Protein Chemistry Research Articles

A Single-Molecule Strategy to Capture Non-native Intramolecular and Intermolecular Protein Disulfide Bridges.

Non-native disulfide bonds are dynamic covalent bridges that form post-translationally between two cysteines within the same protein (intramolecular) or with a neighboring protein (intermolecular), frequently due to changes in the cellular redox potential. The reversible formation of non-native disulfides is intimately linked to alterations in protein function; while they can provide a mechanism to protect against cysteine overoxidation, they are also involved in the early stages of protein multimerization, a hallmark of several protein aggregation diseases. Yet their identification using current protein chemistry technology remains challenging, mainly because of their fleeting reactivity. Here, we use single-molecule spectroscopy AFM and molecular dynamics simulations to capture both intra- and intermolecular disulfide bonds in γD-crystallin, a cysteine-rich, structural human lens protein involved in age-related eye cataracts. Our approach showcases the power of mechanical force as a conformational probe in dynamically evolving proteins and presents a platform to detect non-native disulfide bridges with single-molecule resolution.

Leu22_Leu23 Duplication at the Signal Peptide of PCSK9 Promotes Intracellular Degradation of LDLr and Autosomal Dominant Hypercholesterolemia.

PCSK9 (Proprotein convertase subtilisin/kexin type 9) regulates LDL-C (low-density lipoprotein cholesterol) metabolism by targeting LDLr (LDL receptor) for lysosomal degradation. PCSK9 gain-of-function variants cause autosomal dominant hypercholesterolemia by reducing LDLr levels, the D374Y variant being the most severe, while loss-of-function variants are associated with low LDL-C levels. Gain-of-function and loss-of-function activities have also been attributed to variants occurring in the PCSK9 signal peptide. Among them, L11 is a very rare PCSK9 variant that seems to increase LDL-C values in a moderate way causing mild hypercholesterolemia. Using molecular biology and biophysics methodologies, activities of L8 and L11 variants, both located in the leucine repetition stretch of the signal peptide, have been extensively characterized in vitro. L8 variant is not associated with increased LDLr activity, whereas L11 activity is increased by ≈20% compared with wt PCSK9. The results suggest that the L11 variant reduces LDLr levels intracellularly by a process resulting from impaired cleavage of the signal peptide. This would lead to less efficient LDLr transport to the cell membrane and promote LDLr intracellular degradation. Deletion of a leucine in the signal peptide in L8 variant does not affect PCSK9 activity, whereas the leucine duplication in the L11 variant enhances LDLr intracellular degradation. These findings highlight the importance of deep in vitro characterization of PCSK9 genetic variants to determine pathogenicity and improve clinical diagnosis and therapy of inherited familial hypercholesterolemia disease.

Micromanipulation in Paramecium: From non-mendelian inheritance to the outlook for versatile micromachines.

This review addresses nine areas of knowledge revealed by micromanipulations performed with Paramecium. Microinjection has shown that sexual maturation and senescence of Paramecium caudatum is a programmed process conducted by a specific gene and its product protein. In Paramecium tetraurelia, autogamy was revealed to depend on the number of DNA syntheses rather than the number of cell divisions in clonal aging. The cytoplasmic complementarity test established that microinjection of wild‐type cytoplasm can correct genetic defects of mutants. The concept of complementarity together with protein chemistry revealed compounds that control membrane excitability. In non‐Mendelian inheritance, noncoding small RNAs made from the parental micronucleus regulate the rearrangement of the progeny's macronuclear DNA. The macronucleus has the potential to be used as a factory for genetic engineering. The development and differentiation of progeny's nuclei in mating pairs are controlled by the parental macronucleus. The chemical reaction processes associated with exocytosis have been revealed by microinjection of various enzymes and antibodies. Using the fusion gene of histone H2B and yellow‐fluorescence protein, it was revealed that the fusion gene‐mRNA is transferred between cells during mating. Experiments with endosymbiotic bacteria and the host shed light on the conditions needed to establish sustainable symbiotic relationships.

