Biology subjects

Weiss, K. L.

Publications and source records attributed to Weiss, K. L..

9 recordsLinked to original sources

bioRxiv2026

Critical amino acid residues in the N-terminal domain of NADPH-dependent assimilatory sulfite reductase flavoprotein mediate octameric assembly

How large, flexible enzymes assemble into defined oligomeric architectures remains a central question in biology. NADPH-dependent assimilatory sulfite reductase (SiR) forms a heterododecamer built on an octameric flavoprotein (SiRFP) core, yet the molecular basis for this assembly has been unresolved because of its disordered N-terminus. Here, we use ion mobility mass spectrometry, small-angle neutron scattering, and mutagenesis to define the mechanism of SiRFP oligomerization. We show that SiRFP forms a discrete, stable octamer in solution. We also report that its N-terminal 52-residue segment is necessary and sufficient to mediate assembly, also mediating oligomerization when fused to a heterologous protein. Structure-guided mutagenesis identifies four residues (Gln22, Tyr39, Phe40, and Gln47) whose substitution disrupts the octamer, producing concentration-dependent lower-order species while retaining catalytic activity. These findings define the determinants of SiRFP assembly with broader implications for engineering homomeric protein complexes. ImportanceThis work seeks to understand the basis for oligomerization of a large oxidoreductase that is important for metabolizing sulfur, an essential chemical for all of biology. A 52-residue long leader peptide is necessary and sufficient for assembly into a particularly stable octamer that is resistant to chemical denaturation under diverse conditions.

biophysics↗

bioRxiv2026

Integrating Segmental Deuteration iCM-SANS with SAXS and MD for Dynamical Analysis of Multi-domain Proteins

Multi-domain proteins (MDPs) adopt diverse conformations arising from cooperative inter-domain motions, and such dynamics are intimately coupled to their biological functions. Quantitative characterization of these motions is crucial for elucidating their functional mechanisms. Although small-angle X-ray scattering (SAXS) provides information on overall domain arrangement, the limited experimental constraints hinder reliable discrimination of conformational ensembles derived from molecular dynamics (MD) simulations. To address this limitation, complementary experimental constraints that enable to observe domain-selective structural information are required. Inverse contrast-matching small-angle neutron scattering (iCM-SANS), combined with segmental deuteration, enables selective visualization of individual domains and thus provides such complementary information. However, practical strategies for preparing segmentally deuterated MDPs with arbitrary domain labelling have yet to be established. Here, we develop an experimental protocol that integrates controlled protein deuteration with high-efficiency multi-step protein ligation to generate a segmentally deuterated MDP in high yield. The combined use of SAXS and iCM-SANS yields complementary structural constraints that enhance discrimination of MD-derived conformational ensembles. This protocol expands the applicability of segment-selective visualization and also provides an opportunity for high-precision analysis of dynamics in complex MDPs. SynopsisSegmental deuteration enabled by high-efficiency multi-step protein ligation, combined with inverse contrast-matching SANS and SAXS, provides structural constraints that improve discrimination of molecular dynamics ensembles of multi-domain proteins. IMPORTANTthis document contains embedded data - to preserve data integrity, please ensure where possible that the IUCr Word tools (available from http://journals.iucr.org/services/docxtemplate/) are installed when editing this document.

biophysics↗

bioRxiv2025

Environmental Contributions to Proton Sharing in Protein Low-Barrier Hydrogen Bonds

