Biology subjects

Kurauskas, V.

Publications and source records attributed to Kurauskas, V..

5 recordsLinked to original sources

bioRxiv2025

A highly stable engineered disulfide bond in the dimer interface of E. coli orotate phosphoribosyl transferase

Structural disulfides are very rarely found in cytoplasmic proteins of mesophilic organisms. Nevertheless, disulfides can form in the cytosol if these are stabilized sufficiently by the supporting protein structure. To investigate the redox properties of structural disulfide bonds, we introduced disulfide bonds into the cytosolic enzyme from E. coli, orotate phosphoribosyl transferase (OPRT). Because OPRT is a homodimer the introduction of opposing cysteine residues (R44C and D92C) into separate monomers of the enzyme meant that disulfide bond formation could easily be followed by non-reducing SDS-PAGE. Redox titration experiments in DTT buffers revealed that the designed disulfide bond was exceedingly stable with a redox potential of -314 mV, close to that of dithiothreitol (DTT). This interchain disulfide bond improved global thermostability by 5.9 {degrees}C relative to wild type protein and 21.4 {degrees}C above its reduced form. The catalytic activity of the enzyme in either its thiol or disulfide state was not substantially affected, indicating structural integrity. Combining an inactive subunit with the asymmetric nature of the disulfide bond enabled determining the rates of subunit rearrangement and disulfide formation. The R44C/D92C double mutant resulted in the formation of a symmetric homodimer with two interchain disulfides. However, the global stability of the two-disulfide dimer was not substantially increased relative to the dimer with the single disulfide bond, suggesting that the stabilizing effect was gained by preventing dimer dissociation. Finally, we show that the engineered disulfide bonds are formed to a significant degree in the cytosol of yeast cells, allowing us to determine the redox potential of the cytosol in equilibrium with this substrate to -300 mV. This is slightly lower than that previously determined using a GFP-based sensor, rxYFP in yeast. We find that disulfide bond formation is particularly enhanced in mutants lacking the glutathione reductase, Glr1. Thus, the engineered disulfide provides an alternative method for determining intracellular redox potential in living cells with an extended dynamic range relative to GFP-based sensors.

biochemistry↗

bioRxiv2022

Reassessing the helix bundle crossing model for gating in a non-selective ion channel

A critical part of ion channel function is the ability to open and close in response to stimuli, and thus conduct ions in a regulated fashion. While X-ray diffraction studies of ion channels suggested a general steric gating mechanism located at the helix bundle crossing (HBC), recent functional studies on several channels indicate that the helix bundle crossing is open even in closed, non-conductive channels. Two NaK channel variants were crystallized in very different, open and closed conformations and served as an important model of the HBC gating hypothesis. However, neither of these NaK variants are conductive in liposomes unless phenylalanine 92 is mutated to alanine (F92A). Here we use NMR to probe distances at near-atomic resolution of the two NaK variants in lipid bicelles. We demonstrate that in contrast to the crystal structures, both NaK variants are in a fully open conformation, akin to the well known MthK channel structure were the HBC is widely open. Further inquiry into the gating mechanism suggests that the selectivity filter and pore helix are coupled to the M2 helix below and undergo changes in structure when F92 is mutated. Overall, our data shows that NaK exhibits coupling between the selectivity filter and HBC similar to K+ channels and has a more complex gating mechanism than previously thought.

biochemistry↗

bioRxiv2021

Ion-dependent structure, dynamics, and allosteric coupling in a non-selective cation channel

The selectivity filter (SF) determines which ions are efficiently conducted through ion channel pores. NaK is a non-selective cation channel that conducts Na+ and K+ with equal efficiency. Crystal structures of NaK suggested a rigid SF structure, but later solid-state NMR and MD simulations questioned this interpretation. Here, we use solution NMR to characterize how bound Na+ vs. K+ affects NaK SF structure and dynamics. We find that the extracellular end of the SF is flexible on the ps-ns timescale regardless of bound ion. On a slower timescale, we observe a structural change between the Na+ and K+-bound states, accompanied by increased structural heterogeneity in Na+. We also show direct evidence that the SF structure is communicated to the pore via I88 on the M2 helix. These results support a dynamic SF with multiple conformations involved in non-selective conduction. Our data also demonstrate allosteric coupling between the SF and pore-lining helices in a non-selective cation channel that is analogous to the allosteric coupling previously demonstrated for K+-selective channels, supporting the generality of this model.

biophysics↗

bioRxiv2018

Dynamics and interactions of ADP/ATP transporter AAC3 in DPC detergent are not functionally relevant

A recent study1 used solution-state NMR spectroscopy to examine the interactions and dynamics of the yeast mitochondrial inner-membrane ADP/ATP carrier, yAAC3. Crystal structures of different AACs, including yAAC3, in a conformation locked with a strong inhibitor (CATR) had been determined before. This putative \"c-state\"2,3 is believed to represent one extreme conformation of an alternating access mechanism, which involves a further and yet elusive second state termed \"m-state\". Characterizing the dynamics between these states is of paramount importance to understand the transport mechanism. The authors refolded yAAC3 from inclusion bodies in the detergent dodecylphosphocholine (DPC), and observed micro-to-millisecond (s-ms) motion in part of yAAC3 using CPMG NMR experiments. The authors propose that this asymmetrically distributed dynamics, involving residues located in thr ...

biophysics↗

bioRxiv2017

Slow conformational exchange and overall rocking motion in ubiquitin protein crystals

Proteins perform their functions in solution but their structures are most frequently studied inside crystals. Here we probe how the crystal packing alters microsecond dynamics, using solid-state NMR measurements and multi-microsecond MD simulations of different crystal forms of ubiquitin. In particular, NEar-Rotary-resonance Relaxation Dispersion (NERRD) experiments probe angular backbone motion, while Bloch-McConnell Relaxation Dispersion data report on fluctuations of the local electronic environment. These experiments and simulations reveal that the packing of the protein can significantly alter the thermodynamics and kinetics of local conformational exchange. Moreover, we report small-amplitude reorientational motion of protein molecules in the crystal lattice with a [~]3-5{degrees} amplitude on a tens-of-microseconds time scale in one of the crystals, but not in others. An intriguing possibility arises that overall motion is to some extent coupled to local dynamics. Our study highlights the importance of considering the packing when analyzing dynamics of crystalline proteins.

biophysics↗