Serum autoantibodyome reveals that healthy individuals share common autoantibodies

SUMMARYAutoantibodies are a hallmark of both autoimmune disease and cancer, but they also occur in healthy individuals. Here, we perform a meta-analysis of nine datasets and focus on the common autoantibodies shared by healthy individuals. We report 77 common autoantibodies based on the protein microarray data obtained from probing 182 healthy individual sera on 7,653 human proteins and an additional 90 healthy individual sera on 1,666 human proteins. There is no gender bias; however, the number of autoantibodies increase with age, plateauing around adolescence. We use a bioinformatics pipeline to determine possible molecular-mimicry peptides that can contribute to the elicitation of these common autoantibodies. There is enrichment of intrinsic properties of proteins like hydrophilicity, basicity, aromaticity, and flexibility for common autoantigens. Subcellular localization and tissue-expression analysis reveal that several common autoantigens are sequestered from the circulating autoantibodies.

Transgenic mice encoding modern imaging probes: Properties and applications.

SUMMARYModern biology is increasingly reliant on optical technologies, including visualization and longitudinal monitoring of cellular processes. The major limitation here is the availability of animal models to track the molecules and cells in their natural environment in vivo. Owing to the integrity of the studied tissue and the high stability of transgene expression throughout life, transgenic mice encoding fluorescent proteins and biosensors represent unique tools for in vivo studies in norm and pathology. We review the strategies for targeting probe expression in specific tissues, cell subtypes, or cellular compartments. We describe the application of transgenic mice expressing fluorescent proteins for tracking protein expression patterns, apoptotic events, tissue differentiation and regeneration, neurogenesis, tumorigenesis, and cell fate mapping. We overview the possibilities of functional imaging of secondary messengers, neurotransmitters, and ion fluxes. Finally, we provide the rationale and perspectives for the use of transgenic imaging probes in translational research and drug discovery.

Creative approaches to perform an inclusive faculty search

Names are powerful cues that can trigger unintended bias including ingroup favoritism, a cognitive bias that leads people to overvalue the past accomplishments and future potential of individuals who seem similar to themselves. Research shows that anonymous job searches can significantly increase the chance for applicants from underrepresented (UR) backgrounds to be invited for an interview. This result strongly suggests that in traditional (non‐anonymous) job searches, well‐qualified applicants are overlooked due to bias. I will present the design and results from an unusual tenure‐track Assistant Professor search in the department of Molecular Biophysics and Biochemistry at Yale University. Applicants submitted anonymized research statements—no names of people, places, funding agencies or journals—and a short list of diverse candidates was created based solely on de‐identified documents. Our experiment suggests that anonymous application procedures are one potentially useful tool to combat persistent racial hiring discrimination which could explain the very slow increase of UR scientists among research faculty compared to the growth of UR PhDs.

Modifications of Ribonucleases in Order to Enhance Cytotoxicity in Anticancer Therapy.

Ribonucleases (RNases) are a superfamily of enzymes that have been extensively studied since the 1960s. For a long time, this group of secretory enzymes was studied as an important model for protein chemistry such as folding, stability, and enzymatic catalysis. Since it was discovered that RNases displayed cytotoxic activity against several types of malignant cells, recent investigation has focused mainly on the biological functions and medical applications of engineered RNases. In this review, we describe the structures, functions, and mechanisms of antitumor activity of RNases. They operate at the crossroads of transcription and translation, preferentially degrading tRNA. As a result, this inhibits protein synthesis, induces apoptosis, and causes the death of cancer cells. This effect can be enhanced thousands of times when RNases are conjugated with monoclonal antibodies. Such combinations, called immunoRNases, have demonstrated selective antitumor activity against cancer cells both in vitro and in animal models. This review summarizes the current status of engineered RNases and immunoRNases as promising novel therapeutic agents for different types of cancer. Also, we describe our experimental results from published or previously unpublished research and compare them with other scientific information.

