Session N27: Physics of Proteins: Folding and Function

Dissect the motility mechanism and regulation of the C-terminal kinesin KlpA using single-molecule fluorescence microscopy

C-terminal kinesins such as HSET, Ncd and KlpA are naturally occurring microtubule-based motor proteins that play important roles in eukaryotic cell division by regulating spindle assembly. Microtubules are polar filaments with two distinct ends: the minus end and the plus end. Our previous work showed that while all other C-terminal kinesins exclusively exhibit directional preference for the microtubule minus end, KlpA uniquely exhibit directional preference for the microtubule plus end. In this talk, we will discuss our effort toward understanding the mechanisms underlying KlpA motility and its regulation. Specifically, we found that KlpA relies on a mechanical element called the central stalk (which connects the N-terminal tail and the C-terminal motor domains of KlpA) to achieve plus-end-directed motility on the microtubules. Building upon this finding, we have developed a method for engineering artificial C-terminal kinesins that uniquely exhibit KlpA-type plus-end-directed motility. Furthermore, we found that KlpA has a regulatory protein called TinA, which can directly interact with KlpA to reverse its directionality on the microtubule. Overall, our results are expected to significantly improve current understanding of C-terminal kinesin motors.        

Assessing Mechanical Properties of a Utrophin Fragment with Two Operational Modes

Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by the absence of the protein dystrophin. Utrophin, a dystrophin homologue, is being explored as a protein replacement therapy for DMD. Here, we study mechanical properties of a utrophin fragment encoding the N terminus through spectrin repeat 3 (UtrN-R3) using atomic force microscopy (AFM). We perform single molecule force spectroscopy using two operational modes of AFM, constant speed and constant force. In constant speed mode, the molecule is stretched continually while constant force mode maintains a steady force on the molecule. Under both modes, the bound domains unbound where the force at which the domains unravel and the time of unbinding are recorded under the constant speed and the constant force modes respectively. The Dudko-Hummer-Szabo method [Dudko et. al 2008] relates the statistics of force magnitude data from constant speed mode to the time to unravel statistics of the constant force mode. Our data shows consistent results using constant speed and constant force modes. We also recover parameters of the energy landscape of the domains and perform a homogeneity analysis for UtrN-R3. To further validate our results, Monte Carlo simulations are conducted which corroborate the conclusions drawn from experimental data. We include a Python-based toolbox including a simulation engine and different modeling methods for protein analysis.   

Temperature profiles of beta-amyloid and tau protein using Raman spectroscopy

The rapid aggregation of misfolded proteins leads to various neurodegenerative diseases. Beta-amyloid and tau protein are two proteins associated with Alzheimer's disease. Studies have shown that Raman spectroscopy can reveal important information about protein structures, including misfolding or denaturing processes. Using a Raman microscope with an excitation laser at 532 nm, we studied the vibrational spectrum of different protein aggregations among various stages of fibrillations as a function of temperature, from 20°C to 200°C. Our results show changes in the proteins’ Raman signatures as part of the heating process. Our study demonstrates a label-free and non-destructive technique to investigate amyloid fibril formation and study their secondary and tertiary structure as a function of temperature.  Such data could, one day, enable the design of novel photothermal treatments for Alzheimer's disease.

 

Inter-domain signaling: clues and questions from the photoactive yellow protein family of photoreceptors

Photoactive yellow protein (PYP) is a bacterial photoreceptor protein that has served as a model system to study the biophysics of receptor activation. A next frontier is how the photoactivation of PYP causes a biological signal to be relayed to the next component in signal transduction chains. In general, the principles governing such inter-domain signal relay from an input protein to an output protein remain unclear. Two widely occurring evolutionary processes in Bacteria and Archaea can potentially provide insights into this question: (1) the two proteins involved in some cases are fused into one larger multi-domain protein; and (2) the genes encoding two proteins involved is some cases are encoded adjacent on the DNA in an operon. We cataloged these two processes for the PYP family of photoreceptors and used them to derive clues regarding inter-domain signaling mechanisms. Striking trends in the preference for which likely output domains are found in PYP multidomain proteins (and their C- versus N-terminal location) and which tentative output domains are present in pyp operons. The output domain preference of PYP also appears to be distinct from that for the related photoreceptor LOV. The mechanistic drivers of these differences remain unclear, but have implications for understanding biological signaling and for synthetic biology.   

