Biophysical Society Conference | Tahoe 2023

Conferences

Proton Reactions: From Basic Science to Biomedical Applications Tahoe, California | Granlibakken | August 20–24, 2023

Organizing Committee

Ana Nicoleta Bondar, University of Bucharest, Romania Thomas DeCoursey, Rush University Medical Center, USA

Thank You to Our Sponsors

Thank you to all sponsors for their support.

Proton Reactions: From Basic Science to Biomedical Applications

Meeting Code of Conduct

July 2023

Dear Colleagues, We welcome you to the Biophysical Society Conference on Proton Reactions: From Basic Science to Biomedical Applications. This conference series is an opportunity for scientists from around the world to gather and exchange ideas. For many, this will be one of the first conferences since before the pandemic and we want to acknowledge the impact of how long we have not had the ability to congregate, present, and engage in science in person and at a destination. We strongly hope that the meeting will not only provide a venue for sharing recent and exciting progress, but also to promote fruitful discussions and to foster future collaborations in the search of our molecular understanding of membranes. We have assembled an exciting program, with talks focusing on different aspects related to the properties of protons and proton transporting molecules, and their role in biological processes. We have organized a program with attendees from different fields and countries, promising a truly international and multidisciplinary inspiring environment. Our meeting location, Granlibakken, is in the beautiful Tahoe City, California with lots of outdoor activities. The meeting site has activities focused on adventure, family fun, and relaxation. We hope that you take advantage and enjoy the Treetop Adventure Park, tennis courts, hiking trails, bike paths, spa, and any of the other opportunities in this wonderful meeting site. Thank you all for engaging in the program of this meeting, and we look forward to enjoying biophysics with all of you in Tahoe!

Sincerely,

Ana-Nicoleta Bondar and Tom DeCoursey Co-Chairs, Proton Reactions: From Basic Science to Biomedical Applications

Proton Reactions: From Basic Science to Biomedical Applications

Meeting Code of Conduct

Biophysical Society Code of Conduct, Anti-Harassment Policy The Biophysical Society (BPS) is committed to providing an environment that encourages the free expression and exchange of scientific ideas. As a global, professional Society, the BPS is committed to the philosophy of equal opportunity and respectful treatment for all, regardless of national or ethnic origin, religion or religious belief, gender, gender identity or expression, race, color, age, marital status, sexual orientation, disabilities, veteran status, or any other reason not related to scientific merit. All BPS meetings and BPS-sponsored activities promote an environment that is free of inappropriate behavior and harassment by or toward all attendees and participants of Society events, including speakers, organizers, students, guests, media, exhibitors, staff, vendors, and other suppliers. BPS expects anyone associated with an official BPS-sponsored event to respect the rules and policies of the Society, the venue, the hotels, and the city. Definition of Harassment The term “harassment” includes but is not limited to epithets, unwelcome slurs, jokes, or verbal, graphic or physical conduct relating to an individual’s race, color, religious creed, sex, national origin, ancestry, citizenship status, age, gender or sexual orientation that denigrate or show hostility or aversion toward an individual or group. Sexual harassment refers to unwelcome sexual advances, requests for sexual favors, and other verbal or physical conduct of a sexual nature. Behavior and language that are welcome/acceptable to one person may be unwelcome/offensive to another. Consequently, individuals must use discretion to ensure that their words and actions communicate respect for others. This is especially important for those in positions of authority since individuals with lower rank or status may be reluctant to express their objections or discomfort regarding unwelcome behavior. It does not refer to occasional compliments of a socially acceptable nature. It refers to behavior that is not welcome, is personally offensive, debilitates morale, and therefore, interferes with work effectiveness. The following are examples of behavior that, when unwelcome, may constitute sexual harassment: sexual flirtations, advances, or propositions; verbal comments or physical actions of a sexual nature; sexually degrading words used to describe an individual; a display of sexually suggestive objects or pictures; sexually explicit jokes; unnecessary touching. Attendees or participants who are asked to stop engaging in harassing behavior are expected to comply immediately. Anyone who feels harassed is encouraged to immediately inform the alleged harasser that the behavior is unwelcome. In many instances, the person is unaware that their conduct is offensive and when so advised can easily and willingly correct the conduct so that it does not reoccur. Anyone who feels harassed is NOT REQUIRED to address the person believed guilty of inappropriate treatment. If the informal discussion with the alleged harasser is unsuccessful in remedying the problem or if the complainant does not feel comfortable with such an approach, they can report the behavior as detailed below. Reported or suspected occurrences of harassment will be promptly and thoroughly investigated. Following an investigation, BPS will immediately take any necessary and appropriate action. BPS will not permit or condone any acts of retaliation against anyone who files harassment complaints or cooperates in the investigation of same. Reporting a Violation Violations of this Conduct Policy should be reported immediately. If you feel physically unsafe or believe a crime has been committed, you should report it to the police immediately. To report a violation to BPS:

• You may do so in person at the Annual Meeting at the BPS Business Office in the convention center.

Proton Reactions: From Basic Science to Biomedical Applications

Meeting Code of Conduct

• You may do so in person to BPS senior staff at Thematic Meetings, BPS Conferences, or other BPS events.

• At any time (during or after an event), you can make a report through

http://biophysics.ethicspoint.com or via a dedicated hotline (phone numbers listed on the website) which will collect and relay information in a secure and sensitive manner.

