Session A20: Physics of Proteins: Progress on Structure-Function Relationships I

Lessons from Designing Optogenetic Tools

In the course of trying to engineer proteins to have a particular function, unexpected behaviours sometimes arise. I will describe how following up on some odd behaviour observed while trying to engineer photoactive yellow protein as a light-controlled switch, led to interesting, perhaps, general new insights into the behavior of these remarkable moelcules   

Engineering and application of a biosensor with focused ligand specificity

Microbial cell factories efficiently converting bio-based precursors to chemicals represent an attractive avenue to a more sustainable economy. Yet screening of genetically diverse strain libraries to identify the best-performing whole-cell biocatalysts is a low throughput endeavor. For this reason, transcriptional biosensors attracted attention as they allow screening of vast libraries in combination with fluorescence-activated cell sorting. However, a broad ligand specificity of many transcriptional regulators often prohibits the development of such ultra-high-throughput screens. We here solved the structure of the transcriptional regulator LysG of Corynebacterium glutamicum detecting all three basic amino acids. Semi-rational engineering of LysG using a FACS-based screening/counterscreening approach yielded a new biosensor bearing a regulator insensitive to l-lysine. Molecular dynamics simulations uncovered the underlying structure-function relationships of a crucial amino acid substitution. As proof of principle, we applied the new biosensor and isolated several l-histidine producing strains by FACS.   

Spontaneous Rupture and Entanglement of Human Neuronal Tau Protein Induced by Piconewton Compressive Force

Mechanical force vector fluctuations in the living cells can have a significant impact on protein behavior and functions. We have observed abrupt and spontaneous tau protein ruptures under a compressive force ranging from ∼5 to ∼125 pN, at a biologically available force amplitude range in living cells, using a home-modified atomic force microscopy single-molecule manipulation. The rupture behavior is dependent on the physiological level of the presence of ions, such as K⁺ and Mg²⁺. We observed rupture events in the presence of K⁺ but not in the presence of Mg²⁺ ions. We have also explored the entangled protein state formed following the events of the multiple and simultaneous protein ruptures under crowding. Crowded proteins simultaneously rupture and then spontaneously refold to an entangled folding state, different from either folded or unfolded states of the tau protein, which can be a plausible pathway for the tau protein aggregation that is related to several neurodegenerative diseases, such as the Alzheimer’s and Parkinson’s Diseases.   

Allosteric communications via substrate and linker between domains modulate protein function

The relation between structure and dynamics of biomolecules is important for their functions. Intra-domain dynamics occurring at pico- to milli-second timescales have been shown to correlate with activity. However, the correlation of allosteric communications between domains with biomolecular function is poorly understood. Here we show that inter-domain dynamics of matrix metalloprotease-1 (MMP1) on collagen fibrils are correlated with activity. Using single-molecule FRET, we identified the functionally relevant conformations where the two MMP1 domains are far apart, which were significantly absent for inactive MMP1 and could be modulated by inhibitor and enhancer of activity. All-atom and coarse-grained simulations reproduced the experimental features and revealed that dynamics are similar at pico- and milli-second timescales and substrate-dependent. Functional conformations are accompanied by larger catalytic pocket openings, which are increased by the communications mediated by collagen even if the domain linker is absent. Inter-domain communications are likely important for multidomain proteins in general.   

Disordered Protein Folding and Solubility as a Determinant of Human Disease

A longstanding principle in protein biophysics is that a protein’s sequence dictates its three-dimensional fold, and that structure is inherently linked to function. However, nearly a third of the human proteome contains intrinsically disordered regions that lack a discernible tertiary structure. Many of these proteins, referred to as intrinsically disordered proteins (IDPs), adopt transient conformations in order to carry out a wide-variety of functions, challenging the classical structure-function paradigm. Dysregulation of IDPs can also lead to the pathological accumulation of proteinaceous plaques and fibrils, which are observed in over 22 degenerative diseases and even in many types of cancers. In this talk, I will summarize our efforts to leverage molecular models of IDPs that populate vast free energy landscapes, reconciling how disordered and often soluble protein intermediates contribute to human disease. I will also discuss how these observations can supplement experiments, where transient protein conformations are often extraordinarily difficult to extract.   

Is the Protein Dynamical Transition Useful?

Terahertz Time Domain spectroscopy (THz TDS) has been used to characterize the protein dynamical transition [1, 2] .Typically the THz measurements are performed using solutions, however there is some question as to whether the freezing of the solution effects protein structure or dynamics. To address these questions we performed terahertz dynamical transition measurements in the 100-270 K range and between 0.15 – 2.00 THz on buffered solutions, dry glycerol solutions and minimally hydrated glyercol solutions. Buffer solution measurements using chicken egg white lysozyme and myoglobin protein with concentrations between 2 – 30 mM follow Beer’s law concentration dependence below 15 mM from which terahertz molar absorptivity of the hydrated protein can be extracted. For dry glycerol-protein solutions, the dynamical transition is absent, although it is present for 0.5 g water/g protein indicating that freezing of bulk water does not effect the measurements.
1. He, Y.F., et al., Phys. Rev. Lett., 2008. DOI: 10.1103/PhysRevLett.101.178103.
2. Markelz, A.G., et al., Chem. Phys. Lett., 2007. DOI: 10.1016/j.cplett.2007.05,080.   

