Biophysical Chemistry Research Articles

Statistical Chemical Thermodynamics and Energetic Behavior of Counting: Gibbs' Theory Revisited.

One of the major tenets of Gibbs' equilibrium chemical thermodynamics is "counting the number of particles" and introducing the associated chemical potential. This leads to the concept of chemical energy μdN that becomes a part of the total internal energy change in Gibbs' fundamental thermodynamic relation. However, a pure thermomechanical description of a particle system has only two fundamental forms of energy, namely, mechanical work and heat; Gibbs' μ is an emergent concept. Following this perspective, we present an alternative, generalized Gibbs formalism that serves as a statistical theory of emergence: chemical potential is introduced down to the level of individual meso- or even microstates of a system. The research practice in biophysical chemistry since the 1950s epitomizes this perspective, in which the concept of potential of mean force, or conditional free energy, plays a central role. Our theory suggests a generalized "energetic representation" for scientific data that is in the form of counting frequency, which is measured routinely through equilibrium constants in chemistry laboratories.

Additivity of ionic currents in mixed electrolyte solutions and confined geometries

Electrolyte mixtures in aqueous solutions are central to biophysical chemistry and chemical engineering. In many cases, ions are confined to nanoscale volumes with surface electrical charges. Here, we analyze experimentally the additivity of ionic currents across negatively charged conical nanopores in mixed electrolyte solutions. These pores show an asymmetric axial distribution of carboxylic acid groups on the pore surface, which gives rectified current (I)-voltage (V) curves when a single-pore membrane is bathed by two symmetrical aqueous solutions at neutral pH values. We consider KCl, LiCl, NaCl, CsCl, CaCl2, and K2SO4 pure electrolytes as well as the mixed electrolyte systems LiCl + KCl, NaCl + KCl, CsCl + KCl, CaCl2 + KCl, and K2SO4 + KCl for concentrations ranging from 2 to 500 mM and applied voltages between −2 V and 2 V. The difference between the sum of the pure electrolyte currents and the mixed electrolyte current is studied in terms of the electrolyte concentration, the sign of the applied voltage in the current–voltage curves, and the mixed electrolyte type.

Millisecond Time-Resolved Solid-State NMR Initiated by Rapid Inverse Temperature Jumps

Elucidation of the detailed mechanisms by which biological macromolecules undergo major structural conversions, such as folding, complex formation, and self-assembly, is a central concern of biophysical chemistry that will benefit from new experimental methods. We describe a simple technique for initiating a structural conversion process by a rapid decrease in the temperature of a solution, i.e., a rapid inverse temperature jump. By pumping solutions through copper capillary tubes that are thermally anchored to heated and cooled blocks, solution temperatures can be switched from 95 to 30 °C (or lower) in about 0.8 ms. For time-resolved solid-state nuclear magnetic resonance (ssNMR), solutions can then be frozen rapidly by spraying into cold isopentane after a variable structural evolution time τe. As an initial demonstration, we use this "inverse T-jump" technique to characterize the kinetics and mechanism by which the 26-residue peptide melittin converts from its primarily disordered, monomeric state at 95 °C to its α-helical, tetrameric state at 30 °C. One- and two-dimensional ssNMR spectra of frozen solutions with various values of τe, recorded at 25 K with signal enhancements from dynamic nuclear polarization, show that both helical secondary structure and intermolecular contacts develop on the same time scale of about 6 ms. The dependences on τe of both intraresidue crosspeak patterns and inter-residue crosspeak volumes in two-dimensional spectra can be fit with a unidirectional dimerization model, consistent with dimerization being the rate-limiting step for melittin tetramer formation.

Direct quantification of ligand-induced lipid and protein microdomains with distinctive signaling properties.

