For years, antibodies (Abs) have been used as a paradigm for understanding how protein structure contributes to molecular recognition. However, with the ability to evolve Abs that recognize specific ...chromophores, they also have great potential as models for how protein dynamics contribute to molecular recognition. We previously raised murine Abs to different chromophores and, with the use of three-pulse photon echo peak shift spectroscopy, demonstrated that the immune system is capable of producing Abs with widely varying flexibility. We now report the characterization of the complexes formed between two Abs, 5D11 and 10A6, and the chromophoric ligand that they were evolved to recognize, 8-methoxypyrene-1,3,6-trisulfonic acid (MPTS). The sequences of the Ab genes indicate that they evolved from a common precursor. We also used a variety of spectroscopic methods to probe the photophysics and dynamics of the Ab–MPTS complexes and found that they are similar to each other but distinct from previously characterized anti-MPTS Abs. Structural studies revealed that this difference likely results from a unique mode of binding in which MPTS is sandwiched between the side chain of PheH98, which interacts with the chromophore via T-stacking, and the side chain of TrpL91, which interacts with the chromophore via parallel stacking. The T-stacking interaction appears to mediate relaxation on the picosecond time scale, while the parallel stacking appears to mediate relaxation on an ultrafast, femtosecond time scale, which dominates the response. The anti-MPTS Abs thus not only demonstrate the simultaneous use of the two limiting modes of stacking for molecular recognition, but also provide a unique opportunity to characterize how dynamics might contribute to molecular recognition. Both types of stacking are common in proteins and protein complexes where they may similarly contribute to dynamics and molecular recognition.
We designed β-strand peptides that stabilize integral membrane proteins (IMPs). β-strand peptides self-assemble in solution as filaments and become restructured upon association with IMPs; resulting ...IMP-β-strand peptide complexes resisted aggregation when diluted in detergent-free buffer and were visible as stable, single particles with low detergent background in electron micrographs. β-strand peptides enabled clear visualization of flexible conformations in the highly dynamic ATP-binding cassette (ABC) transporter MsbA.
Unspecific peroxygenases (UPO, EC 1.11.2.1) secreted by fungi open an efficient way to selectively oxyfunctionalize diverse organic substrates, including less‐activated hydrocarbons, by transferring ...peroxide‐borne oxygen. We investigated a cell‐free approach to incorporate epoxy and hydroxyl functionalities directly into the bulky molecule testosterone by a novel unspecific peroxygenase (UPO) that is produced by the ascomycetous fungus Chaetomium globosum in a complex medium rich in carbon and nitrogen. Purification by fast protein liquid chromatography revealed two enzyme fractions with the same molecular mass (36 kDa) and with specific activity of 4.4 to 12 U mg−1. Although the well‐known UPOs of Agrocybe aegerita (AaeUPO) and Marasmius rotula (MroUPO) failed to convert testosterone in a comparative study, the UPO of C. globosum (CglUPO) accepted testosterone as substrate and converted it with total turnover number (TTN) of up to 7000 into two oxygenated products: the 4,5‐epoxide of testosterone in β‐configuration and 16α‐hydroxytestosterone. The reaction performed on a 100 mg scale resulted in the formation of about 90 % of the epoxide and 10 % of the hydroxylation product, both of which could be isolated with purities above 96 %. Thus, CglUPO is a promising biocatalyst for the oxyfunctionalization of bulky steroids and it will be a useful tool for the synthesis of pharmaceutically relevant steroidal molecules.
Oxyfunctionalized testosterone: CglUPO, a novel peroxygenase from the ubiquitous ascomycetous fungus C. globosum, facilitates the direct incorporation of epoxy and hydroxy functionalities into the sterically demanding molecule testosterone. The reaction requires only hydrogen peroxide as co‐substrate. This peroxygenase and related enzymes might be useful tools for the synthesis of pharmaceutically relevant steroidal molecules.