My Journey with cAMP‐dependent Protein Kinase

I was first introduced to cAMP‐dependent protein kinase (PKA) when I was just a young scientist at the beginning of my career, and it has remained as a lifelong companion. PKA has taken me from protein chemistry to atomic structures to brain imaging, and the quest for new knowledge is still ongoing. PKA was the second kinase to be discovered, and we solved the first protein kinase structure in 1991. In the subsequent decades we have come to appreciate the highly dynamic features that drive this molecular allosteric switch. In cells the activity of the PKA catalytic subunit is typically packaged as a holoenzyme complex with functionally non‐redundant cAMP‐binding regulatory (R) subunits, and the holoenzymes are localized to specific sites in the cell in close proximity to dedicated substrates. Many dedicated “cAMP signaling islands” exist within one cell, some at the plasma membrane but others at organelles such as the mitochondria and Golgi and in the nucleus, and each is tightly regulated. Nowhere is PKA signaling more complex than in the brain. PKA is ubiquitous in every mammalian cell, and in so many ways has served as the “poster child” for the kinome that contains over 500 proteins. How then can we explain why the Cb subunit lies near the bottom of the Dark Kinome where important protein kinase are buried but not yet studied? The Cb family contains several isoforms that all differ in only their first exon and includes many splice variants. While Cb1 is expressed ubiquitously in most cells at low levels, Cb2 is expressed in lymphoid tissues while the Cb3/4 isoforms are expressed almost exclusively in brain. Indeed ~50% of PKA signaling in the brain is mediated by Cb isoforms. Using the retina with its well‐organized and terminally differentiated neurons as a “window into the brain” we find that Cb isoforms are highly expressed and that Cb is localized differently from Ca. In addition, we find that Cb is localized to mitochondria in contrast to Ca, RIIa, and RIIb, which are all excluded from mitochondria. Four distinct mutations in Cb lead to a complex phenotype that includes cardiac and skeletal defects as well as a polydactyl presentation. These mutations also inhibit Sonic hedgehog (Shh) signaling adding further support to the importance of Cb in neuronal signaling and development. We are our interdisciplinary strategy to explore this new frontier for PKA signaling in the brain.

Walter B. Gratzer (1932–2021) – molecular scientist and science writer

Walter Gratzer (1932–2021) arrived in England as a child refugee from Nazism, without speaking English, and became one of the most successful and influential science writers. His principal career was in molecular biophysics at King’s College London, researching the structure of nucleic acids and proteins.

Reductive site-selective atypical C,Z-type/N2-C2 cleavage allows C-terminal protein amidation.

Biomolecule environments can enhance chemistries with the potential to mediate and modulate self-modification (e.g., self-cleavage). While these enhanced modes are found in certain biomolecules (e.g., RNA ribozymes), it is more rare in proteins. Targeted proteolytic cleavage is vital to physiology, biotechnology, and even emerging therapy. Yet, purely chemically induced methods for the site-selective cleavage of proteins remain scarce. Here, as a proof of principle, we designed and tested a system intended to combine protein-enhanced chemistry with tag modification to enable synthetic reductive protein chemistries promoted by diboron. This reductively driven, single-electron chemistry now enables an operationally simple, site-selective cleavage protocol for proteins directed to readily accessible dehydroalanine (Dha) residues as tags under aqueous conditions and in cell lysates. In this way, a mild, efficient, enzyme-free method now allows not only precise chemical proteolysis but also simultaneous use in the removal of affinity tags and/or protein-terminus editing to create altered N- and C-termini such as protein amidation (─CONH2).

Uniting diversity to create a more inclusive academic environment.

Uniting diversity to create a more inclusive academic environment.

B12-induced reassembly of split photoreceptor protein enables photoresponsive hydrogels with tunable mechanics.

Although the tools based on split proteins have found broad applications, ranging from controlled biological signaling to advanced molecular architectures, many of them suffer from drawbacks such as background reassembly, low thermodynamic stability, and static structural features. Here, we present a chemically inducible protein assembly method enabled by the dissection of the carboxyl-terminal domain of a B12-dependent photoreceptor, CarHC. The resulting segments reassemble efficiently upon addition of cobalamin (AdoB12, MeB12, or CNB12). Photolysis of the cofactors such as AdoB12 and MeB12 further leads to stable protein adducts harboring a bis-His-ligated B12. Split CarHC enables the creation of a series of protein hydrogels, of which the mechanics can be either photostrengthened or photoweakened, depending on the type of B12. These materials are also well suited for three dimensional cell culturing. Together, this new protein chemistry, featuring negligible background autoassembly, stable conjugation, and phototunability, has opened up opportunities for designing smart materials.