Lipid rafts are ordered lipid domains that are enriched in saturated lipids, such as the ganglioside GM1. While lipid rafts are believed to exist in cells and to serve as signaling platforms through their enrichment in signaling components, they have not been directly observed in the plasma membrane without treatments that artificially cluster GM1 into large lattices. Here, we report that microscopic GM1-enriched domains can form, in the plasma membrane of live mammalian cells expressing the EphA2 receptor tyrosine kinase in response to its ligand ephrinA1-Fc. The GM1-enriched microdomains form concomitantly with EphA2-enriched microdomains. To gain insight into how plasma membrane heterogeneity controls signaling, we quantify the degree of EphA2 segregation and study initial EphA2 signaling steps in both EphA2-enriched and EphA2-depleted domains. By measuring dissociation constants, we demonstrate that the propensity of EphA2 to oligomerize is similar in EphA2-enriched and -depleted domains. However, surprisingly, EphA2 interacts preferentially with its downstream effector SRC in EphA2-depleted domains. The ability to induce microscopic GM1-enriched domains in live cells using a ligand for a transmembrane receptor will give us unprecedented opportunities to study the biophysical chemistry of lipid rafts.

Walter Bruno Gratzer (1932–2021): In his own words, as far as possible

Miranda Robertson commemorates the life, remarkable times and witty and erudite writing of protein chemist and biophysicist Walter Gratzer.

The Von Willebrand factor-ADAMTS-13 axis: a two-faced Janus in bleeding and thrombosis

Von Willebrand factor (VWF), a blood multimeric protein with a very high molecular weight, plays a crucial role in the primary hemostasis, the physiological process characterized by the adhesion of blood platelets to the injured vessel wall. Hydrodynamic forces are responsible for the VWF multimers conformational transitions from a globular to a stretched linear conformation. These characteristics render this protein a valuable object to be investigated by mechanochemistry, the biophysical chemistry branch that studies the effects of shear forces on protein conformation. This review will focus on the structural elements of the VWF molecule involved in the biochemical response to shear forces. The stretched VWF conformation favors the interaction with the platelet GpIb and at the same time with ADAMTS-13, the zinc-protease that cleaves VWF in the A2 domain, limiting its prothrombotic capacity. It is important to consider the level or the function of VWF or ADAMTS-13 always in relation each other, keeping in mind that in many thrombotic forms of microangiopathies the reduction of the ratio between the ADAMTS-13 activity and the VWF level (lower than 0.5) can be a valuable parameter to predict a real thrombotic risk. Hence, a significant increase in VWF level alone, even without any reduction of ADAMTS-13 concentration, would still be responsible for a significant reduction of the ADAMTS-13/VWF ratio, which ultimately could reflect or predict a prothrombotic risk. Future studies will have to validate the concept whether ADAMTS-13/VWF ratio could a valuable and reliable biomarker to predict or confirm the presence of thrombotic risk in several morbid conditions.

On “Structural implications of the analysis of electron paramagnetic resonance spectra of natural and artificial copper proteins” by J. Peisach and W.E. Blumberg

The title article serves as a lasting product of the 25-year scientific collaboration between Jack Peisach, a biophysical chemist at the Albert Einstein College of Medicine, and William E. Blumberg, a physicist at Bell Telephone Laboratories.

The neurosciences at the Max Planck Institute for Biophysical Chemistry in Göttingen

ABSTRACT The Max Planck Institute (MPI) for Biophysical Chemistry (Karl-Friedrich Bonhoeffer Institute) was founded in 1971 in Göttingen. Two of the 11 departments at the institute had a neuroscientific focus. Otto D. Creutzfeldt (1927–1992) and Victor P. Whittaker (1919–2016) were directors of the Neurobiological and Neurochemical Departments, respectively. Creutzfeldt’s department researched the structure and function of the cerebral cortex, and Whittaker’s department concentrated on the biochemical analysis of synapses and synaptic vesicles. Creutzfeldt and Whittaker were already internationally respected scientists when they were appointed to Göttingen. The next generation of departmental directors, Erwin Neher and Bert Sakmann, were “home-grown” researchers from the institute and, during their time as junior group leaders, they developed the so-called patch clamp technique, with which they were able to measure single ion channels in nerve cells. This technique revolutionized neurophysiology, and Neher and Sakmann were awarded the 1991 Nobel Prize in Physiology or Medicine for their work in this area. Neher was appointed director of the Membrane Biophysics Department in 1983 and, since then, his department has mainly examined the role of Ca2+ in the release of neurotransmitters at synapses and in the secretion of catecholamines from chromaffin cells. From 1985, Sakmann was director of the Cell Physiology Department, and his laboratory concentrated on the molecular and physiological characterization of transmitter receptors in postsynaptic membranes. In 1989, he was appointed to the MPI for Medical Research in Heidelberg. Reinhard Jahn became director of the Neurobiology Department in 1997, researching the molecular mechanisms of the release of neurotransmitters from the presynaptic terminals, and he discovered several proteins associated with the synaptic vesicles. With their work, Neher, Sakmann, and Jahn have made the MPI for Biophysical Chemistry one of the world’s leading research centers for the transmission of signals at synapses.

