Biology subjects

Sachleben, J. R.

Publications and source records attributed to Sachleben, J. R..

5 recordsLinked to original sources

bioRxiv2025

NMR Spectroscopy for the Validation of AlphaFold2 Structures

The introduction of AlphaFold has fundamentally changed our ability to predict the structure of proteins from their primary sequence of amino acids. As machine learning (ML) and artificial intelligence (AI) based protein prediction continues to advance, we examine the potential of hybrid techniques that combine experiment and computation that may yield more accurate structures than AI alone with significantly reduced experimental burden. We have developed heuristics comparing N-edited NOESY spectra and AlphaFold predicted structures that seek to determine whether the predicted structure reasonably describes the structure of the protein which generated the NOESY. These heuristics are fundamentally different than those used before in that they concentrate on interresidue contacts and not specific HH pairs. We present a large collection of data connecting entries across the BMRB, PDB and AlphaFold Database that includes experimentally derived structures and corresponding spectra, establishing it as a means to develop and test hybrid methods utilizing AlphaFold and NMR spectra to perform structure determination. These data test the new heuristics ability to identify inaccurate AlphaFold structures. A support vector machine was developed to test the consistency of NMR data with predicted structure and we show its application to the structure of an unsolved engineered protein, LoTOP.

biophysics↗

bioRxiv2023

Molecular insight into how the position of an abasic site and its sequence environment influence DNA duplex stability and dynamics

Local perturbations to DNA base-pairing stability from lesions and chemical modifications can alter the stability and dynamics of an entire oligonucleotide. End effects may cause the position of a disruption within a short duplex to influence duplex stability and structural dynamics, yet this aspect of nucleic acid modifications is often overlooked. We investigate how the position of an abasic site (AP site) impacts the stability and dynamics of short DNA duplexes. Using a combination of steady-state and time-resolved spectroscopy and molecular dynamics simulations, we unravel an interplay between AP-site position and nucleobase sequence that controls energetic and dynamic disruption to the duplex. The duplex is disrupted into two segments by an entropic barrier for base pairing on each side of the AP site. The barrier induces fraying of the short segment when an AP site is near the termini. Shifting the AP site inward promotes a transition from short-segment fraying to fully encompassing the barrier into the thermodynamics of hybridization, leading to further destabilization the duplex. Nucleobase sequence determines the length scale for this transition by tuning the barrier height and base-pair stability of the short segment, and certain sequences enable out-of-register base pairing to minimize the barrier height.

biophysics↗

bioRxiv2023

Direct monitoring of the thermodynamics and kinetics of DNA and RNA dinucleotide dehybridization from gaps and overhangs

Hybridization of short nucleic acid segments (<4 nucleotides) to single-strand templates occurs as a critical intermediate in processes such as non-enzymatic nucleic acid replication and toehold-mediated strand displacement. These templates often contain adjacent duplex segments that stabilize base pairing with single-strand gaps or overhangs, but the thermodynamics and kinetics of hybridization in such contexts are poorly understood due to experimental challenges of probing weak binding and rapid structural dynamics. Here we develop an approach to directly measure the thermodynamics and kinetics of DNA and RNA dinucleotide dehybridization using steady-state and temperature-jump infrared spectroscopy. Our results suggest that dinucleotide binding is stabilized through coaxial stacking interactions with the adjacent duplex segments as well as from potential non-canonical base pairing configurations and structural dynamics of gap and overhang templates revealed using molecular dynamics simulations. We measure timescales for dissociation ranging from 0.2 to 40 {micro}s depending on the template and temperature. Dinucleotide hybridization and dehybridization involves a significant free energy barrier with characteristics resembling that of canonical oligonucleotides. Together, our work provides an initial step for predicting the stability and kinetics of hybridization between short nucleic acid segments and various templates.

biophysics↗

bioRxiv2022

Sequential Activation and Local Unfolding Control Poly(A)-Binding Protein Condensation

Eukaryotic cells form biomolecular condensates to sense and adapt to their environment1,2. Poly(A)-binding protein (Pab1), a canonical stress granule marker3,4, condenses upon heat shock or starvation, promoting adaptation5. The molecular basis of condensation has remained elusive due to a dearth of techniques to probe structure directly in condensates. Here we apply hydrogen-deuterium exchange/mass spectrometry (HDX-MS) to investigate the molecular mechanism of Pab1s condensation. We find that Pab1s four RNA recognition motifs (RRMs) undergo different levels of partial unfolding upon condensation, and the changes are similar for thermal and pH stresses. Although structural heterogeneity is observed, the ability of MS to describe individual subpopulations allows us to identify which regions become partially unfolded and contribute to the condensates interaction network. Our data yield a clear molecular picture of Pab1s stress-triggered condensation, which we term sequential activation, wherein each RRM becomes activated at a temperature where it partially unfolds and associates with other likewise activated RRMs to form the condensate. This model thus implies that sequential activation is dictated by the underlying free energy surface, an effect we refer to as thermodynamic specificity. Our study represents a methodological advance for elucidating the interactions that drive biomolecular condensation that we anticipate will be widely applicable. Furthermore, our findings demonstrate how condensation can use thermodynamic specificity to perform an acute response to multiple, stresses, a potentially general mechanism for stress-responsive proteins.

biophysics↗

bioRxiv2018

Aβ Fibrils Can Act as Aqueous Pores: a Molecular Dynamics Study

Aggregation of A{beta} peptides is important in the etiology of Alzheimers Disease (AD), an increasingly prevalent neurodegenerative disease. We ran multiple [~] 300 ns all-atom explicit solvent molecular dynamics (MD) simulations starting from three NMR-based structural models of A{beta}(1-40 residues) fibrils having 2-fold (pdb code 2LMN) or 3-fold rotational symmetry (2LMP, and 2M4J). The 2M4J structure is based on an AD brain-seeded fibril whereas 2LMP and 2LMN represent two all-synthetic fibrils. Fibrils are constructed to contain either 6 or an infinite number of layers made using periodic images. The 6 layer fibrils partially unravel over the simulation time, mainly at their ends, while infinitely long fibrils do not. Once formed, the D23-K28 salt bridges are very stable and form within and between chains. Fibrils tend to retain (2LMN and 2LMP) or develop (2M4J) a \"stagger\" or register shift of {beta}-strands along the fibril axis. The brain-seeded fibril rapidly develops gaps at the sides of the fibril, which allows bidirectional flow of water and ions from the bulk phase in and out the central longitudinal core of the fibril. Similar but less marked changes were also observed for the 2LMP fibrils. The residues defining the gaps largely coincide with those demonstrated to have relatively rapid Hydrogen-Deuterium exchange in solid state NMR studies. These observations suggest that A{beta}(1-40 residues) fibrils may act as aqueous pores that might disrupt water and ion fluxes if inserted into a cell membrane.

neuroscience↗