Biology subjects

Goluguri, R. R.

Publications and source records attributed to Goluguri, R. R..

5 recordsLinked to original sources

bioRxiv2026

The Dilated Cardiomyopathy E525K β-Myosin Mutation Causes Hypocontractility in Cardiomyocytes Without Altering Crossbridge Cycling

The cardiac {beta}-myosin (MYH7) mutation E525K was identified in 2012 in a patient with dilated cardiomyopathy (DCM). Work using engineered constructs has shown that this mutation stabilizes the interacting heads motif (IHM) of myosin and increases the ATPase activity of mutant motor S1 heads. However, no measurements have been made in myofilaments or cardiomyocytes to determine its effect on contractile function. Here, we present force and contractile kinetics measurements from induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) engineered for heterozygous expression of E525K. Single-cell contraction for E525K hiPSC-CMs decreased by 65%, and isometric twitch force in engineered heart tissues (EHTs) decreased by 39%. In contrast, maximal Ca2+ activated isometric force in isolated myofibrils increased by 45% and sub-maximal Ca2+ activated force was similar to WT myofibrils. Structural analysis revealed reduced myofibril content (13.7% decrease) and decreased organization (increased z-disk dispersion angle) in E525K cells. We confirmed that E525K S1 myosin has higher actin affinity than WT S1 and elevated ATPase activity. However, there was no change in S1 ADP release rate. There was also no change in either the rate of force development or relaxation in myofibrils, cells, or EHTs. These findings suggest that E525K myosin crossbridge cycling is not altered during loaded contractions. To understand how twitch force of myocytes was reduced but maximal isometric force was increased, we used a spatially explicit sarcomere model. The results were explained by changing three rates: reduced recruitment from the OFF/IHM state, and increased rates of actin binding and Pi release. Additional force deficits in cells and EHTs likely result from the myofibrillar disorganization. This study demonstrates the value of multi-scale analysis and coupled, computational modeling to understand the molecular mechanisms of sarcomere mutations in cardiomyocytes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/733270v2_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1a64b4aorg.highwire.dtl.DTLVardef@98f053org.highwire.dtl.DTLVardef@c9f839org.highwire.dtl.DTLVardef@1c795c6_HPS_FORMAT_FIGEXP M_FIG C_FIG A model for how the E525K mutation impacts contracting myofibrils Here, we show that the E525K mutation impacts contraction in multiple ways: (1) Decreased sarcomere organization in cells and tissues drives decreased force generation. (2) Increased binding affinity of E525K myosin for actin and faster Pi release contributes to increased force generation in isolated myofibrils. (3) Increased IHM stability reduces twitch force. (4) The rate-limiting step of loaded crossbridge cycling, ADP release, is unchanged by the E525K mutation, and the rates of loaded contraction and relaxation are unchanged at all scales of contraction.

biophysics↗

bioRxiv2025

The G256E HCM mutation prolongs relaxation via altered nucleotide handling

Mutations in myosin alter its motor functions in diverse ways by affecting different structural and chemo-mechanical events. Multidisciplinary strategies can be used to understand how varying alterations in motor function converge to common phenotypes like hypercontractility and hypertrophic cardiomyopathy (HCM). Here, we combined molecular dynamics (MD) simulations with protein biochemical and myofibril mechanical analyses to study the HCM-causing myosin variant G256E. MD simulations demonstrated that G256E induces structural changes that increase the work required to displace ADP.Mg2+ from actomysoin complex. Stopped-flow biochemical analysis demonstrated increased ADP affinity for actomyosin and single myofibril mechanics analysis demonstrated increased force generation and reduced ADP sensitivity of the early, slow phase of relaxation. Together, these results demonstrate that slower ADP release from myosin during contraction is a significant contributor to pathological contractile nature of the G256E mutation. This study highlights the importance of detailed chemo-mechanical analysis of mutations associated with hereditary cardiac diseases.