Deciphering the sensing of α-amyrin acetate with hs-DNA: a multipronged biological probe.

In this study, we focus on the biomimetic development of small molecules and their biological sensing with DNA. The binding of herring sperm deoxyribonucleic acid (hs-DNA) with naturally occurring bioactive small molecule α-amyrin acetate (α-AA), a biomimetic – isolated from the leaves of Ficus (F.) arnottiana is investigated. Collective information from various imaging, spectroscopic and biophysical experiments provides evidence that α-AA is a minor groove sensor of hs-DNA and preferentially binds to the A–T-rich regions. Interactions of different concentrations of small molecule α-AA with hsDNA were evaluated via various analytical techniques such as UV-Vis, circular dichroism (CD) and fluorescence emission spectroscopy. Fluorescence emission spectroscopy results suggest that α-AA decreases the emission level of hsDNA. DNA minor groove sensor Hoechst 33258 and intercalative sensor EB, melting transition analysis (TM) and viscosity analysis clarified that α-AA binds to hs-DNA via a groove site. Biophysical chemistry and molecular docking studies show that hydrophobic interactions play a major role in this binding. The present research deals with a natural product biosynthesis-linked chemical–biology interface sensor as a biological probe for α-AA: hs-DNA.

PELDOR Measurements on Nitroxide-Labeled Oligonucleotides.

In the past decades, pulsed dipolar electron paramagnetic resonance spectroscopy (PDS) has emerged as a powerful tool in biophysical chemistry to study the structure, dynamics, and function of biomolecules like oligonucleotides and proteins. Structural information is obtained from PDS methods in form of a distribution of distances between spin centers. Such spin centers can either be intrinsically present paramagnetic metal ions and organic radicals or may be attached to the biomolecule by means of site-directed spin labeling. The most common PDS experiment for probing interspin distances in the nanometer range is pulsed electron-electron double resonance (PELDOR or DEER). In the protocol presented here, we provide a step-by-step workflow on how to set up a PELDOR experiment on a commercially available pulsed EPR spectrometer, outline the data analysis, and highlight potential pitfalls. We suggest PELDOR measurements on nitroxide-labeled oligonucleotides to study the structure of either RNA-cleaving DNAzymes in complex with their RNA targets or modified DNAzymes with different functions and targets, in which deoxynucleotides are substituted by nitroxide-labeled nucleotides.

Stress signaling convergence and nutrient crosstalk determine zinc-mediated amelioration against cadmium toxicity in rice