Polyglutamylation: biology and analysis

Polyglutamylation is a posttranslational modification (PTM) that adds several glutamates on glutamate residues in the form of conjugated peptide chains by a family of enzymes known as polyglutamylases. Polyglutamylation is well documented in microtubules. Polyglutamylated microtubules consist of different α- and β-tubulin subunits with varied number of added glutamate residues. Kinetic control and catalytic rates of tubulin modification by polyglutamylases influence the polyglutamylation pattern of functional microtubules. The recent studies uncovered catalytic mechanisms of the glutamylation enzymes family, particularly tubulin tyrosine ligase-like (TTLL). Variable length polyglutamylation of primary sequence glutamyl residues have been mapped with a multitude of protein chemistry and proteomics approaches. Although polyglutamylation was initially considered a tubulin-specific modification, the recent studies have uncovered a calmodulin-dependent glutamylase, SidJ. Nano-electrospray ionization (ESI) proteomic approaches have identified quantifiable polyglutamylated sites in specific substrates. Indeed, conjugated glutamylated peptides were used in nano-liquid chromatography gradient delivery due to their relative hydrophobicity for their tandem mass spectrometry (MS/MS) characterization. The recent polyglutamylation characterization has revealed three major sites: E445 in α-tubulin, E435 in β-tubulin, and E860 in SdeA. In this review, we have summarized the progress made using proteomic approaches for large-scale detection of polyglutamylated peptides, including biology and analysis.

Molecular Modeling is an Enabling Approach to Complement and Enhance Channelopathy Research.

Hundreds of human membrane proteins form channels that transport necessary ions and compounds, including drugs and metabolites, yet details of their normal function or how function is altered by genetic variants to cause diseases are often unknown. Without this knowledge, researchers are less equipped to develop approaches to diagnose and treat channelopathies. High-resolution computational approaches such as molecular modeling enable researchers to investigate channelopathy protein function, facilitate detailed hypothesis generation, and produce data that is difficult to gather experimentally. Molecular modeling can be tailored to each physiologic context that a protein may act within, some of which may currently be difficult or impossible to assay experimentally. Because many genomic variants are observed in channelopathy proteins from high-throughput sequencing studies, methods with mechanistic value are needed to interpret their effects. The eminent field of structural bioinformatics integrates techniques from multiple disciplines including molecular modeling, computational chemistry, biophysics, and biochemistry, to develop mechanistic hypotheses and enhance the information available for understanding function. Molecular modeling and simulation access 3D and time-dependent information, not currently predictable from sequence. Thus, molecular modeling is valuable for increasing the resolution with which the natural function of protein channels can be investigated, and for interpreting how genomic variants alter them to produce physiologic changes that manifest as channelopathies. © 2022 American Physiological Society. Compr Physiol 12:3141-3166, 2022.

Glycosylation is key for enhancing drug recognition into spike glycoprotein of SARS-CoV-2

The emergence of COVID-19 caused by SARS-CoV-2 and its spread since 2019 represents the major public health problem worldwide nowadays, which has generated a high number of infections and deaths. The spike protein (S protein) is the most studied protein of SARS-CoV-2, and key to host-cell entry through ACE2 receptor. This protein presents a large pattern of glycosylations with important roles in immunity and infection mechanisms. Therefore, understanding key aspects of the molecular mechanisms of these structures, during drug recognition in SARS-CoV-2, may contribute to therapeutic alternatives. In this work, we explored the impact of glycosylations on the drug recognition on two domains of the S protein, the receptor-binding domain (RBD) and the N-terminal domain (NTD) through molecular dynamics simulations and computational biophysics analysis. Our results show that glycosylations in the S protein induce structural stability and changes in rigidity/flexibility related to the number of glycosylations in the structure. These structural changes are important for its biological activity as well as the correct interaction of ligands in the RBD and NTD regions. Additionally, we evidenced a roto-translation phenomenon in the interaction of the ligand with RBD in the absence of glycosylation, which disappears due to the influence of glycosylation and the convergence of metastable states in RBM. Similarly, glycosylations in NTD promote an induced fit phenomenon, which is not observed in the absence of glycosylations; this process is decisive for the activity of the ligand at the cryptic site. Altogether, these results provide an explanation of glycosylation relevance in biophysical properties and drug recognition to S protein of SARS-CoV-2, which must be considered in the rational drug development and virtual screening targeting S protein.