Vibrational spectroscopy provides a direct route to the physicochemical characterization of molecules. While both IR and Raman spectroscopy have been used for decades to provide detailed ...characterizations of small molecules, similar studies with proteins are largely precluded due to spectral congestion. However, the vibrational spectra of proteins do include a “transparent window”, between ∼1800 and ∼2500 cm–1, and progress is now being made to develop site-specifically incorporated carbon–deuterium (C–D), cyano (CN), thiocyanate (SCN), and azide (N3) “transparent window vibrational probes” that absorb within this window and report on their environment to facilitate the characterization of proteins with small molecule-like detail. This Review opens with a brief discussion of the advantages and limitations of conventional vibrational spectroscopy and then discusses the strengths and weaknesses of the different transparent window vibrational probes, methods by which they may be site-specifically incorporated into peptides and proteins, and the physicochemical properties they may be used to study, including electrostatics, stability and folding, hydrogen bonding, protonation, solvation, dynamics, and interactions with inhibitors. The use of the probes to vibrationally image proteins and other biomolecules within cells is also discussed. We then present four case studies, focused on ketosteroid isomerase, the SH3 domain, dihydrofolate reductase, and cytochrome c, where the transparent window vibrational probes have already been used to elucidate important aspects of protein structure and function. The Review concludes by highlighting the current challenges and future potential of using transparent window vibrational probes to understand the evolution and function of proteins and other biomolecules.
Electrostatic and conformational heterogeneity make central contributions to protein function, but their experimental characterization requires a combination of spatial and temporal resolution that ...is challenging to achieve. Src homology 3 (SH3) domains mediate protein–protein interactions, and NMR studies have demonstrated that most possess conformational heterogeneity, which could be critical for their function. Here, we use the IR absorptions of carbon–deuterium (C–D) bonds site-selectively incorporated throughout the N-terminal SH3 domain from the murine adapter protein Crk-II to characterize its different microenvironments with high spatial and temporal resolution. The C–D absorptions are only differentiated in the folded state of the protein where they show evidence of significant environmental heterogeneity. However, the spectra of the folded state are independent of temperature, and upon thermal denaturation the protein undergoes a single, global unfolding transition. While some evidence of conformational heterogeneity is found within the peptide backbone, the majority of the environmental heterogeneity appears to result from electrostatics.
Hydrogen-bonds (H-bonds) between backbone N–H donors and CO acceptors are central to our understanding of protein structure and stability. However, while interactions between backbone N atoms and the ...N–H of the following residue are also common, they have been ignored as potential H-bonds due to their bent geometry and the assumption that the amide N is a poor H-bond acceptor. Recently, we reported indirect experimental evidence that these interactions constitute functional H-bonds. We now report a combined atoms in molecules and noncovalent interaction theoretical analysis of electron density that unambiguously supports the characterization of these interactions as H-bonds. The calculations further suggest that the N i+1–H···N i H-bonds are largely electrostatic in nature and, importantly, that they make a significant contribution to stability. Thus, given their apparently frequent occurrence, N i+1–H···N i H-bonds likely make critical, but previously unrecognized, contributions to protein structure and function.
The production of antibodies that selectively bind virtually any foreign compound is the hallmark of the immune system. While much is understood about how sequence diversity contributes to this ...remarkable feat of molecular recognition, little is known about how sequence diversity impacts antibody dynamics, which is also expected to contribute to molecular recognition. Toward this goal, we examined a panel of antibodies elicited to the chromophoric antigen fluorescein. On the basis of isothermal titration calorimetry, we selected six antibodies that bind fluorescein with diverse binding entropies, suggestive of varying contributions of dynamics to molecular recognition. Sequencing revealed that two pairs of antibodies employ homologous heavy chains that were derived from common germline genes, while the other two heavy chains and all six of the light chains were derived from different germline genes and are not homologous. Interestingly, more than half of all the somatic mutations acquired during affinity maturation among the six antibodies are located in positions unlikely to contact fluorescein directly. To quantify and compare the dynamics of the antibody−fluorescein complexes, three-pulse photon echo peak shift and transient grating spectroscopy were employed. All of the antibodies exhibited motions on three distinct time scales, ultrafast motions on the <100 fs time scale, diffusive motions on the picosecond time scale, and motions that occur on time scales longer than nanoseconds and thus appear static. However, the exact frequency of the picosecond time scale motion and the relative contribution of the different motions vary significantly among the antibody−chromophore complexes, revealing a high level of dynamic diversity. Using a hierarchical model, we relate the data to features of the antibodies’ energy landscapes as well as their flexibility in terms of elasticity and plasticity. In all, the data provide a consistent picture of antibody flexibility, which interestingly appears to be correlated with binding entropy as well as with germline gene use and the mutations introduced during affinity maturation. The data also provide a gauge of the dynamic diversity of the antibody repertoire and suggest that this diversity might contribute to molecular recognition by facilitating the recognition of the broadest range of foreign molecules.