Consumption of rice (Oryza sativa L.) is one of the major pathways for heavy metal bioaccumulation in humans over time. Understanding the molecular responses of rice to heavy metal contamination in agriculture is useful for eco-toxicological assessment of cadmium (Cd) and its interaction with zinc (Zn). In certain crops, the impacts of Cd stress or Zn nutrition on the biophysical chemistry and gene expression have been widely investigated, but their molecular interactions at transcriptomic level, particularly in rice roots, are still elusive. Here, hydroponic investigations were carried out with two rice genotypes (Yinni-801 and Heizhan-43), varying in Cd contents in plant tissues to determine their transcriptomic responses upon Cd15 (15 µM) and Cd15+Zn50 (50 µM) treatments. High throughput RNA-sequencing analysis confirmed that 496 and 2407 DEGs were significantly affected by Cd15 and Cd15+Zn50, respectively, among which 1016 DEGs were commonly induced in both genotypes. Multitude of DEGs fell under the category of protein kinases, such as calmodulin (CaM) and calcineurin B-like protein-interacting protein kinases (CBL), indicating a dynamic shift in hormonal signal transduction and Ca2+ involvement with the onset of treatments. Both genotypes expressed a mutual regulation of transcription factors (TFs) such as WRKY, MYB, NAM, AP2, bHLH and ZFP families under both treatments, whereas genes econding ABC transporters (ABCs), high affinity K+ transporters (HAKs) and Glutathione-S-transferases (GSTs), were highly up-regulated under Cd15+Zn50 in both genotypes. Zinc addition triggered more signaling cascades and detoxification related genes in regulation of immunity along with the suppression of Cd-induced DEGs and restriction of Cd uptake. Conclusively, the effective integration of breeding techniques with candidate genes identified in this study as well as economically and technologically viable methods, such as Zn nutrient management, could pave the way for selecting cultivars with promising agronomic qualities and reduced Cd for sustainable rice production.

Science writing versus scientists writing for ourselves: Why Walter Gratzer mattered.

We are all aware of journalists whose craft is the nontechnical dissection of scientific advances for a broad readership. Two sterling examples for me are Dennis Overbye and Gina Kolata, both writing mostly for the New York Times. There are also engaging biographers of scientists, among whom my favorite is Georgina Ferry. But then there is the phenotype of scientists who themselves write for broader audiences, aside from their primary scientific publications. Notable among them were Thomas Huxley on evolution, Louis Pasteur on vaccination, Carl Sagan on the cosmos, and Steven J. Gould, also on evolution.1 Other domains of biology and medical science were powerfully brought to the public by the scientist-authors Paul de Kruif,2 Lewis Thomas,3 and Oliver Sacks.4 Among the most gifted at work today is the oncologist Siddharta Mukherjee.5 There is a third guild, small in number. These are the scientists who write for scientists themselves, but not just within their domain of expertise. This is perhaps the most daring craft of the three. The science journalists access extensive sources, and scientists writing for the public about their expertise bring that. But for a scientist to write for scientists outside their immediate expertise, well—that takes grit and skill. One of the all-time best at this, Walter Gratzer (Figure 1), died on October 20, 2021, at 89. As is typical of these rare pens, Walter was not afraid of antagonizing those in whatever field he was writing about, and the reason was that he was of extraordinary breadth and erudition. At the journal Nature, he was for years sought out by its editors for his judgment on several manuscripts every week, with their confidence that his grasp was uncommonly broad (and his nose for hyperbole exceptionally acute). But it was in Walter's own writing that many came to know and admire him. He could be rough, but never uninformed. His training was in biophysical chemistry, and his research was mainly on erythrocyte cell biology. But his mind captured everything, and he never forgot anything. One of many examples is the book he wrote on the history of us humans gathering, consuming, and eventually cooking our food.6 His mastery of every facet of this intriguing topic was stunning, and with each sentence crafted with uncommon skill, as in all his writing. Another favorite of mine was his review of a biography of Francis Crick, in which he added so much beyond the book.7 Several years ago, a friend of mine published a biography of the Philadelphia naturalist, Joseph Leidy. I received my signed copy with gratitude but questioned the author about the subtitle: The Last Man Who Knew Everything. I asked him: “How sure can we be about that?” My friend smiled—there was the answer. License was taken, of course. I recently received a copy of an evocative letter sent to Gratzer's widow, containing the line: “I used to suspect Walter knew everything.” I did, too. His friendship and writing elegance inspired me. When we scientists can reach one another, beyond our standard research papers and just within our given specialization, we add to the joyful vibrancy of this wonderful profession we have been lucky enough to find. This is why Walter Gratzer mattered. We shall not see the likes of him anytime soon.

