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Electron bifurcation (BF) is an evolutionarily ancient energy coupling mechanism in anaerobes, whose associated enzymatic machinery remains enigmatic. In BF-flavoenzymes, a chemically high-potential electron forms in a thermodynamically favorable fashion by simultaneously dropping the potential of a second electron before its donation to physiological acceptors. The cryo-EM and spectroscopic analyses of the BF-enzyme Fix/EtfABCX from Thermotoga maritima suggest that the BF-site contains a special flavin-adenine dinucleotide and, upon its reduction with NADH, a low-potential electron transfers to ferredoxin and a high-potential electron reduces menaquinone. The transfer of energy from high-energy intermediates must be carefully orchestrated conformationally to avoid equilibration. Herein, anaerobic size exclusion-coupled small-angle X-ray scattering (SEC-SAXS) shows that the Fix/EtfAB heterodimer subcomplex, which houses BF- and electron transfer (ET)-flavins, exists in a conformational equilibrium of compacted and extended states between flavin-binding domains, the abundance of which is impacted by reduction and NAD(H) binding. The conformations identify dynamics associated with the T. maritima enzyme and also recapitulate states identified in static structures of homologous BF-flavoenzymes. Reduction of Fix/EtfABCX's flavins alone is insufficient to elicit domain movements conducive to ET but requires a structural "trigger" induced by NAD(H) binding. Models show that Fix/EtfABCX's superdimer exists in a combination of states with respect to its BF-subcomplexes, suggesting a cooperative mechanism between supermonomers for optimizing catalysis. The correlation of conformational states with pathway steps suggests a structural means with which Fix/EtfABCX may progress through its catalytic cycle. Collectively, these observations provide a structural framework for tracing Fix/EtfABCX's catalysis.

The replication transcription complex (RTC) from the virus SARS-CoV-2 is responsible for recognizing and processing RNA for two principal purposes. The RTC copies viral RNA for propagation into new virus and for ribosomal transcription of viral proteins. To accomplish these activities, the RTC mechanism must also conform to a large number of imperatives, including RNA over DNA base recognition, basepairing, distinguishing viral and host RNA, production of mRNA that conforms to host ribosome conventions, interfacing with error checking machinery, and evading host immune responses. In addition, the RTC will discontinuously transcribe specific sections of viral RNA to amplify certain proteins over others. Central to SARS-CoV-2 viability, the RTC is therefore dynamic and sophisticated. We have conducted a systematic structural investigation of three components that make up the RTC: Nsp7, Nsp8, and Nsp12 (also known as RNA-dependent RNA polymerase). We have solved high-resolution crystal structures of the Nsp7/8 complex, providing insight into the interaction between the proteins. We have used small-angle x-ray and neutron solution scattering (SAXS and SANS) on each component individually as pairs and higher-order complexes and with and without RNA. Using size exclusion chromatography and multiangle light scattering-coupled SAXS, we defined which combination of components forms transient or stable complexes. We used contrast-matching to mask specific complex-forming components to test whether components change conformation upon complexation. Altogether, we find that individual Nsp7, Nsp8, and Nsp12 structures vary based on whether other proteins in their complex are present. Combining our crystal structure, atomic coordinates reported elsewhere, SAXS, SANS, and other biophysical techniques, we provide greater insight into the RTC assembly, mechanism, and potential avenues for disruption of the complex and its functions.

The refractive index gradient of the eye lens is controlled by the concentration and distribution of its component crystallin proteins, which are highly enriched in polarizable amino acids. The current understanding of the refractive index increment ([Formula: see text]) of proteins is described using an additive model wherein the refractivity and specific volume of each amino acid type contributes according to abundance in the primary sequence. Here we present experimental measurements of [Formula: see text] for crystallins from the human lens and those of aquatic animals under uniform solvent conditions. In all cases, the measured values are much higher than those predicted from primary sequence alone, suggesting that structural factors also contribute to protein refractive index.