Reported or suspected occurrences of harassment will be promptly and thoroughly investigated per the procedure detailed below. Following an investigation, BPS will immediately take any necessary and appropriate action. BPS will not permit or condone any acts of retaliation against anyone who files harassment complaints or cooperates in the investigation of same. Investigative Procedure All reports of harassment or sexual harassment will be treated seriously. However, absolute confidentiality cannot be promised nor can it be assured. BPS will conduct an investigation of any complaint of harassment or sexual harassment, which may require limited disclosure of pertinent information to certain parties, including the alleged harasser. Once a complaint of harassment or sexual harassment is received, BPS will begin a prompt and thorough investigation. Please note, if a complaint is filed anonymously, BPS may be severely limited in our ability to follow-up on the allegation. • An impartial investigative committee, consisting of the current President, President-Elect, and Executive Officer will be established. If any of these individuals were to be named in an allegation, they would be excluded from the committee. • The committee will interview the complainant and review the written complaint. If no written complaint exists, one will be requested. • The committee will speak to the alleged offender and present the complaint. • The alleged offender will be given the opportunity to address the complaint, with sufficient time to respond to the evidence and bring his/her own evidence. • If the facts are in dispute, the investigative team may need to interview anyone named as witnesses. • The investigative committee may seek BPS Counsel’s advice. • Once the investigation is complete, the committee will report their findings and make recommendations to the Society Officers. • If the severity of the allegation is high, is a possible repeat offense, or is determined to be beyond BPS’s capacity to assess claims and views on either side, BPS may refer the case to the alleged offender’s home institution (Office of Research Integrity of similar), employer, licensing board, or law enforcement for their investigation and decision. Disciplinary Actions Individuals engaging in behavior prohibited by this policy as well as those making allegations of harassment in bad faith will be subject to disciplinary action. Such actions range from a written warning to ejection from the meeting or activity in question without refund of registration fees, being banned from participating in future Society meetings or Society-sponsored activities, being expelled from membership in the Society, and reporting the behavior to their employer or calling the authorities. In the event that the individual is dissatisfied with the results of the investigation, they may appeal to the President of the Society. Any questions regarding this policy should be directed to the BPS Executive Officer or other Society Officer.

Proton Reactions: From Basic Science to Biomedical Applications

Table of Contents

Table of Contents

General Information……………………………………………………………………………....1 Program Schedule..……………………………………………………………………………….3 Speaker Abstracts………………………………………………………………………………...9 Poster Sessions…………………………………………………………………………………...45

Proton Reactions: From Basic Science to Biomedical Applications

General Information

GENERAL INFORMATION

Registration/Information Location and Hours On Sunday and Monday venue check-in to obtain your room key will be located at the Main Lodge Front Desk at Granlibakken Tahoe, 725 Granlibakken Road, Tahoe City, CA 96145. An Information Desk to pick up meeting materials will be located at the Ballroom Pre-Function at the following times: Sunday, August 20 3:30 PM – 6:30 PM Monday, August 21 8:00 AM - 6:30 PM Tuesday, August 22 8:00 AM - 2:00 PM Wednesday, August 23 8:00 AM - 5:00 PM Instructions for Presentations (1) Presentation Facilities: A data projector will be available in the Ballroom. Speakers are required to bring their own laptops and adaptors. It is recommended to have a backup of the presentation on a USB drive in case of any unforeseen circumstances. Speakers are advised to preview their final presentations before the start of each session. (2) Poster Session: 1) All poster sessions will be held in the Ballroom. 2) A display board measuring 243 cm wide x 121 cm high (8 feet wide x 4 feet high) will be provided for each poster. Poster boards are numbered according to the same numbering scheme as listed in the e-book. 3) Poster boards require pushpins or thumbtacks for mounting. Authors are expected to bring their own mounting materials. 4) There will be formal poster presentations on Monday and Tuesday. Posters will be available for viewing during their scheduled presentation date only. Presenting authors with odd-numbered poster boards should present from 8:00 PM – 8:30 PM and those with even-numbered poster boards should present from 8:30 PM – 9:00 PM. 5) During the assigned poster presentation sessions, presenters are requested to remain in front of their poster boards to meet with attendees. 6) All posters left uncollected at the end of the meeting will be disposed.

1

Proton Reactions: From Basic Science to Biomedical Applications

General Information

Note Pads/Pens Pens will be available, however please bring your own notepad. Covid Precautions

While there are no formal covid protocols, it is suggested that you should test in advance, stay home if you are ill, and isolate and test if you experience symptoms at the meeting. Masks are not mandatory but encouraged for all indoor spaces. Meals and Coffee Breaks Breakfast, Lunch, and Dinner will be served at the Granhall. Coffee Breaks will be held at the Ballroom Pre-Function area. Smoking Please be advised that smoking is not permitted at Granlibakken Tahoe. Name Badges Name badges will be given to you when you arrive at the check-in desk to receive your room keys. Badges are required to enter all scientific sessions, poster sessions, and social functions. Please wear your badge throughout the conference. Internet Wifi will be provided at the venue. Contact If you have any further requirements during the meeting, please contact the meeting staff at the registration desk from August 20-23 during registration hours. In case of emergency, you may contact the following: Dorothy Chaconas Phone : 301-785-0802 Email: dchaconas@biophysics.org Adam Vincent Email: adamvincent@granlibakken.com

2

Proton Reactions: From Basic Science to Biomedical Applications

Daily Schedule

Proton Reactions: From Basic Science to Biomedical Applications Tahoe, California August 20-24, 2023 All scientific sessions will be held in the Mountain Ballroom unless otherwise noted. PROGRAM

Sunday, August 20, 2023

3:30 PM – 6 :30 PM

Registration/Information

Ballroom Pre-Function

6:30 PM – 8:00 PM

Dinner

Granhall/Garden Deck

Session I

Protons in Physics and Biology Co-Chairs: Ana-Nicoleta, Bondar, University of Bucharest, Romania Thomas Decoursey, Rush University Medical Center, USA

8:00 PM – 8:10 PM

Welcome and Opening Remarks

8:10 PM – 8:40 PM

Keynote Lecture Robert Gennis, University of Illinois at Urbana-Champaign, USA Structure and Proposed Mechanism of the Nicotinamide Nucleotide Transhydrogenase from E. Coli. Keynote Lecture Gregory Voth, University of Chicago, USA Proton Transport in Biomolecular Systems: A Remarkably Complex and Collective Phenomenon

8:40 PM – 9:20 PM

9:20 PM – 10:00 PM

Keynote Lecture Menachem Gutman, Tel Aviv University, Israel The Many-Faces Nature of Proton Transfer Reactions