Hydrogen bonds (H-bonds) are central to biomolecular structure and dynamics. Although H-bonds are typically characterized by well-defined proton positions, proton delocalization can play a key role in facilitating enzyme catalysis and allostery in some systems. Experimentally locating protons is difficult, hampering the study of the proton mobility in H-bonds. We used neutron crystallography and large quantum mechanics/molecular mechanics-Born Oppenheimer molecular dynamics (QM/MM-BOMD) simulations of human DJ-1 and its bacterial homolog YajL to validate atomic resolution X-ray crystallographic bond length analysis, directly visualizing the shared deuteron in a low-barrier hydrogen bond (LBHB) between Glu14-Asp23 in YajL that is a conventional H-bond in DJ-1. In addition, X-ray bond length analysis of protiated and perdeuterated DJ-1 and YajL shows no significant effect of deuteron substitution on these carboxylic acid-carboxylate H-bonds but does reveal an effect at the active site glutamic acid. Residues in the vicinity of Glu14-Asp23 that might favor LBHB formation in YajL were interrogated by mutagenesis of homologous residues in DJ-1. X-ray bond length analysis and QM/MM-BOMD simulations demonstrate that a distal I21T DJ-1 substitution increases proton delocalization in the Glu-Asp H-bond. In addition, simulations show that the extent of proton mobility in the H-bond influences correlated dimer-spanning motions in YajL and DJ-1. In total, we show that mutations within extended H-bonding networks can modulate proton transfer barriers in carboxylic acid-carboxylate H-bonds, allowing proton delocalization to be engineered using combined bioinformatic, structural, and computational information.

biochemistry↗

bioRxiv2024

The role of Tyr34 in proton-coupled electron transfer of human manganese superoxide dismutase

Human manganese superoxide dismutase (MnSOD) plays a crucial role in controlling levels of reactive oxygen species (ROS) by converting superoxide (O2*-) to molecular oxygen (O2) and hydrogen peroxide (H2O2) with proton-coupled electron transfers (PCETs). The reactivity of human MnSOD is determined by the state of a key catalytic residue, Tyr34, that becomes post-translationally inactivated by nitration in various diseases associated with mitochondrial dysfunction. We previously reported that Tyr34 has an unusual pKa due to its proximity to the Mn metal and undergoes cyclic deprotonation and protonation events to promote the electron transfers of MnSOD. To shed light on the role of Tyr34 MnSOD catalysis, we performed neutron diffraction, X-ray spectroscopy, and quantum chemistry calculations of Tyr34Phe MnSOD in various enzymatic states. The data identifies the contributions of Tyr34 in MnSOD activity that support mitochondrial function and presents a thorough characterization of how a single tyrosine modulates PCET catalysis.

biochemistry↗

bioRxiv2024

Revealing the atomic and electronic mechanism of human manganese superoxide dismutase product inhibition

Human manganese superoxide dismutase (MnSOD) is a crucial oxidoreductase that maintains the vitality of mitochondria by converting O2[bullet]- to O2 and H2O2 with proton-coupled electron transfers (PCETs). Since changes in mitochondrial H2O2 concentrations are capable of stimulating apoptotic signaling pathways, human MnSOD has evolutionarily gained the ability to be highly inhibited by its own product, H2O2. A separate set of PCETs is thought to regulate product inhibition, though mechanisms of PCETs are typically unknown due to difficulties in detecting the protonation states of specific residues that coincide with the electronic state of the redox center. To shed light on the underlying mechanism, we combined neutron diffraction and X-ray absorption spectroscopy of the product-bound, trivalent, and divalent states to reveal the all-atom structures and electronic configuration of the metal. The data identifies the product-inhibited complex for the first time and a PCET mechanism of inhibition is constructed.

biochemistry↗

bioRxiv2022

Structural characterization of an intrinsically disordered protein complex using integrated small-angle neutron scattering and computing

Characterizing the structural ensembles of intrinsically disordered proteins (IDPs) is essential for studying structure-function relationships as conformational dynamics govern proteins biological functions. Due to the notable difference between the neutron scattering lengths of hydrogen and deuterium, selective labeling and contrast matching in small-angle neutron scattering (SANS) becomes an effective tool to study dynamic structures of disordered systems. However, the experimental timescale typically results in measurements averaged over multiple conformations, leaving complex SANS data for disentanglement. We hereby demonstrate an integrated method to elucidate the structural ensemble of a protein complex formed by two IDP domains, the NCBD/ACTR complex, using data from selective labeling SANS experiments, microsecond all-atom molecular dynamics (MD) simulations with four molecular mechanics force fields, and an autoencoder-based deep learning (DL) algorithm. By incorporating structural metrics derived from the SANS experiments as constraints in MD structure classification, we describe a structural ensemble that captures the experimental SANS and, in addition, NMR data better than MD ensembles generated by one single force field. Based on structural similarity, DL reveals three clusters of distinct conformations in the ensemble. Our results demonstrate a new integrated approach for characterizing structural ensembles of IDPs.