The tunicate (Ciona intestinalis) βγ-crystallin represents an intermediate case between the calcium-binding proteins ancestral to the vertebrate βγ-crystallin fold and the vertebrate structural crystallins. Unlike the structural βγ-crystallins in the vertebrate eye lens, this βγ-crystallin strongly binds Ca2+. Furthermore, Ca2+ binding greatly stabilizes the protein, an effect that has previously been observed in microbial βγ-crystallins but not in those of vertebrates. This relationship between binding and protein stabilization makes the tunicate βγ-crystallin an interesting model for studying the evolution of the human βγ-crystallin. We also compare and contrast the binding sites of tunicate βγ-crystallin with those of other βγ-crystallins to develop hypotheses about the functional origin of the lack of Ca2+-binding sites in human crystallins.

Shelterin, a six-member complex, protects telomeres from nucleolytic attack and regulates their elongation by telomerase. Here, we have developed a strategy, called MICro-MS (Mapping Interfaces via Crosslinking-Mass Spectrometry), that combines crosslinking-mass spectrometry and phylogenetic analysis to identify contact sites within the complex. This strategy allowed identification of separation-of-function mutants of fission yeast Ccq1, Poz1, and Pot1 that selectively disrupt their respective interactions with Tpz1. The various telomere dysregulation phenotypes observed in these mutants further emphasize the critical regulatory roles of Tpz1-centered shelterin interactions in telomere homeostasis. Furthermore, the conservation between fission yeast Tpz1-Pot1 and human TPP1-POT1 interactions led us to map a human melanoma-associated POT1 mutation (A532P) to the TPP1-POT1 interface. Diminished TPP1-POT1 interaction caused by hPOT1-A532P may enable unregulated telomere extension, which, in turn, helps cancer cells to achieve replicative immortality. Therefore, our study reveals a connection between shelterin connectivity and tumorigenicity.

The γS1- and γS2-crystallins, structural eye lens proteins from the Antarctic toothfish (Dissostichus mawsoni), are homologues of the human lens protein γS-crystallin. Although γS1 has the higher thermal stability of the two, it is more susceptible to chemical denaturation by urea. The lower thermodynamic stability of both toothfish crystallins relative to human γS-crystallin is consistent with the current picture of how proteins from organisms endemic to perennially cold environments have achieved low-temperature functionality via greater structural flexibility. In some respects, the sequences of γS1- and γS2-crystallin are typical of psychrophilic proteins; however, their amino acid compositions also reflect their selection for a high refractive index increment. Like their counterparts in the human lens and those of mesophilic fish, both toothfish crystallins are relatively enriched in aromatic residues and methionine and exiguous in aliphatic residues. The sometimes contradictory requirements of selection for cold tolerance and high refractive index make the toothfish crystallins an excellent model system for further investigation of the biophysical properties of structural proteins.

The γS1- and γS2-crystallins,structural eye lens proteinsfrom the Antarctic toothfish (Dissostichus mawsoni), are homologues of the human lens protein γS-crystallin.Although γS1 has the higher thermal stability of the two, itis more susceptible to chemical denaturation by urea. The lower thermodynamicstability of both toothfish crystallins relative to human γS-crystallinis consistent with the current picture of how proteins from organismsendemic to perennially cold environments have achieved low-temperaturefunctionality via greater structural flexibility. In some respects,the sequences of γS1- and γS2-crystallin are typical ofpsychrophilic proteins; however, their amino acid compositions alsoreflect their selection for a high refractive index increment. Liketheir counterparts in the human lens and those of mesophilic fish,both toothfish crystallins are relatively enriched in aromatic residuesand methionine and exiguous in aliphatic residues. The sometimes contradictoryrequirements of selection for cold tolerance and high refractive indexmake the toothfish crystallins an excellent model system for furtherinvestigation of the biophysical properties of structural proteins. [ABSTRACT FROM AUTHOR]