Monday, August 21, 2023 7:00 AM – 8:30 AM

Breakfast

Granhall

8:00 AM – 6:30 PM

Registration/Information

Ballroom Pre-Function

Session II

Molecular Ion Transporters Chair: Petra Hellwig, University of Strasbourg, France

9:00 AM – 9:30 AM

Jessica Swanson, University of Utah, USA Kinetic Selection in Transporters: Teasing Out the Roles of the Electrical and Chemical Gradients Petra Hellwig, University of Strasbourg, France Electrocatalytic and Spectroscopic Studies on Cytochrome C Oxidase, a Highly Diverse Bacterial Membrane Protein

9:30 AM – 10:00 AM

3

Proton Reactions: From Basic Science to Biomedical Applications

Daily Schedule

10:00 AM – 10:30 AM

Keiichi Inoue, University of Tokyo, Japan The Role of Proton Transfer in Ion-Transporting Microbial Rhodopsins

10:30 AM – 10:50 AM

Coffee Break

Ballroom Pre-Function

Session III

The Hv1 Proton Channel Chair: Tom DeCoursey, Rush University Medical Center, USA Carlos L. Gonzalez, University of Miami, USA * Molecular Determinants of Voltage Sensor in CIHv1 Channel Francesco Tombola, University of California, Irvine, USA Pulling the Strings of Proton Conduction: Mechanical Regulation of Voltage-Gated Proton Channels Jana Shen, University of Maryland, USA Mechanism of pH Sensing in the Human Voltage-Gated Proton Channel Hv1 Ian Ramsey, Virginia Commonwealth University, USA Insights into the Structural Basis of pH-Dependent Gating and H + Permeation Mechanisms in the Hv1 Voltage-Gated Proton Channel Reactions at Membrane Interfaces Chair: Marylin Gunner, City College of New York, USA Balarama Sridhar Dwadasi, University of Calgary, Canada * Constant pH Molecular Dynamics Simulations of the Anion Exchanger-1 (AE1) Karen Fleming, Johns Hopkins University, USA Membrane Protein Thermodynamic Measurements Enabled by Protons Ana-Nicoleta Bondar, University of Bucharest, Romania Dynamic Hydrogen-Bond Networks for Proton Binding and Proton Transfer at Bio-Membrane Interfaces Thomas DeCoursey, Rush University Medical Center, USA Deciphering Apparent Cholesterol Effects on Voltage-Gated Proton Channels Lunch

10:50 AM – 11:00 AM

11:00AM – 11:30 AM

11:30 AM – 12:00 PM

12:00 PM – 12:30 PM

12:30 PM – 1:30 PM

Granhall/Garden Deck

Session IV

1:50 PM – 2:00 PM

2:00 PM – 2:30 PM

2:30 PM – 3:00 PM

3:00 PM – 3:30 PM

3:30 PM – 4:00 PM

Coffee Break

Ballroom Pre-Function

Session V

Mechanisms and Evolutionary Diversity of Molecular Proton Transporters Chair: Keiichi Inoue, Tokyo University, Japan

4:00 PM – 4:30 PM

Boris Musset, University of Nürenberg, Germany The Voltage-Gated Proton Channel Discovers its Family Marilyn Gunner, City College of New York, USA How the Multiplicity of Protonation States in Proteins Supports Proton Transfers

4:30 PM – 5:00 PM

4

Proton Reactions: From Basic Science to Biomedical Applications

Daily Schedule

Session VI

Proton Transfer Reactions at Heterogeneous Interfaces Chair: Alexei Stuchebrukhov, University of California, Davis, USA

5:00 PM – 5:30 PM

Paul Champion, Northeastern University, USA * Quantum Mechanical Tunneling in Proton Wires

5:30 PM – 5:50 PM

Florian Brünig, Freie Universität Berlin, Germany (Young Investigator Talk) Spectral Signatures of Excess-Proton Waiting and Transfer-Path Dynamics Nadav Amdursky, Israel Institute of Technology, Israel New Insights on Proton Transport in Biosystems Using a Light- Triggered Proton Donor and Solid-State Protonic Devices

5:50 PM – 6:20 PM

6:30 PM – 8:00 PM

Dinner

Granhall/Garden Deck

8:00 PM – 9:00 PM

Poster Session I

Tuesday, August 22, 2023 7:00 AM – 8:30 AM

Breakfast

Granhall

8:00 AM – 2:00 PM

Registration/Information

Ballroom Pre-Fucntion

Session VII

pH- and Voltage-Dependent Proton Reactions at Membrane Interfaces Chair: Kalina Hristova, Johns Hopkins University, USA Zhi Yue, University of Chicago, USA * Conformational Changes and the Role of Internal Glutamate in the C1-H+ Antiporters

8:50 AM – 9:00 AM

9:00 AM – 9:30 AM

William Wimley, Tulane University, USA Peptide Nanopores are Stabilized by a Cooperative Hydrogen-Bond Network Yasushi Okamura, Osaka University, Japan Activities of Voltage-Gated Proton Channels on Intracellular Membranes

9:30 AM – 10:00 AM

10:00 AM – 10:30 AM

Peter Pohl, University of Linz, Austria Interfacial Proton Diffusion

10:30 AM – 11:00 AM

Coffee Break

Ballroom Pre-Function

Session VI II

Regulation of pH Dynamics in Cells, and pH-Regulated Membrane Reactions Chair: Helmut Grubmüller, Max Plank Institute, Germany

11:00 AM – 11:30 AM

Kalina Hristova, Johns Hopkins University, USA Charge-Induced Bias in EGFR Signal Transduction Across the Plasma Membrane Diane Barber, University of California, San Francisco, USA How Intracellular pH Dynamics Regulate Cell Behaviors