Remembrance of Petre T. Frangopol (1933–2020) the promoter of scientometrics in Romania

The article is an obituary aimed to pay tribute to Petre T. Frangopol, a brilliant Romanian scientist (chemist and biophysicist). He had many fields of interest: Chemistry, Radiochemistry, Physical Organic Chemistry, Biophysical Chemistry, Biophysics, Medical Physics, Archaeometry, Oceanography, History of Chemistry in Romania. However, I consider Petre T. Frangopol as the main promotor of Scientometrics in Romania since he published a lot of articles and books in this field. The achievements of Petre T. Frangopol were recognized by many prizes and distinctions received throughout his life, including the election in The Romanian Academy. He was also a man of great character and an unforgettable friend (Petrache).

Stationary and Time-Dependent Carbon Monoxide Stretching Mode Features in Carboxy Myoglobin: A Theoretical-Computational Reappraisal.

The stationary and time-dependent infrared spectrum (IR) of the CO stretching mode (νCO) in carboxymyoglobin (MbCO), a longstanding problem of biophysical chemistry, has been modeled through a theoretical-computational method specifically designed for simulating quantum observables in complex atomic-molecular systems and based on a combined application of long time scale molecular dynamics simulations and quantum-chemical calculations. This study is basically focused on two aspects: (i) the origin of the stationary IR substates (termed as A0, A1, and A3) and (ii) the modeling and the interpretation of the νCO energy relaxation. The results, strengthened by a more than satisfactory agreement with the experimental data, concisely indicate that (i) the conformational His64-FeCO relevant substates, i.e., characterized by the formation-disruption of the H-bond between the above moieties, are the main responsible of the presence of two distinct and well separated (A0 and A1/A3) spectroscopic regions; (ii) the characteristic bimodal shape of the A1/A3 spectral region, according to our model, is the result of the fluctuation of the electric field pattern as provided by the protein-solvent framework perturbing the bound His64-CO-Heme complex; and (iii) the electric field pattern, in conjunction with the relatively high density of MbCO vibrational states, is also the main determinant of the νCO energy relaxation, characterizing its kinetic efficiency.

Instructional Design for an Undergraduate Laboratory Course in Molecular Biophysics

ABSTRACT In this article, an approach to teaching molecular biophysics is described. The organization and course content has been carefully chosen and curated so that fundamental ideas in molecular biophysics can be taught effectively to upper classmen in higher education. Three general topic areas are introduced along with accompanying experiments that illustrate major principles related to each topic area. This article outlines an approach to organizing chosen course material and suggests multiple teaching activities within each major topic area: thermodynamics, kinetics, and structural biology. Subtopics are presented along with suggested laboratory experiments. The experiments are outlined in a way that they can be readily adopted by educators teaching a biophysical chemistry lab. The accompaniment of workshop exercises as an additional teaching modality is a component of the course intended to enhance the development of important problem-solving skills and comprehension of new content. Finally, a reflection on student feedback and course outcomes along with targeted learning goals is discussed.

A different spin on NMR

Not that kind of instrument “ Resonance” is right there in the name of nuclear magnetic resonance spectroscopy, but the technique doesn’t make most chemists think of music. Ayyalusamy Ramamoorthy , a biophysical chemist at the University of Michigan, thinks it should. Wanting a reliable way to teach NMR to students, he and graduate student Srikar Munukutla translated the NMR spectra of organic compounds into musical notes. In traditional proton—or 1 H—NMR, chemists use the chemical shifts of hydrogen atoms to help identify molecular structures. Normally reported in parts per million, these shifts are related to the electromagnetic frequencies that resonate with the spins of electrons on hydrogen atoms inside a big magnet . With a standard equation, chemists can convert the shifts into hertz—a measure of frequency in cycles per second. Music notes are also in hertz , so NMR spectra can be made into music. The team took

How to Model for a Living: The CSGF as a Catalyst for Supermodels.

Models are ubiquitous and uniting tools for computational scientists across disciplines. As a computational biophysical chemist, I apply multiple models to understand and predict how molecules recognize and interact with each other in complex, dynamic biological environments. The Department of Energy Computational Science Graduate Fellowship (DOE CSGF) cultivates interest in engaging in models from an multidisciplinary perspective and enables junior scientists to see how computational modeling is a creative and collaborative process. Below, I describe ways, based in part on my own experiences as a CSGF recipient, in which modeling can be used both to understand the molecular world and to excite others about computational science.

