) regulates light harvesting in cyanobacteria by acting as a photoreceptor in its resting form, OCP O , and by effecting the quenching of bilin excitons upon binding to the core of the phycobilisome in its photoactivated red form, OCP R . We show herein using fluorescence anisotropy measurements and the action spectra for the rate constants of the two light-driven steps in the mechanism that the photoactivation of the OCP from Synechocystis sp. PCC 6803 is triggered by excited-state motions of the canthaxanthin chromophore that yield a twisted and bent conformation. Well-tempered metadynamics simulations reveal that a bicycle-pedal configuration, due to twisting of the two adjacent C=C bonds at the C13−C14 and C15−C15′ positions in the center of canthaxanthin’s π-conjugated isoprenoid backbone, can be accommodated by the binding site in the OCP, with the energy of a captured photon required to cross the local activation energy barriers from the dark equilibrium structure. The bicycle-pedal configuration breaks the conserved hydrogen-bonding interactions between the carbonyl substituent of the β-ionone end ring of canthaxanthin and the adjacent W288 and Y201 residues in the C-terminal domain. The action spectra are modulated by the vibronic excitation prepared by absorption transitions to the S 2 state, indicating that the photoactivation reactions are triggered by the canthaxanthin chromophore well prior to vibrational equilibration. These findings show that an ultrafast structural response of the OCP protein to the excited-state motions of the canthaxanthin chromophore controls the photoactivation yield and the sensing of blue light.

Fluorescence↗
DOE OSTI2021

Bioactive scaffolds with enhanced supramolecular motion promote recovery from spinal cord injury

The signaling of cells by scaffolds of synthetic molecules that mimic proteins is known to be effective in the regeneration of tissues. Here, we describe peptide amphiphile supramolecular polymers containing two distinct signals and test them in a mouse model of severe spinal cord injury. One signal activates the transmembrane receptor β1-integrin and a second one activates the basic fibroblast growth factor 2 receptor. By mutating the peptide sequence of the amphiphilic monomers in nonbioactive domains, we intensified the motions of molecules within scaffold fibrils. This resulted in notable differences in vascular growth, axonal regeneration, myelination, survival of motor neurons, reduced gliosis, and functional recovery. Here, we hypothesize that the signaling of cells by ensembles of molecules could be optimized by tuning their internal motions.

36 MATERIALS SCIENCE↗
DOE OSTI2020

Quantitative comparison between sub-millisecond time resolution single-molecule FRET measurements and 10-second molecular simulations of a biosensor protein

Molecular Dynamics (MD) simulations seek to provide atomic-level insights into conformationally dynamic biological systems at experimentally relevant time resolutions, such as those afforded by single-molecule fluorescence measurements. However, limitations in the time scales of MD simulations and the time resolution of single-molecule measurements have challenged efforts to obtain overlapping temporal regimes required for close quantitative comparisons. Achieving such overlap has the potential to provide novel theories, hypotheses, and interpretations that can inform idealized experimental designs that maximize the detection of the desired reaction coordinate. Here, we report MD simulations at time scales overlapping with in vitro single-molecule Förster (fluorescence) resonance energy transfer (smFRET) measurements of the amino acid binding protein LIV-BP SS at sub-millisecond resolution. Computationally efficient all-atom structure-based simulations, calibrated against explicit solvent simulations, were employed for sampling multiple cycles of LIV-BP SS clamshell-like conformational changes on the time scale of seconds, examining the relationship between these events and those observed by smFRET. The MD simulations agree with the smFRET measurements and provide valuable information on local dynamics of fluorophores at their sites of attachment on LIV-BP SS and the correlations between fluorophore motions and large-scale conformational changes between LIV-BP SS domains. We further utilize the MD simulations to inform the interpretation of smFRET data, including Förster radius (R 0 ) and fluorophore orientation factor (κ 2 ) determinations. The approach we describe can be readily extended to distinct biochemical systems, allowing for the interpretation of any FRET system conjugated to protein or ribonucleoprotein complexes, including those with more conformational processes, as well as those implementing multi-color smFRET.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Disruption of electrostatic contacts in the HNH nuclease from a thermophilic Cas9 rewires allosteric motions and enhances high-temperature DNA cleavage