11:30 AM – 12:00 PM

5

Proton Reactions: From Basic Science to Biomedical Applications

Daily Schedule

12:00 PM – 12:10 PM

Ulrike Alexiev, Freie Universitäte Berlin, Germany * From Proton Reactions to Advanced Biophysical Methods for Biomedical Application Daniel Kortzak, Forschungszentrum Jülich, Germany * Molecular Dynamics Simulations of a Vescular Gutamate Transporter in Different Protonation States Chenghan Li, University of Chicago, USA * Insight into the Quantitative Mechanism of Ion Selectivity of the Human Voltage-Gated Proton Channel Hv1 Proton Reactions in the Time of Exascale Computing Chair: Jana Shen, University of Maryland, USA Sharon Hammes Schiffer, Yale University, USA Proton-Coupled Electron Transfer in Proteins Helmut Grubmüller, Max Plank Institute, Germany Biomolecular Simulations at Constant pH with GPU-Accelerated Fast Multipoles Electrostatics Juergen Kreiter and Amy Nava, Stanford University, USA * Cryo-EM, Molecular Dynamics Simulations and Functional Assays Reveal Unanticipated Insights into the Chloride/Proton Antiport Mechanism of CLC-EC1 Lunch Free Time

12:10 PM – 12:20 PM

12:20 PM – 12:30 PM

12 :30 PM – 2:00 PM

Granhall/Garden Deck

2:00 PM – 5 :00 PM

Session IX

5:00 PM – 5:30 PM

5:30 PM – 6:00 PM

6:00 PM – 6:20 PM

6:30 PM – 8:00 PM

Dinner

Granhall/Garden Deck

Poster Session II

8:00 PM – 9:00 PM

9:00 PM – 10:00 PM

Get Together

Wednesday, August 23, 2023

7:00 AM – 8:30 AM

Breakfast

Granhall

8:00 AM – 5:00 PM

Registration/Information

Ballroom Pre-Fucntion

Session X

Water in Membrane Reactions Chair: Leonid Brown, University of Guelph, Canada

8:50 AM – 9:00 AM

Valeria Baranauskaite, Ben Gurion University of Negev, Israel * Exploring Protonation Reactions and Stability of Intact Carbonic Acid in Alcohol Solvent Mixtures Leonid Brown, University of Guelph, Canada Conserved Hydrogen-Bond Motifs as Hubs of Stability for Human Water Channels

9:00 AM – 9:30 AM

6

Proton Reactions: From Basic Science to Biomedical Applications

Daily Schedule

9:30 AM – 10:00 AM

Gebhard Schertler, Paul Scherer Institute and ETH Zurich, Switzerland The Role of Water Molecules in the Activation Mechanism of Retinal- Binding Membrane Proteins and G Protein-Coupled Receptors Paolo Carloni, Forschungszentrum Jülich, Germany Massively Parallel QM/M Simulations of Proton Transfer Processes in Different Environment

10:00 AM – 10:30 AM

10:30 AM – 10:50 AM

Coffee Break

Ballroom Pre-Function

Session XI

Protons in Cell Physiology and Disease Chair: Paolo Carloni, Forschungszentrum Jülich, Germany

10:50 AM – 11:00 AM

Bhav Kapur, Böhringer Ingelheim, Germany * Protons Taken Hostage

11:00 AM – 11:30 AM

Polina Lishko, University of California, Berkley, USA Protons in Reproduction, Contraception, and Heat Production Mark D. Parker, University of Buffalo, USA The Role of NH3 Promoting H+/OH- Flux Through the Alkali-Activated Channel Protein SLC4A11

11:30 AM – 12:00 PM

12:00 PM – 12:30 PM

Elena Pohl, University of Vienna, Austria Mitochondrial ATP/ADP Carrier - Proton or Fatty Acid Anion Transporter?

12:30 PM – 12:50 PM

Group Photo

12:50 PM – 2:00 PM

Lunch

Granhall/Garden Deck

Session XII

pH Sensing and Pharmacology Chair: Diana Barber, University of California, San Francisco, USA

2:00 PM – 2:30 PM

Dan Isom, University of Miami, USA Proton Gating of GPCR Signaling

2:30 PM – 3:00 PM

Christoph Stein, Charité Berlin, Germany Biophysics Meets Medicine: pH-Dependent Opioid Pain Killers

3:00 PM – 3:20 PM

Kota Katayama, Nagoya Institute of Technology, Japan (Young Investigator Talk) Photoactivation Mechanism of Cone Opsins Regulated by Proton Transfer Reaction

3:20 PM – 3:50 PM

Coffee Break

Ballroom Pre-Function

Session XIII

Gates and Protons Chair: Kota Katayama, Nagoya Institute of Technology, Japan Gustavo Chavez, KNMS, Paracelsus Medical University, Germany * Biophysical Properties of Newly Found Voltage-Gated Proton Channels Christophe Jardin, KNMS, Paracelsus Medical University, Germany * Learning About Properties of the Voltage-Gated Proton Channel From in Silico Methods

3:50 PM – 4:00 PM

4:00 PM – 4:10 PM

7

Proton Reactions: From Basic Science to Biomedical Applications

Daily Schedule

4:10 PM – 4:30 PM

Shizhen Wang, University of Missouri-Kansas City, USA (Young Investigator Talk) Mechanisms of Ligand Regulation in Human Voltage-Gated Proton Channel HHV1

4:30 PM – 5:00 PM

Emily Liman, University of Southern California, USA Gating Mechanisms of OTOP Proton Channels

6:00 PM – 7:30 PM

Dinner

Granhall/Garden Deck

Session XIV

Protons in Biology, Material Sciences and Medicine: What the Future Holds Chair: Gebhard Schertler, Paul Scherer Institute and ETH Zürich, Switzerland

7:30 PM – 8:00 PM

Peter Tieleman, University of Calgary, Canada Transport Mechanisms of SLC4 Anion Transporters Alexey Stuchebrukhov, University of California, Davis, USA Proton Transport in Biosystems

8:00 PM – 8:30 PM

8:30 PM – 8:45 PM Closing Remarks *Contributed talks selected from among submitted abstracts