biophysics↗

bioRxiv2024

A FRET assay to quantitate levels of the human β-cardiac myosin interacting heads motif based on its near-atomic resolution structure

In cardiac muscle, many myosin molecules are in a resting or "OFF" state with their catalytic heads in a folded structure known as the interacting heads motif (IHM). Many mutations in the human {beta}-cardiac myosin gene that cause hypertrophic cardiomyopathy (HCM) are thought to destabilize (decrease the population of) the IHM state. The effects of pathogenic mutations on the IHM structural state are often studied using indirect assays, including a single-ATP turnover assay that detects the super-relaxed (SRX) biochemical state of myosin functionally. Here we develop and use a fluorescence resonance energy transfer (FRET) based sensor for direct quantification of the IHM state in solution. The FRET sensor was able to quantify destabilization of the IHM state in solution, induced by (a) increasing salt concentration, (b) altering proximal S2 tail length, or (c) introducing the HCM mutation P710R, as well as stabilization of the IHM state by introducing a dilated cardiomyopathy-causing mutation (E525K). Our FRET sensor conclusively showed that these perturbations indeed alter the structural IHM state. These results establish that the structural IHM state is one of the structural correlates of the biochemical SRX state in solution.

biophysics↗

bioRxiv2024

Hypertrophic cardiomyopathy mutations Y115H and E497D disrupt the folded-back state of human beta-cardiac myosin allosterically

At the molecular level, clinical hypercontractility associated with many hypertrophic cardiomyopathy (HCM)-causing mutations in {beta}-cardiac myosin appears to be driven by their disruptive effect on the energy-conserving, folded-back, super relaxed (SRX) OFF-state of myosin. A pathological increase in force production results from release of heads from this OFF-state, which results in an increase in the number of heads free to interact with actin and produce force. Pathogenic mutations in myosin can conceivably disrupt the OFF-state by (1) directly affecting the intramolecular interfaces stabilizing the folded-back state, or (2) allosterically destabilizing the folded-back state via disruption of diverse conformational states of the myosin motor along its chemomechanical cycle. However, very little is understood about the mutations that fall in the latter group. Here, using recombinant human {beta}-cardiac myosin, we analysed the biomechanical properties of two such HCM-causing mutations, Y115H (in the transducer) and E497D (in the relay helix), neither of which falls in the regions that interact to stabilize myosins folded-back state. We find these mutations have diverse effects on the contractility parameters of myosin, yet the primary hypercontractile change in both cases is the destabilization of the OFF-state of myosin and increased availability of active myosin heads for actin-binding. Experimental data and molecular dynamics simulations indicate that these mutations likely destabilize the pre-powerstroke state of myosin, the conformation the motor adopts in the inactive folded-back state. We propose that destabilization of the folded-back state of myosin, directly and/or allosterically, is the molecular basis of hypercontractility in HCM in a far greater number of pathogenic mutations than currently thought.

biochemistry↗

bioRxiv2023

How to Scan DNA using Promiscuous Recognition and No Sliding Clamp: A Model for Pioneer Transcription Factors

DNA scanning proteins slide on the DNA assisted by a clamping interface and uniquely recognize their cognate sequence motif. The transcription factors that control cell fate in eukaryotes must forgo these elements to gain access to both naked DNA and chromatin, so whether or how they scan DNA is unknown. Here we use single-molecule techniques to investigate DNA scanning by the Engrailed homeodomain (enHD) as paradigm of promiscuous recognition and open DNA interaction. We find that enHD scans DNA as fast and extensively as conventional scanners and 10,000,000 fold faster than expected for a continuous promiscuous slide. Our results indicate that such supercharged scanning involves stochastic alternants between local sequence sweeps of [~]85 bp and very rapid deployments to locations [~]500 bp afar. The scanning mechanism of enHD reveals a strategy perfectly suited for the highly complex environments of eukaryotic cells that might be generally used by pioneer transcription factors. TeaserEukaryotic transcription factors can efficiently scan DNA using a rather special mechanism based on promiscuous recognition.

biophysics↗