Improving cryo-EM grids for amyloid fibrils using interface-active solutions and spectator proteins.

Preparation of cryoelectron microscopy (cryo-EM) grids for imaging of amyloid fibrils is notoriously challenging. The human islet amyloid polypeptide (hIAPP) serves as a notable example, as the majority of reported structures have relied on the use of nonphysiological pH buffers, N-terminal tags, and seeding. This highlights the need for more efficient, reproducible methodologies that can elucidate amyloid fibril structures formed under diverse conditions. In this work, we demonstrate that the distribution of fibrils on cryo-EM grids is predominantly determined by the solution composition, which is critical for the stability of thin vitreous ice films. We discover that, among physiological pH buffers, HEPES uniquely enhances the distribution of fibrils on cryo-EM grids and improves the stability of ice layers. This improvement is attributed to direct interactions between HEPES molecules and hIAPP, effectively minimizing the tendency of hIAPP to form dense clusters in solutions and preventing ice nucleation. Furthermore, we provide additional support for the idea that denatured protein monolayers forming at the interface are also capable of eliciting a surfactant-like effect, leading to improved particle coverage. This phenomenon is illustrated by the addition of nonamyloidogenic rat IAPP (rIAPP) to a solution of preaggregated hIAPP just before the freezing process. The resultant grids, supplemented with this "spectator protein", exhibit notably enhanced coverage and improved ice quality. Unlike conventional surfactants, rIAPP is additionally capable of disentangling the dense clusters formed by hIAPP. By applying the proposed strategies, we have resolved the structure of the dominant hIAPP polymorph, formed in vitro at pH 7.4, to a final resolution of 4 Å. The advances in grid preparation presented in this work hold significant promise for enabling structural determination of amyloid proteins which are particularly resistant to conventional grid preparation techniques.

Protein cohabitation: long-term immunoglobulin G storage at room temperature.

Long-term functional storage of therapeutic proteins at room temperature has been an eternal challenge. Inspired by the cellular cooperativity of proteins, we have taken a step forward to address this challenge by cohabitating Immunoglobulin G (IgG1) with a food protein gelatin in the solid-state at room temperature. Interestingly, IgG1 remained functionally active for a record 14 months revealed from the western-blot assay. Further quantification by HP-LC analysis showed 100% structural integrity of IgG1 with no degradation in the gelatin matrix during this period. The developed formulation has a direct application in oral medical nutrition therapy to cure gastrointestinal microbial infections. Also the strategy provides a robust energy economic alternative to the protein engineering methods for long-term functional storage of therapeutic proteins at room temperature.

Ground-state heterogeneity and vibrational energy redistribution in bacterial phytochrome observed with femtosecond 2D IR spectroscopy.

Phytochromes belong to a group of photoreceptor proteins containing a covalently bound biliverdin chromophore that inter-converts between two isomeric forms upon photoexcitation. The existence and stability of the photocycle products are largely determined by the protein sequence and the presence of conserved hydrogen-bonding interactions in the vicinity of the chromophore. The vibrational signatures of biliverdin, however, are often weak and obscured under more intense protein bands, limiting spectroscopic studies of its non-transient signals. In this study, we apply isotope-labeling techniques to isolate the vibrational bands from the protein-bound chromophore of the bacterial phytochrome from Deinococcus radiodurans. We elucidate the structure and ultrafast dynamics of the chromophore with 2D infra-red (IR) spectroscopy and molecular dynamics simulations. The carbonyl stretch vibrations of the pyrrole rings show the heterogeneous distribution of hydrogen-bonding structures, which exhibit distinct ultrafast relaxation dynamics. Moreover, we resolve a previously undetected 1678 cm-1 band that is strongly coupled to the A- and D-ring of biliverdin and demonstrate the presence of complex vibrational redistribution pathways between the biliverdin modes with relaxation-assisted measurements of 2D IR cross peaks. In summary, we expect 2D IR spectroscopy to be useful in explaining how point mutations in the protein sequence affect the hydrogen-bonding structure around the chromophore and consequently its ability to photoisomerize to the light-activated states.

Transient IR spectroscopy identifies key interactions and unravels new intermediates in the photocycle of a bacterial phytochrome.

Phytochromes are photosensory proteins in plants, fungi, and bacteria, which detect red- and far-red light. They undergo a transition between the resting (Pr) and photoactivated (Pfr) states. In bacterial phytochromes, the Pr-to-Pfr transition is facilitated by two intermediate states, called Lumi-R and Meta-R. The molecular structures of the protein in these states are not known and the molecular mechanism of photoconversion is not understood. Here, we apply transient infrared absorption spectroscopy to study the photocycle of the wild-type and Y263F mutant of the phytochrome from Deinococcus radiodurans (DrBphP) from nano- to milliseconds. We identify two sequentially forming Lumi-R states which differ in the local structure surrounding the carbonyl group of the biliverdin D-ring. We also find that the tyrosine at position 263 alters local structure and dynamics around the D-ring and causes an increased rate of Pfr formation. The results shed new light on the mechanism of light-signalling in phytochrome proteins.

Ultrafast Chemical Exchange Dynamics of Hydrogen Bonds Observed via Isonitrile Infrared Sensors: Implications for Biomolecular Studies.

Local probes are indispensable to study protein structure and dynamics with site-specificity. The isonitrile functional group is a highly sensitive and H-bonding interaction-specific probe. Isonitriles exhibit large spectral shifts and transition dipole moment changes upon H-bonding while being weakly affected by solvent polarity. These unique properties allow a clear separation of distinct subpopulations of interacting species and an elucidation of their ultrafast dynamics with two-dimensional infrared (2D-IR) spectroscopy. Here, we apply 2D-IR to quantify the picosecond chemical exchange dynamics of solute-solvent complexes forming between isonitrile-derivatized alanine and fluorinated ethanol, where the degree of fluorination controls their H-bond-donating ability. We show that the molecules undergo faster exchange in the presence of more acidic H-bond donors, indicating that the exchange process is primarily dependent on the nature of solvent-solvent interactions. We foresee isonitrile as a highly promising probe for studying of H-bonds dynamics in the active site of enzymes.