8

Proton Reactions: From Basic Science to Biomedical Applications

Speaker Abstracts

SPEAKER ABSTRACTS

9

Proton Reactions: From Basic Science to Biomedical Applications

Sunday Speaker Abstracts

STRUCTURE AND PROPOSED MECHANISM OF THE NICOTINAMIDE NUCLEOTIDE TRANSHYDROGENASE FROM E. COLI Robert B. Gennis 3 ; Jiao Li 1,2 ; Jonathan Zoller 4 ; Lici Schurig-Briccio 3 ; Sangjin Hong 3 ; Martin Eisinger 5 ; Malcolm Anderson 6 ; Melanie Radloff 5 ; kristina desch 4 ; Kai Zhang 2 ; Julian Langer 4 ; Jiapeng Zhu 1 ; 1 Nanjing University, School of Medicine and Integrative Holistic Medicine, Nanjing, China 2 Yale University, Department of Molecular Biophysics and Biochemistry, New Haven, CT, USA 3 University of Illinois at Urbana-Champaign, Biochemistry, Urbana, IL, USA 4 Max Planck Institute of Biophysics, Proteomics, Frankfurt am Main, Germany 5 Max Planck Institute of Biophysics, Molecular Membrane Biology, Frankfurt am Main, Germany 6 Waters Corporation, Winslow, United Kingdom The nicotinamide nucleotide transhydrogenase is present in the mitochondrial inner membrane and in the cytoplasmic membranes of many bacteria. The enzyme couples the proton motive force across the membrane to the hydride transfer between NAD(H) and NADP(H). Under most circumstances the enzyme generates NADPH and for each NADPH formed, one proton is translocated across the membrane from the outside (electrically positive) to the inside (electrically negative). The enzyme consists of three domains: domain I binds NAD(H); domain III binds NADP(H); and domain II which contains multiple transmembrane helices and a proton channel. In this work, the structure of the transhydrogenase from E. coli has been determined by cryo-electron microscopy, revealing major conformational changes depending on the presence/absence of nucleotides. These conformational changes were further characterized using hydrogen-deuterium exchange with mass spectrometry (HDX-MS). Results show coupling of the conformation of the helices comprising the transmembrane proton channel in domain II to the binding of either NADPH or NADP+ to domain III. The data are generally consistent with the mechanism previously proposed by Kampjut and Sazanov [Nature (2019) 573, 291] based on studies of the ovine transhydrogenase. The proton channel contains a single protonatable histidine which can be exposed either to the periplasmic side or the cytoplasmic side of the membrane. The side to which the histidine is exposed as well as its pK a is determined by whether the reactant (NADP+) or product (NADPH) is bound to domain III. The transhydrogenase functions essentially as a ligand-gated proton transporter.

10

Proton Reactions: From Basic Science to Biomedical Applications

Sunday Speaker Abstracts

PROTON TRANSPORT IN BIOMOLECULAR SYSTEMS: A REMARKABLY COMPLEX AND COLLECTIVE PHENOMENON Gregory A Voth 1,2,3 ; 1 The University of Chicago, Department of Chemistry, Chicago, IL, USA 2 The University of Chicago, Chicago Center for Theoretical Chemistry, Chicago, IL, USA 3 The University of Chicago, Institute for Biophysical Dynamics, Chicago, IL, USA The hydrated excess proton (aka "hydronium cation") is critical in many areas of chemistry, biology, and materials science. Despite playing a central role in fundamental chemical (e.g., acid–base) and biological (e.g., bioenergetics) processes, the nature of the excess proton remains mysterious, surprising, and sometimes misunderstood. In this presentation, our longstanding efforts to characterize proton solvation and transport in biomolecular systems will be described. These studies employ a novel, accurate, and computationally efficient multiscale reactive molecular dynamics method combined with large scale computer simulation. The methodology allows for the treatment of explicit (Grotthuss) proton shuttling and charge defect delocalization, which strongly influences proton solvation and transport in proteins such as transmembrane proton channels, pumps, and transporters. The unique electrostatics related to the dynamic delocalization of the excess proton charge defect in water chains and amino acid residues will be elaborated, as well as the effects of these complex electrostatics on the proton transport and selectivity properties. The often opposing and asymptotic viewpoints related to electrostatics on one hand and Grotthuss proton shuttling on the other will be reconciled and unified into a single conceptual framework. The intrinsically coupled nature of the excess proton translocation and the water hydration can also be elaborated through these computer simulations. It is found that a prior existing "water wire" is not necessary for excess protons to transport through hydrophobic spaces in proteins via water mediated Grotthuss shuttling. The proton translocation process can sometimes create its own transient water wire as needed and can be quantified via the definition of a proper collective variable. Specific simulation results will be given for proton transport in the SERCA calcium pump, the ClC Cl – /H + antiporter, the PiPT phosphate transporter, and the Hv1 voltage gated proton channel, as time allows, along with a comparison to experimental results.

11

Proton Reactions: From Basic Science to Biomedical Applications

Sunday Speaker Abstracts

THE MANY-FACES NATURE OF PROTON TRANSFER REACTIONS  M. GUTMAN AND ESTHER NACHLIEL LASER LABORATORY FOR FAST REACTIONS BIOCHEMISTRY TEL AVIV UNIVERSITY. TEL AVIV ISRAEL Menachem Gutman ; menachem gutman 1 ; 1 Tel Aviv university, biochemistry, Tel Aviv, Israel In 1979 we published a manuscript titled “Rapid pH and deltamuH+ jump by short laser pulse” describing how pulse excitation of naphthol derivatives can perturb the acid-base equilibrium of aqueous solutions and even build a proton motive force across a lipid membrane. Now, with the hindsight of ~40 years, we can evaluate the various aspects and significance of the photo-ejection of protons from excited molecules. The most initial event is the discharge of the proton to the solvent, a reaction that reflects the nature of the excited molecule ΦOH* and the composition of the solvent. The time constant of the proton ejection depends on the pK* of the excited molecule and the availability of the acceptor. Once the proton was released into the solvent, it can ei ther recombine with the parent molecule ΦO -* or diffuse to the bulk. The recombination reaction is observed as a slowdown of the ΦOH* emission decay -reflecting the re-protonation of ΦO -*. This reaction takes place in the ps-ns time frame. The quantitative analysis reflects the reaction space's geometry, diffusion coefficient, ionic strength, and the dielectric constant of the immediate environment. Once the perturbation expands beyond the Coulomb Cage (ns-µs), the system is in a state of acid-base disequilibrium. Equilibrium is regained by a large number of parallel diffusion-controlled reactions among all reactants present in the system. A set of differential rate equations that includes all possible interactions among all components of the system, together with the Genetic Algorithm assures the uniqueness of the derived rate constants. This analysis yields the rate constants of all first and second-order reactions that participate in the perturbed system.