biophysics↗

bioRxiv2021

Inhibitor Binding Modulates Protonation States in the Active Site of SARS-CoV-2 Main Protease

The main protease (3CL Mpro) from SARS-CoV-2, the virus that causes COVID-19, is an essential enzyme for viral replication with no human counterpart, making it an attractive drug target. Although drugs have been developed to inhibit the proteases from HIV, hepatitis C and other viruses, no such therapeutic is available to inhibit the main protease of SARS-CoV-2. To directly observe the protonation states in SARS-CoV-2 Mpro and to elucidate their importance in inhibitor binding, we determined the structure of the enzyme in complex with the -ketoamide inhibitor telaprevir using neutron protein crystallography at near-physiological temperature. We compared protonation states in the inhibitor complex with those determined for a ligand-free neutron structure of Mpro. This comparison revealed that three active-site histidine residues (His41, His163 and His164) adapt to ligand binding, altering their protonation states to accommodate binding of telaprevir. We suggest that binding of other -ketoamide inhibitors can lead to the same protonation state changes of the active site histidine residues. Thus, by studying the role of active site protonation changes induced by inhibitors we provide crucial insights to help guide rational drug design, allowing precise tailoring of inhibitors to manipulate the electrostatic environment of SARS-CoV-2 Mpro.

biophysics↗

bioRxiv2020

Direct detection of coupled proton and electron transfers in human manganese superoxide dismutase

Human manganese superoxide dismutase (MnSOD) is a critical oxidoreductase found in the mitochondrial matrix. Concerted proton and electron transfers (CPETs) are used by the enzyme to rid the mitochondria of O2 *-, a precursor to other harmful reactive oxygen and nitrogen species. The mechanisms of CPET-utilizing enzymes are typically unknown due to the difficulties in detecting the protonation states of specific residues and solvent molecules involved in catalysis while controlling the redox state of the enzyme. Here, neutron diffraction of redox-controlled MnSOD crystals revealed the all-atom structures of Mn3+SOD and Mn2+SOD delivering unique data on sites that change protonation state. A novel mechanism is proposed from the direct observation of glutamine deprotonation, the involvement of Tyr and His with altered pKas, and four unusual strong-short hydrogen bonds, including a low barrier hydrogen bond, that change with the oxidation state of the metal. Quantum calculations provide insight into the electronic modulation of the observed structures and the enzymatic mechanism.

biochemistry↗

bioRxiv2020

Protonation states in SARS-CoV-2 main protease mapped by neutron crystallography

The main protease (3CL Mpro) from SARS-CoV-2, the etiological agent of COVID-19, is an essential enzyme for viral replication, possessing an unusual catalytic dyad composed of His41 and Cys145. A long-standing question in the field has been what the protonation states of the ionizable residues in the substrate-binding active site cavity are. Here, we present the room-temperature neutron structure of 3CL Mpro from SARS-CoV-2, which allows direct determination of hydrogen atom positions and, hence, protonation states. The catalytic site natively adopts a zwitterionic reactive state where His41 is doubly protonated and positively charged, and Cys145 is in the negatively charged thiolate state. The neutron structure also identified the protonation states of other amino acid residues, mapping electrical charges and intricate hydrogen bonding networks in the SARS-CoV-2 3CL Mpro active site cavity and dimer interface. This structure highlights the ability of neutron protein crystallography for experimentally determining protonation states at near-physiological temperature - the critical information for structure-assisted and computational drug design.

biochemistry↗