Schizophrenia is a devastating disease that arises on the background of genetic predisposition and environmental risk factors, such as early life stress (ELS). In this study, we show that ELS-induced ...schizophrenia-like phenotypes in mice correlate with a widespread increase of histone-deacetylase 1 (Hdac1) expression that is linked to altered DNA methylation. Hdac1 overexpression in neurons of the medial prefrontal cortex, but not in the dorsal or ventral hippocampus, mimics schizophrenia-like phenotypes induced by ELS. Systemic administration of an HDAC inhibitor rescues the detrimental effects of ELS when applied after the manifestation of disease phenotypes. In addition to the hippocampus and prefrontal cortex, mice subjected to ELS exhibit increased Hdac1 expression in blood. Moreover, Hdac1 levels are increased in blood samples from patients with schizophrenia who had encountered ELS, compared with patients without ELS experience. Our data suggest that HDAC1 inhibition should be considered as a therapeutic approach to treat schizophrenia.
Modulation of frontal-midline theta by neurofeedback Enriquez-Geppert, Stefanie; Huster, René J.; Scharfenort, Robert ...
Biological psychology,
January 2014, 2014-Jan, 2014-01-00, 20140101, Letnik:
95
Journal Article
Recenzirano
•We demonstrated the trainability of frontal-midline theta by neurofeedback.•We developed an individual, eight-session, gap-spaced neurofeedback training.•Fm-theta power was significantly enhanced ...compared to an active control group.•We report subjects’ self-reported motivation, commitment and training difficulty.
Cortical oscillations demonstrate a relationship with cognition. Moreover, they also exhibit associations with task performance and psychiatric mental disorders. This being the case, the modification of oscillations has become one of the key interests of neuroscientific approaches for cognitive enhancement. For such kind of alterations, neurofeedback (NF) of brain activity constitutes a promising tool. Concerning specific higher cognitive functions, frontal-midline theta (fm-theta) has been suggested as an important indicator of relevant brain processes. This paper presents a novel approach for an individualized, eight-session NF training to enhance fm-theta. An individual's dominant fm-theta frequency was determined based on experiments tapping executive functions. Effects of the actual NF training were compared to a pseudo-NF training. Participants of the pseudo-NF training experienced a comparable degree of motivation and commitment as the subjects of the actual NF training, but found the “training” slightly easier. In comparison to the pseudo-NF training, proper NF training significantly enhanced fm-theta amplitude in the actual training sessions, as well as during the whole course of training. However, unspecific changes in the alpha and beta frequency ranges found with both the actual NF and the pseudo-NF training groups emphasize the relevance of active control groups for neurofeedback studies.
While adaptive mutations can bestow new functions on proteins via the introduction or optimization of reactive centers, or other structural changes, a role for the optimization of protein dynamics ...also seems likely but has been more difficult to evaluate. Antibody (Ab) affinity maturation is an example of adaptive evolution wherein the adaptive mutations may be identified and Abs may be raised to specific targets that facilitate the characterization of protein dynamics. Here, we report the characterization of three affinity matured Abs that evolved from a common germline precursor to bind the chromophoric antigen (Ag), 8-methoxypyrene-1,3,6-trisulfonate (MPTS). In addition to characterizing the sequence, molecular recognition, and structure of each Ab, we characterized the dynamics of each complex by determining their mechanical response to an applied force via three-pulse photon echo peak shift (3PEPS) spectroscopy and deconvoluting the response into elastic, anelastic, and plastic components. We find that for one Ab, affinity maturation was accomplished via the introduction of a single functional group that mediates a direct contact with MPTS and results in a complex with little anelasticity or plasticity. In the other two cases, more mutations were introduced but none directly contact MPTS, and while their effects on structure are subtle, their effects on anelasticity and plasticity are significant, with the level of plasticity correlated with specificity, suggesting that the optimization of protein dynamics may have contributed to affinity maturation. A similar optimization of structure and dynamics may contribute to the evolution of other proteins.
PDF