12

Proton Reactions: From Basic Science to Biomedical Applications

Monday Speaker Abstracts

KINETIC SELECTION IN TRANSPORTERS: TEASING OUT THE ROLES OF THE ELECTRICAL AND CHEMICAL GRADIENTS Jessica Swanson University of Utah, USA No Abstract

ELECTROCATALYTIC AND SPECTROSCOPIC STUDIES ON CYTOCHROME BD OXIDASE, A HIGHLY DIVERSE BACTERIAL MEMBRANE PROTEN Iryna Makarchuk 1 ; Jan Kägi 2 ; Frédéric Melin 1 ; Thorsten Friedrich 2 ; Petra Hellwig ; 1 University of Strasbourg, UMR 7140 , Strasbourg, France 2 University of Freiburg, Institute for Biochemistry, Freiburg im Breisgau, Germany The selective reduction of oxygen to water is crucial to life and a central process in aerobic organisms. It is catalyzed by several different enzymes, including cytochrome bd oxidases that are solely present in prokaryotes, including several pathogens. In addition, these enzymes play a crucial role in protection against oxidative stress, in virulence, adaptability and antibiotics resistance. The reduction of O 2 occurs at the high spin D-type heme in all cytochrome bd oxidases, that is also the binding site for several ligands from signaling processes, including NO, H 2 S and CO. Here we present the electrocatalytic study of the cytochrome bd I and bd II oxidases from Escherichia coli as well on other related bd oxidases. Structural parameters that are crucial for the reactivity towards oxygen are analyzed. The pH dependency of the binding and release of NO, an important signaling factor is presented. The influence of mutants in the proton channel on the NO release is discussed.

13

Proton Reactions: From Basic Science to Biomedical Applications

Monday Speaker Abstracts

THE ROLE OF PROTON TRANSFER IN ION-TRANSPORTING MICROBIAL RHODOPSINS Keiichi Inoue 1 ; 1 The University of Tokyo, The Institute for Solid State Physics, Kashiwa, Japan Microbial rhodopsins are a superfamily of photoreceptive membrane proteins consisting of a seven transmembrane-helical architecture and a retinal chromophore. While microbial rhodopsins exhibit diverse biological functions in a light-dependent manner, ion channel and ion pump rhodopsins are widely used in optogenetics to regulate neural activity by light. Despite their importance in optogenetic application, the mechanistic elements essential for pump and channel functions are not completely understood. Here we studied the dynamics of proton transfer and the conformation change of the retinal chromophore by laser flash photolysis, time resolved Raman spectroscopy, and laser electrophysiology. Microbial rhodopsins exhibit the photocyclic reaction including several photo-intermediates. In contrast to the best characterized outward proton pumping rhodopsin (bacteriorhodopsin), most elementary processes between the respective photo-intermediates of inward proton pumping rhodopsins (xenorhodopsin and schizorhodopsin) were slowed down in D 2 O solvent compared with in H 2 O solvent. These kinetic isotope effects (KIE) indicate that most elementary processes of inward proton pumping rhodopsins are rate-limited by the proton transfer in the protein [1,2]. In contrast, whereas the channel opening of cation (C1C2 and ChRmine) and anion (GtACR1) channelrhodopsins do not show significant KIE, the channel closing rate became slower in D 2 O solvent than in H 2 O solvent [3,4]. Interestingly, the channel opening of C1C2 was induced by the unique twisting of the retinal polyene chain. Our results indicate that the conformation changes critical for the functions of ion-transporting rhodopsins are tightly coupled with intramolecular proton transfer events and artificial modification of their rates would be useful for the development of next generation optogenetic tools. 1. Tahara, S. et al.: J. Phys. Chem. Lett., 6, 4481-4486 (2015).2. Kawasaki, Y. et al.: Biochim. Biophys. Acta Biomembr., 1864, 184016 (2022).3. Shibata, K. et al.: J. Am. Chem. Soc., published on the web, doi:10.1021/jacs.3c01879 (2023).4. Shibata, K. et al.: Manuscript in preparation.