Evolutionary selection of proteins with two folds

Although most globular proteins fold into a single stable structure, an increasing number have been shown to remodel their secondary and tertiary structures in response to cellular stimuli. State-of-the-art algorithms predict that these fold-switching proteins adopt only one stable structure, missing their functionally critical alternative folds. Why these algorithms predict a single fold is unclear, but all of them infer protein structure from coevolved amino acid pairs. Here, we hypothesize that coevolutionary signatures are being missed. Suspecting that single-fold variants could be masking these signatures, we developed an approach, called Alternative Contact Enhancement (ACE), to search both highly diverse protein superfamilies–composed of single-fold and fold-switching variants–and protein subfamilies with more fold-switching variants. ACE successfully revealed coevolution of amino acid pairs uniquely corresponding to both conformations of 56/56 fold-switching proteins from distinct families. Then, we used ACE-derived contacts to predict two experimentally consistent conformations of a candidate protein with unsolved structure and develop a blind prediction pipeline for fold-switching proteins. The discovery of widespread dual-fold coevolution indicates that fold-switching sequences have been preserved by natural selection, implying that their functionalities provide evolutionary advantage and paving the way for predictions of diverse protein structures from single sequences.   

Structural Mechanism of TCR-pMHC Catch Bonding

T cells perform the remarkable task of determining whether an antigen presenting cell (APC) is healthy or unhealthy by probing only small peptide fragments displayed on the APC’s surface. Recent experiments have found that T cell receptors (TCRs) form catch-slipbonds with some peptides displayed by pMHC ligands, but slip bonds with other peptides. These experiments suggest that T cells may utilize the catch-slip behaviors of TCR-pMHC to achieve high fidelity peptide discrimination by applying (pN scale) forces to TCR-pMHC bonds. Recent studies have made significant progress in elucidating the mechanism of TCR-pMHC catch-slip bonding, but a physical model is still lacking. We have developed a mathematical framework of protein-ligand binding under force, showing how catch bonding is induced by singularities in the “flow field” that describes force-induced molecular deformations. By applying this mathematical framework to a free energy landscape model of TCR-pMHC, we show how minor changes in the peptide binding interaction convert the bond between slip and catch-slip behaviors.   

Functional clustering of proteins to map out the protein shape space

Understanding the relationship between sequence, structure, and function in proteins is a persistent challenge with implications throughout science and medicine. Within this issue, predicting function for structures that are not well known remains a difficult task, and a primary obstacle is in identifying critical sites that contribute to a protein’s function. Here we present a method for protein function classification and prediction of functional sites. Given a query structure, our scheme identifies high classifying residues corresponding to functional sites using feature-space embeddings extracted from a pre-trained network for protein structures. This network respects essential physical symmetries, yielding maximally informative and rotationally invariant embeddings. Candidate proteins are then clustered based on their similarity to these classifying residues. Specifically, we study protein kinase families and exploit established CATH superfamilies to validate the output clusters. We demonstrate that our approach accurately clusters structures from unseen families for a large group of protein kinases. This work can be used to shed light on the structure of the protein universe, with potential for grouping formerly uncharacterized structures and aiding in efforts to describe the landscape of protein shape-space.     

Understanding and design of beta-sheet forming antimicrobial peptides

Antimicrobial resistance is a growing problem that must be combatted via the design of new antimicrobial agents. A difficult prospect, such design requires both searching rationally through uncharted areas in biomolecular search space and a heightened understanding of the molecular-level mechanisms by which different agents act to inhibit bacterial growth. An attractive line of research in both of these lines is antimicrobial peptides (AMPs), short amphiphilic peptides, many of which are produced by the innate immune systems of diverse organisms, and many of which are thought to act via membrane destabilization. While a great deal of attention has been paid to helix-forming AMPs, there are other important categories, including AMPs that aggregate into beta sheets on the surface of cell membranes and potentially indiscriminately strain the membrane, rather than forming pores. In this talk, I will report on our work in devising a workflow for the active learning design of beta-sheet-forming AMPs. I will discuss how we have employed emergent deep learning techniques in biomolecular de novo design to devise an initial estimate of a rational search space for biomolecular design and how in conjunction we have employed multi-scale molecular dynamics to study variants of a synthetic beta-sheet forming antimicrobial peptide at an atomistic level and at a coarse-grained level. We show charge-dependent self-interactions and how those interactions dictate the behavior in conjunction with a membrane. Overall, this work sheds light on methods for combatting antimicrobial resistance through a rational exploration of biomolecular search spaces.   