Allosteric signaling within multidomain proteins is a driver of communication between spatially distant functional sites. Understanding the mechanism of allosteric coupling in large multidomain proteins is the most promising route to achieving spatial and temporal control of the system. The recent explosion of CRISPR-Cas9 applications in molecular biology and medicine has created a need to understand how the atomic level protein dynamics of Cas9, which are the driving force of its allosteric crosstalk, influence its biophysical characteristics. In this study, we used a synergistic approach of nuclear magnetic resonance (NMR) and computation to pinpoint an allosteric hotspot in the HNH domain of the thermostable GeoCas9. We show that mutation of K597 to alanine disrupts a salt-bridge network, which in turn alters the structure, the timescale of allosteric motions, and the thermostability of the GeoHNH domain. This homologous lysine-to-alanine mutation in the extensively studied mesophilic S. pyogenes Cas9 similarly alters the dynamics of the SpHNH domain. We have previously demonstrated that the alteration of allostery via mutations is a source for the specificity enhancement of SpCas9 (eSpCas9). Hence, this may also be true in GeoCas9.

Chemistry↗
DOE OSTI2022

Structural basis for the mechanisms of human presequence protease conformational switch and substrate recognition

Presequence protease (PreP), a 117kDa mitochondrial M16C metalloprotease vital for mitochondrial proteostasis, degrades presequence peptides cleaved off from nuclear-encoded proteins and other aggregation-prone peptides, such as amyloid β (Aβ). PreP structures have only been determined in a closed conformation; thus, the mechanisms of substrate binding and selectivity remain elusive. Here, we leverage advanced vitrification techniques to overcome the preferential denaturation of one of two ~55 kDa homologous domains of PreP caused by air-water interface adsorption. Thereby, we elucidate cryoEM structures of three apo-PreP open states along with Aβ- and citrate synthase presequence-bound PreP at 3.3–4.6 Å resolution. Together with integrative biophysical and pharmacological approaches, these structures reveal the key stages of the PreP catalytic cycle and how the binding of substrates or PreP inhibitor drives a rigid body motion of the protein for substrate binding and catalysis. Together, our studies provide key mechanistic insights into M16C metalloproteases for future therapeutic innovations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Mycobacterial DNA polymerase I: activities and crystal structures of the POL domain as apoenzyme and in complex with a DNA primer-template and of the full-length FEN/EXO–POL enzyme

Mycobacterial Pol1 is a bifunctional enzyme composed of an N-terminal DNA flap endonuclease/5' exonuclease domain (FEN/EXO) and a C-terminal DNA polymerase domain (POL). Here we document additional functions of Pol1: FEN activity on the flap RNA strand of an RNA:DNA hybrid and reverse transcriptase activity on a DNA-primed RNA template. We report crystal structures of the POL domain, as apoenzyme and as ternary complex with 3'-dideoxy-terminated DNA primer-template and dNTP. The thumb, palm, and fingers subdomains of POL form an extensive interface with the primer-template and the triphosphate of the incoming dNTP. Progression from an open conformation of the apoenzyme to a nearly closed conformation of the ternary complex entails a disordered-to-ordered transition of several segments of the thumb and fingers modules and an inward motion of the fingers subdomain—especially the O helix—to engage the primer-template and dNTP triphosphate. Distinctive structural features of mycobacterial Pol1 POL include a manganese binding site in the vestigial 3' exonuclease subdomain and a non-catalytic water-bridged magnesium complex at the protein-DNA interface. We report a crystal structure of the bifunctional FEN/EXO–POL apoenzyme that reveals the positions of two active site metals in the FEN/EXO domain.

59 BASIC BIOLOGICAL SCIENCES↗