14

Proton Reactions: From Basic Science to Biomedical Applications

Monday Speaker Abstracts

MOLECULAR DETERMINANTS OF VOLTAGE SENSOR IN CIHV1 CHANNEL Carlos L. Gonzalez 1,2,3 ; Miguel Fernandez 2,3 ; Juan Alvear-Arias 2,3 ; Emerson M Carmona 4 ; Josè A Garate 3,5,6 ; 1 University of Miami, Department of Physiology and Biophysics, Miller School of Medicine, Miami, FL, USA 2 Universidad de Valparaíso, Centro Interdisciplinario de Neurociencia de Valparaíso, Valparaiso, Chile 3 Universidad de Valparaíso, Millennium Nucleus in NanoBioPhysics, Valparaiso, Chile 4 Texas Tech University , Health Sciences Center, Lubbock, TX, USA 5 Universidad San Sebastián, acultad de Ingeniería y Tecnología,, Santiago, Chile 6 Centro Científico y Tecnológico de Excelencia Ciencia y Vida, Santiago, Chile The Ciona intestinalis proton channel (Hv1) is a membrane protein with the voltage sensing, pH, and permeation pathway located in the same structural region. In the mutant ΔNΔC -N264R, we observed a reduction of conductance and a fast activation kinetics accompanied by a robust ON gating current and diminished macroscopic proton current. We named the decrease of the OFF gating-charge component upon repolarization as trapping. We used patch-clamp gating currents and molecular dynamics experiments to elucidate the mechanism of voltage sensor movement during channel activation. The trapped charge disappears when selectivity filter (D160) is mutated (D160N) on the ΔNΔC monomeric background, indicating that trapping is not an intrinsic featur e of the channel. However, the double mutant (ΔNΔC -D160N N264R) showed only partial trapping, revealing that electrostatic effects are not the only driving force. Increasing the D160 hydrophobicity increases the trapping phenomenon. Molecular dynamics simulations showed that this effect causes electrostatic repulsion towards the arginines (258 and 261) of the voltage sensor, allowing them to move upwards, which facilitates salt bridge formation with D160, trapping the voltage sensor in its active state. Gating charge trapping can be mimicked by blocking a specific inhibitor (2GBI) or increasing proton concentration (positive ΔpH), indicating that changes in the electrostatic-hydrophobicity environment can induce a delay in the voltage sensor returning to the resting position. In conclusion, the OFF-gating charges delay on the N264R mutant is due to a trapped voltage sensor in the active state during channel activation, where residues D160 and N264 are essential in Hv1 voltage sensor displacement molecular mechanism.

15

Proton Reactions: From Basic Science to Biomedical Applications

Monday Speaker Abstracts

PULLING THE STRINGS OF PROTON CONDUCTION: MECHANICAL REGULATION OF VOLTAGE-GATED PROTON CHANNELS Chang Zhao 1 ; Parker D Webster 1 ; Alexis De Angeli 2 ; Francesco Tombola 1 ; 1 University of California, Irvine, Department of Physiology & Biophysics, Irvine, CA, USA 2 University of Montpellier, CNRS, INRAE, 2IPSiM, Institut Agro, Montpellier, France Swelling-induced potentiation of Hv1-mediated proton current was previously proposed to contribute to brain damage after ischemic stroke through excessive activation of the NADPH oxidase in microglial cells. The mechanism underlying Hv1 potentiation is unknown, but it is believed to involve a transition from a state with normal activity to a state with facilitated activation, induced by the mechanical stimulus. Here, we describe an Hv homolog in the angiosperm plant Arabidopsis thaliana that gates with a unique modality as it is activated by an electrical stimulus if it is first exposed to membrane stretch in a process that we call priming. The homolog from another angiosperm, T. cacao, shares the requirement for mechanical priming, whereas homologs from the non-flowering plants P. sitchensis and S. moellendorffii do not, as they can be activated by the electrical stimulus alone. Guided by AI-generated structural models of plant Hv proteins, we swapped protein regions and individual residues between Hvs from A. thaliana and P. sitchensis, and measured the response of the resulting channels to mechanical stimulation. We identified a set of residues that play a crucial role in mechanical priming and propose that Hvs from angiosperm plants require priming because they contain a network of hydrophilic/charged residues that locks the channels in a silent resting state. The mechanical stimulus destabilizes the network allowing the conduction pathway to turn on. Such network appears to be fragmented, and therefore constitutively destabilized, in Hv channels from non flowering plants. Conformations similar to the silent state of Arabidopsis Hv might exist in animal homologs. These states may be sufficiently stable to inhibit channel activation by membrane depolarization but not stable enough to prevent opening altogether. The mechanical stimulus could then destabilize these states, as it is proposed for Arabidopsis Hv, resulting in a facilitated opening.

16

Proton Reactions: From Basic Science to Biomedical Applications

Monday Speaker Abstracts

MECHANISM OF PH SENSING IN THE HUMAN VOLTAGE-GATED PROTON CHANNEL HV1 Jana Shen 1 ; Yandong Huang 2 ; 1 University of Maryland, Pharmaceutical Sciences, Baltimore, MD, USA 2 Jimei University, Computer Science, Xiamen, China Hv1 is an important model system for understanding voltage-gated proton channels. It has been studied for nearly four decades; however, the mechanism of gating and proton selectivity remains poorly understood. I will discuss our recent findings from the continuous constant pH molecular dynamics simulations based on the hyperpolarized and depolarized structural models of human Hv1. The analysis of conformational dynamics coupled to proton titration offers an explanation of the existing experimental data (e.g., threshold voltage shifts due to the neutralizing mutations), and more importantly, it leads to a hypothesis regarding the pH sensing residues.

17

Proton Reactions: From Basic Science to Biomedical Applications

Monday Speaker Abstracts

INSIGHTS INTO THE STRUCTURAL BASIS OF PH-DEPENDENT GATING AND H* PERMEATION MECHANISMS IN THE HV1 VOLTAGE-GATED PROTON

CHANNEL Ian Ramsey Virginia Commonwealth University, USA No Abstract

CONSTANT PH MOLECULAR DYNAMICS SIMULATIONS OF THE ANION EXCHANGER-1 (AE1) Balarama Sridhar Dwadasi 1 ; Hristina Zhekova 1 ; Sergei Y Noskov 1 ; D. Peter Tieleman 1 ; 1 University Of Calgary, Department of Biological Sciences, Centre for Molecular Simulation, Calgary, AB, Canada The Solute Carrier 4 (SLC4) family of transporters perform an important function of maintaining the acid-base homeostasis in the body. In particular, the Anion Exchanger 1 (AE1) is involved in the exchange of chloride/bicarbonate ions in the erythrocytes and kidneys. The binding pockets and ion permeation pathways of AE1 contain several titratable residues which may play an important role in the transport of ions. Experimental evidence suggests that E681, an acidic residue in the central binding site S1 of the AE1 pocket, when protonated facilitates the transport of divalent anions (1). The acid-base equilibrium of bicarbonate/carbonate further complicates this scenario. Molecular dynamics simulations conducted by our group in prior studies revealed that protonation of E681 impacts the frequency and longevity of ion binding events at site S1 (2). However, these methods are limited by assignment of discrete protonation states throughout the simulation, which do not change in response to potentially important environmental effects. Protonation states are affected by environmental factors such as pH, ionic strength, and nearby amino acids. Constant pH Molecular Dynamics (CpHMD) techniques simulate dynamic protonation events in molecular systems and we adopt them in the present study for assessment of the protonation states of residues of importance in the binding pocket of AE1. We will present the pKa values of the key titratable residues of the transporter and the bicarbonate anion. References 1. Jennings, M. L. (2021) Cell physiology and molecular mechanism of anion transport by erythrocyte band 3/AE1. Am. J. Physiol. Physiol. 321, C1028–C1059 2. Zhekova, H. R., Pushkin, A., Kayık, G., Kao, L., Azimov, R., Abuladze, N., Kurtz, D., Damergi, M., Noskov, S. Y., and Kurtz, I. (2021) Identification of multiple substrate binding sites in SLC4 transporters in the outward-facing conformation: Insights into the transport mechanism. J. Biol. Chem. 296, 100724