Anti-cancer drug containing apolipoprotein B refolding through first-order-like phase transition process

First-order-like phase transition process is a promising approach for protein refolding into functional state. However, lipid binding protein, apolipoprotein B (apoB), reconstitution is challenging until our previous study. At the same time, the reconstituted apoB lipoparticle (rABL) may be an idea vehicle for drug delivery. In this study, we refolded apoB with anti-cancer drug from denatured state to functional state follow a first-order-like state transition model. By characterizing the reconstituted drug loaded rABL (drug@rABL) with circular dichroism (CD), dynamic light scattering (DLS), zeta potential and transmission electron microscope(TEM), we found drug@rABL is similar to rABL and native LDL. Moreover, cell viability assay indicated drug@rABL demonstrated highly anti-cancer efficacy in low concentration. Futhermore, the anti-cancer mechanism of the drug can be revealed, too.   

Integrative structural biology: insights from photoactive yellow protein

The integration of computational approaches with multiple different experimental techniques offers an exciting avenue to address open questions in protein science. We report such an integrative structural biology approach using a bacterial photoreceptor (photoactive yellow protein) as an accessible model system. A coarse-grained model of PYP in its initial pG state and its light-activate pB intermediate was calibrated based on a range of experimental techniques and was used to obtain mechanistic and molecular insights into the structure and energetics of the pB intermediate. Engineered disulfide bonds were used to limit specific molecular motions in PYP. This approach revealed that the pB state is highly glassy, with multiple substates in which the N-terminal region of PYP adopts quite distinct conformations, which in turn greatly alter the lifetime of the pB intermediate. These results reconcile apparent contradictions in published literature regarding the structure of pB, demonstrate that the N-terminal region undergoes Functionally Important Motions (FIMs) during the PYP photocycle, and explains the classic observation that the N-terminal region of PYP modulates pB lifetime.   

Decoupling between translation al and rotation al motions of water in the proximity of a protein molecule

The interaction between water and bio macromolecules is of fundamental interest in biophysics and biochemistry. By performing neutron scattering on a perdeuterated protein at various hydration levels, we characterized the dynamics of water in each molecular layer surrounding the biomolecule . We found that the translation al motion of the interfacial water is slowed down more significantly by protein than its rotation, and the retardation effect extends to the second hydration layer for translation while being limited in the first layer for rotation. Molecular dynamics simulation revealed that the observed translation rotation decoupling in hydration water results from that the translation of water is more correlated over space and highly restrained by the spatial confinement on protein. More importantly, water molecules around the structurally most stable protein residues exhibit the most retarded translation while maintaining their rotational freedom. Restraining translation of water around these residues protect the protein stability while maintaining the rotational mobility renders local flexibility to the biomolecule.   

Probing Temperature-Dependent Dynamics of Hemoglobin and Water Molecules in the Hydration Shells Using High Sensitivity Dielectric Spectroscopy

Hemoglobin, an iron-containing protein in red blood cells, has diverse biological functions including oxygen/carbon-dioxide transport and storage. The functions and behaviors of hemoglobin proteins strongly depend on environmental conditions and temperatures. The complicated quaternary structure of hemoglobin and the hydrogen-bond network in aqueous environments make it a challenge of identifying the conformational dynamics. Employing high-sensitivity dielectric megahertz-to-terahertz frequency-domain spectroscopy, we have systematically examined the dynamics of hemoglobin and the water molecules in their hydration shells as a function of temperature at the molecular level. Combining experiments with molecular dynamics simulations, we have determined the conformational dynamics of hemoglobin and water in aqueous solutions. The results help us identify hemoglobin dynamics and hemoglobin-water interactions that determine the biochemical functions and reactivity of hemoglobin proteins.   

Single Molecule Studies of DNA-Histone Interactions

Mechanically-induced unfolding of protein-compacted-DNA molecules is a powerful tool for studying protein-DNA interactions at the single-molecule level. We perform single molecule experiments using horizontal magnetic tweezers on histones complexed with tethered DNA under tension. Native or hyperacetylated histones purified from cultured cells were used. Our experiments allowed us to characterize the step-length distribution for nucleosomes reconstituted from native histones allowing for a comparison to data from hyperacetylated histones. Here we present preliminary data characterizing the mechanical properties of DNA molecules bound to native and post-translationally modified histones.