Concerted Motions and Molecular Function: What Physical Chemistry We Can Learn from Light-Driven Ion-Pumping Rhodopsins.

Transmembrane ion gradients are generated and maintained by ion-pumping proteins in cells. Light-driven ion-pumping rhodopsins are retinal-containing proteins found in archaea, bacteria, and eukarya. Photoisomerization of the retinal chromophore induces structural changes in the protein, allowing the transport of ions in a particular direction. Understanding unidirectional ion transport by ion-pumping rhodopsins is an exciting challenge for biophysical chemistry. Concerted changes in ion-binding affinities of the ion-binding sites in proteins are key to unidirectional ion transport, as is the coupling between the chromophore and the protein moiety to drive the concerted motions regulating ion-binding affinities. The commonality of ion-pumping rhodopsin protein structures and the diversity of their ion-pumping functions suggest universal principles governing ion transport, which would be widely applicable to molecular systems. In this Perspective, I review the insights obtained from previous studies on rhodopsins and discuss future perspectives.

Evaluation of the Method of Binding Tamoxifen to DNA Experimentally and Computationally

Background and Aim: Tamoxifen is a group of drugs of selective estrogen receptor modulators, and is one of the drugs effective in the prevention and treatment of some cancers (such as breast cancer). In this study, the interaction of tamoxifen with DNA is investigated experimentally. Also, the electronic structure (at atomic scale) of the molecular system of tamoxifen was theoretically investigated, using atom in molecule (AIM) theory. Methods & Materials: First, in the experimental section of this study, the interaction of Tamoxifen with DNA were investigated by UV-ViS technique and hydrodynamic method (Viscometry). In addition, the analysis of the experimental results shows the obvious effect of concentration on the mechanism of how the tamoxifen molecule binds to DNA. Then, in the theoretical part of this research, using computational biophysical chemistry methods, some properties of tamoxifen molecular system, such as electronic Density of States (DOS), boundary orbital’s energy (HOMO/LUMO), Electrostatic Potential Energy (EPS) and electronic contour maps of the electron density and its Laplacian, will be calculated. Ethical Considerations: This article is a meta-analysis with animal sample. Results: Result of the UV-ViS spectroscopy technique and viscometry indicated hyperchromism and hypochromism effect. In addition, the result were depend on the concentration of the drug and affected the kind of binding of Tamoxifen to DNA. the analysis of computational studies on the drug tamoxifen suggests that the mechanism of the local charge/energy distribution in the molecular system of tamoxifen plays an important role in how this drug binds to DNA. Conclusion: Based on the experimental results of UV-ViS technique and viscometry, as well as the electronic/vibrational properties of the tamoxifen molecular system, it was defined that the Tamoxifen interacts significantly with all the binding sites of DNA.

Dissecting Multiple Pathways in the Relaxation Dynamics of Helix Coil Transitions with Optimum Dimensionality Reduction.

Fast kinetic experiments with dramatically improved time resolution have contributed significantly to understanding the fundamental processes in protein folding pathways involving the formation of a-helices and b-hairpin, contact formation, and overall collapse of the peptide chain. Interpretation of experimental results through application of a simple statistical mechanical model was key to this understanding. Atomistic description of all events observed in the experimental findings was challenging. Recent advancements in theory, more sophisticated algorithms, and a true long-term trajectory made way for an atomically detailed description of kinetics, examining folding pathways, validating experimental results, and reporting new findings for a wide range of molecular processes in biophysical chemistry. This review describes how optimum dimensionality reduction theory can construct a simplified coarse-grained model with low dimensionality involving a kinetic matrix that captures novel insights into folding pathways. A set of metastable states derived from molecular dynamics analysis generate an optimally reduced dimensionality rate matrix following transition pathway analysis. Analysis of the actual long-term simulation trajectory extracts a relaxation time directly comparable to the experimental results and confirms the validity of the combined approach. The application of the theory is discussed and illustrated using several examples of helix <==> coil transition pathways. This paper focuses primarily on a combined approach of time-resolved experiments and long-term molecular dynamics simulation from our ongoing work.