18

Proton Reactions: From Basic Science to Biomedical Applications

Monday Speaker Abstracts

MEMBRANE PROTEIN THERMODYNAMIC MEASUREMENTS ENABLED BY PROTONS Karen Fleming John Hopkins University, USA No Abstract DYNAMIC HYDROGEN-BOND NETWORKS FOR PROTON BINDING AND PROTON TRANSFER AT BIO-MEMBRANE INTERFACES Eva Bertalan 1 ; Honey Jain 2,3 ; Joseph Matthew Rodrigues 4 ; Gebhard F Schertler 4 ; Leonid Brown 5 ; Ana-Nicoleta Bondar 2,6 ; 1 Physikzentrum, RWTH-Aachen University, Aachen, Germany 2 University of Bucharest, Faculty of Physics, Bucharest-Magurele, Romania 3 Freie Universität Berlin, Physics Department, Berlin, Germany 4 Paul Scherer Institut, Laboratory of Biomolecular Research, Villigen, Switzerland 5 University of Guelph, Department of Physics, Guelph, ON, Canada 6 Forschungszentrum Jülich, Institute for Computational Biomedicine (IAS-5/INM-9), Jülich, Germany Membrane proteins that bind and transfer protons to and from the bulk often rely on water mediated hydrogen-bond networks that contain titratable sidechains and couple to other functionally important sites of the protein. As such networks may extend throughout a significant region of the protein, the identity of the proton-binding sites and the response of the protein to protonation change can be difficult to predict. We develop and apply graph-based algorithms to compute and dissect hydrogen-bond networks in proton-binding membrane proteins and at the interfaces of lipid membranes with different lipid composition. From analyses of datasets of membrane protein structures from structural biology and simulations, we identify structural and sequence motifs of proton-binding hydrogen-bond networks. Depending on the identity of their headgroups, lipids can directly participate in dynamic hydrogen-bond networks with titratable sidechains at proton uptake/release sites of proton-coupled membrane transporters. Research was supported in part by the European Union’s Horizon 2020 Research and Innovation Program under the Marie Sklodowska-Curie grant agreement No 860592, Innovative Training Network ‘Proton and proton-coupled transport’, and by computing time from the Physics Department of the Freie Universität Berlin and from the Forschungszentrum Jülich. DECIPHERING APPARENT CHOLESTEROL EFFECTS ON VOLTAGE-GATED PROTON CHANNELS

Thomas DeCoursey Rush University, USA No Abstract

19

Proton Reactions: From Basic Science to Biomedical Applications

Monday Speaker Abstracts

THE VOLTAGE-GATED PROTON CHANNEL DISCOVERS ITS FAMILY Boris Musset 1 ; 1 PMU Nuremberg, Center of Physiology, Pathophysiology and Biophysics, Nuremberg, Germany A little more than four decades ago, the first voltage-clamp recording of the voltage-gated proton channel triggered a novel research field. Almost two and a half decades later, the discovery of the channel's gene initiated further progress in elucidating the biophysical properties, structure, function, and physiology of this unique channel. Despite this, the proton channel field continues to evolve, presenting numerous unanswered questions. One of the hallmarks of the voltage-gated proton channel was that each species so far detected held solely one gene coding for the proton channel. Here, we present the discovery and characterization of three independent genes coding for distinct proton channels, in a single species. Aplysia californica, a classical model organism of neural plasticity, harbours these genes. Additionally, we present our advances in the structure and function of the proton channel, by iteratively combining molecular dynamics simulations with patch-clamp recordings, specifically targeting the proton channel gating mechanism. Lastly, we suggest a potential path for the evolution of the voltage-gated proton channel. HOW A MULTIPLICITY OF PROTONATION STATES IN PROTEINS SUPPORTS PROTON TRANSFERS Marilyn Gunner 1 ; Junjun Mao 1 ; Umesh Khaniya 1 ; Gehan Ranapura 1 ; Rongmei Wei 2 ; Jose Ortiz-Soto 2 ; Md. Raihan Uddin 3 ; 1 City College of New York CUNY, Physics, New York, NY, USA 2 City College of New York CUNY, Chemistry, New York, NY, USA 3 City College of New York CUNY, Biochemistry, New York, NY, USA Proteins are known to exist in a distribution of conformations, but the complexity of the distribution of protonation states in the equilibrium ensemble is under-appreciated. Recent developments in the MCCE program have enabled the analysis of protonation and conformation microstates in Monte Carlo sampling, providing insight into the diversity of protonation states at equilibrium. The individual microstates, which define the protonation states and conformation of each residue and ligand are akin to an MD snapshot. Analysis of these states reveals how protons move within complex clusters of buried protonatable residues that make up proton loading sites. The proton coupled electron transfers in photosynthetic reaction centers, cytochrome c oxidase and complex I provide examples of how the protonation and conformation microstates influence site redox potentials, proton affinities of proton loading sites and the connectivity of extended proton transfer pathways.

20