A rapid transition in the unfolding of ubiquitin at high SDS concentrations

Protein interactions with surfactants are crucial in various contexts, from enzymatic applications in detergent formulations to the formulation of protein pharmaceuticals and industrial processes. Sodium dodecyl sulfate (SDS), an anionic surfactant, is a well-known denaturant capable of inducing changes in protein conformation. Understanding the intricacies of this interaction is crucial for unraveling the dynamics of protein-surfactant complexes.  

This study focuses on ubiquitin (Ubi) as a model protein and employs small-angle X-ray Scattering (SAXS) and molecular dynamics (MD) simulations to investigate its behavior in the presence of SDS. Earlier controversies surrounding the nature of the Ubi-SDS complex have been resolved by these SAXS studies, confirming that Ubi conforms to the canonical core-shell model upon exposure to SDS.  

We made the surprising observation that Ubi unfolding rate accelerates rapidly when crossing a certain SDS concentration threshold far above the critical micelle concentration of 5 mM SDS. At room temperature, Ubi unfolds slowly in the presence of SDS at concentrations from the cmc to around 60 mM, showing that it is kinetically stable against denaturation in SDS. However, within the concentration range of 60-70 mM SDS, there is a pronounced acceleration in unfolding, regardless of temperature and independent of ionic strength. A similar trend is observed in all-atom MD simulations performed at elevated temperatures (450K), which reveal the unfolding mechanisms to be the intercalation of SDS molecules inside the protein, resulting in the breakage of the secondary structure from the inside. In the concentration range above 50mM, the unfolding rate increases several times, as with higher concentrations, we observe a higher degree of clustering of SDS molecules inside the protein. It is worth noting that the resulting Ubi-SDS complexes after cooling down to 303.15K, closely resemble that of the canonical core-shell structures shown by previous SAXS studies.    

Designing Protein Stabilizers: Small Molecule Effects on Protein Folding are Driven by Direct Interactions and Solvent Rearrangement

Proteins are marginally stable macromolecules, prone to denaturation when exposed to environmental stressors. This susceptibility burdens the development, storage, and transportation of biological therapeutics. Thus, biological formulations are generally accompanied with small molecule excipients – including amino acids and sugars – that stabilize the native state of proteins. While empirically these effects are well known, the molecular features of excipients that drive stability remain unclear. Additionally, due to the chemical heterogeneity of proteins, it is difficult to resolve contributions arising from many competing forces involved in folding. To establish a molecular picture, we have utilized molecular dynamics simulations to explore the effects of various excipient solutions on miniprotein folding. We observe that residue-additive interactions, along with perturbations of the water structure surrounding the miniproteins, are the primary determinants of stability. These results bring into focus the molecular motifs that act as determinants of stability, and provide insights towards protein folding manipulation via small molecule additives. The insights will provide molecular design principles to discover novel molecules for stabilizing biological therapeutics.   

Effects of Lipids during Insertion Process of pH Low Insertion Peptide into Membrane

Membrane system consists of two phases separated by the interfacial region. These two regions are hydrophobic lipid tail region and hydrophilic lipid head plus aqueous region. The interfacial attributes such as surface tension and elastic properties are particularly important while we deal with membrane-peptide association and integrations. Pressure profile plays the key role on determining such properties of the bilayer. The elastic properties of chains of lipid tail affects the entropy of the system. Inclusion and insertion of hydrophobic peptide into a membrane stiffen the lipid chains in its immediate vicinity. Therefore, peptide inclusion into a membrane lowers the conformation entropy of the system. This talk aims to focus on lipid effects to the free energy cost for insertion of pH Low Insertion Peptide into membrane.