Probing the microscopic structure and flexibility of oxidized DNA by molecular simulations

The oxidative damage of DNA is a compelling issue in molecular biophysics as it plays a vital role in the epigenetic control of gene expression and is believed to be associated with mutagenesis, carcinogenesis, and ageing. To understand the microscopic structural changes in physical properties of DNA and the resulting influence on its function due to oxidative damage of its nucleotide bases, we have conducted all-atom molecular dynamic simulations of double-stranded DNA (dsDNA) with its guanine bases being oxidized. The guanine bases are more prone to oxidative damage due to the lowest value of redox potential among all nucleobases. We have analyzed the local as well as global mechanical properties of native and oxidized dsDNA and explained those results by microscopic structural parameters and thermodynamic calculations. Our results show that the oxidative damage of dsDNA does not deform the Watson-Crick geometry; instead, the oxidized DNA structures are found to be better stabilized through electrostatic interactions. Moreover, oxidative damage changes the mechanical, helical, and groove parameters of dsDNA. The persistence length, stretch modulus, and torsional stiffness are found to be 48.87 nm, 1239.26 pN, and 477.30 pN.nm^2, respectively, for native dsDNA, and these values are 61.31 nm, 659.91 pN, and 407.79 pN.nm^2, respectively, when all the guanine bases of the dsDNA are oxidized. Compared to the global mechanical properties, the changes in helical and groove properties are found to be more prominent, concentrated locally at the oxidation sites, and causing the transition of the backbone conformations from BI to BII at the regions of oxidative damage.

Quantifying biomolecular diffusion with a “spherical cow” model

The dynamics of biological polymers, including proteins, RNA, and DNA, occur in very high-dimensional spaces. Many naturally occurring polymers can navigate a vast phase space and rapidly find their lowest free energy (folded) state. Thus, although the search process is stochastic, it is not completely random. Instead, it is best described in terms of diffusion along a downhill energy landscape. In this context, there have been many efforts to use simplified representations of the energetics, for which the potential energy is chosen to be a relatively smooth function with a global minimum that corresponds to the folded state. That is, instead of including every type of physical interaction, the broad characteristics of the landscape are encoded in approximate energy functions. We describe a particular class of models, called structure-based models, that can be used to explore the diffusive properties of biomolecular folding and conformational rearrangements. These energy functions may be regarded as the spherical cow for modeling molecular biophysics. We discuss the physical principles underlying these models and provide an entry-level tutorial, which may be adapted for use in curricula for physics and non-physics majors.

First person – Jianhui Li

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Jianhui Li is first author on ‘ Selective microautophagy of proteasomes is initiated by ESCRT-0 and is promoted by proteasome ubiquitylation’, published in JCS. Jianhui is a postdoc in the lab of Mark Hochstrasser at the Department of Molecular Biophysics and Biochemistry, Yale University, CT, USA, investigating how cellular signaling pathways and autophagy regulate nucleocytoplasmic trafficking and quality control of the proteasome under changing nutrient conditions.

Peptides before and during the nucleotide world: an origins story emphasizing cooperation between proteins and nucleic acids.

Recent developments in Origins of Life research have focused on substantiating the narrative of an abiotic emergence of nucleic acids from organic molecules of low molecular weight, a paradigm that typically sidelines the roles of peptides. Nevertheless, the simple synthesis of amino acids, the facile nature of their activation and condensation, their ability to recognize metals and cofactors and their remarkable capacity to self-assemble make peptides (and their analogues) favourable candidates for one of the earliest functional polymers. In this mini-review, we explore the ramifications of this hypothesis. Diverse lines of research in molecular biology, bioinformatics, geochemistry, biophysics and astrobiology provide clues about the progression and early evolution of proteins, and lend credence to the idea that early peptides served many central prebiotic roles before they were encodable by a polynucleotide template, in a putative ‘peptide-polynucleotide stage’. For example, early peptides and mini-proteins could have served as catalysts, compartments and structural hubs. In sum, we shed light on the role of early peptides and small proteins before and during the nucleotide world, in which nascent life fully grasped the potential of primordial proteins, and which has left an imprint on the idiosyncratic properties of extant proteins.

Structure determination and optimization of a cysteine-less intein with NMR spectroscopy and molecular dynamics simulations

Inteins are a unique class of proteins that are capable of stitching other proteins together in a process known as protein splicing. Inteins are valuable tools for protein chemists as they allow for selective isotopic labelling in NMR experiments and the assembly of proteins with novel properties. Most efficient inteins to date rely on cysteine chemistry to carry out the splicing reaction. This means that a key cysteine residue has to be present at the desired protein's split site. As cysteines are relatively rare in proteins, this often requires mutagenesis at the split junction.