Alignment and Defect Structures in Oriented Phosphatidylcholine Multilayers

The alignment of dilauryl-, dimyristoyl-, and dipalmitoylphosphatidylcholine at various water concentrations into large oriented monodomain multilayers by annealing at elevated temperatures (Powers and Clark, 1975, Proc. Natl. Acad. Sci. U.S.A. 72:840; Powers and Pershan. 1977. Biophys. J. 20:137) is accompanied by the formation and subsequent dissolution of various defect structures. Some of these defects appear similar to those observed in thermotropic and other lyotropic liquid crystals, reflecting the lamellar structure of these materials. The formation and evolution of defects during the alignment of the lipids into the defect-free, monodomain, multilamellar geometry is studied using polarized microscopy. A combination of polarized and dark-field microscopy facilitated characterization of the defects; specific structural models are proposed. A new alignment technique involving compression and dilation of the lipid, which effects sample alignment at temperatures that are lower than those required by the Powers technique, is described. Lower temperature alignment avoids thermal decomposition that will sometimes occur if the lipid is maintained at elevated temperatures for prolonged periods. With this technique, samples $(80 \mu m thick)$ of dilaurylphosphatidylcholine with 20% water by weight were aligned at room temperature.Engineering and Applied Science

A Soluble C1b Protein and Its Regulation of Soluble Type 7 Adenylyl Cyclase†

Adenylyl cyclase (AC) is a prototypical cell-signaling molecule expressed in virtually all organisms from bacteria to man. While C1b, a poorly conserved region within mammalian AC, has been implicated in numerous isoform-specific regulatory properties, no one has purified the C1b region as a functional protein to homogeneity in order to study its role in enzyme function. We hypothesize that C1b is an internal regulatory subunit. To pursue this hypothesis, we constructed several soluble C1b proteins from type VII AC, arriving at one, 7C1b-S, which can be expressed and purified from Escherichia coli. 7C1b-S is relatively stable, as demonstrated by limited proteolytic analysis, circular dichroism, and UV Raman spectroscopy. Using size-exclusion chromatography and co-immunoprecipitation we demonstrate that 7C1b-S interacts with a cardinal activator of AC (Gsα) and with the conserved first catalytic domain (C1a) of type VII AC. We show that 7C1b-S inhibits Gsα-stimulated and Gsα-forskolin stimulated activity in our soluble ACVII model system. On the basis of these results, we suggest that 7C1b-S meets basic criteria to serve as a model protein for the C1b region and may be used as a prototype to develop other isoform C1b soluble model proteins to further investigate the role of this domain in isoform-specific regulation of adenylyl cyclase

Peptide secondary structure folding reaction coordinate: Correlation between uv raman amide iii frequency, psi ramachandran angle, and hydrogen bonding

We used UV resonance Raman (UVRR) spectroscopy to quantitatively correlate the peptide bond AmIII 3 frequency to its Ψ Ramachandran angle and to the number and types of amide hydrogen bonds at different temperatures. This information allows us to develop a family of relationships to directly estimate the Ψ Ramachandran angle from measured UVRR AmIII 3 frequencies for peptide bonds (PBs) with known hydrogen bonding (HB). These relationships ignore the more modest Φ Ramachandran angle dependence and allow determination of the Ψ angle with a standard error of (8°, if the HB state of a PB is known. This is normally the case if a known secondary structure motif is studied. Further, if the HB state of a PB in water is unknown, the extreme alterations in such a state could additionally bias the Ψ angle by (6°. The resulting ability to measure Ψ spectroscopically will enable new incisive protein conformational studies, especially in the field of protein folding. This is because any attempt to understand reaction mechanisms requires elucidation of the relevant reaction coordinate(s). The Ψ angle is precisely the reaction coordinate that determines secondary structure changes. As shown elsewhere (Mikhonin et al. J. Am. Chem. Soc. 2005, 127, 7712), this correlation can be used to determine portions of the energy landscape along the Ψ reaction coordinate. Introduction The various techniques of molecular spectroscopy constitute the toolset used by scientists for investigating molecular conformations and reaction mechanisms. These various spectroscopic techniques require quantitative correlations between the spectral parameters measured and the molecular conformational parameters. NMR and especially multidimensional NMR techniques are certainly the most powerful spectroscopic methods for solution studies. 1928 J. Phys. Chem. B 2006, 110, 1928-1943 10 by C R -D. We are optimistic that these relationships will be very useful for protein conformational studies, especially in the field of protein folding. This is because any attempt to understand reaction mechanisms, such as, for example, protein folding, requires elucidation of the relevant reaction coordinate(s). The Ψ angle is precisely the reaction coordinate that determines secondary structure changes. As shown elsewhere 60 the correlation we propose can be used to experimentally determine features of the energy landscape along this Ψ reaction coordinate. Such an experimental insight into a protein conformation and energy landscape is crucially needed, since there are still a lot of unresolved questions regarding the theoretical modeling of protein folding despite remarkable recent achievments. As described elsewhere, 43 the 21-residue alanine-based peptide AAAAA(AAARA) 3 A (AP) was prepared (HPLC pure) at the Pittsburgh Peptide Facility by using the solid-state peptide synthesis method. The AP solutions in water contained 1 mg/ mL concentrations of AP, and 0.2 M concentrations of sodium perchlorate, which was used as an internal intensity and frequency standard. UV Resonance Raman Instrumentation. The Raman instrumentation has been described in detail elsewhere. Results and Discussion Dependence of AmIII 3 Frequency on Ramachandran Angles and Hydrogen Bonding. The amide III (AmIII) band region is complex. We recently examined this spectral region in detail and identified a band, which we call AmIII 3 and which is most sensitive to the peptide bond conformation. Peptide Secondary Structure Folding Reaction Coordinate J. Phys. Chem. B, Vol. 110, No. 4, 2006 1929 gives rise to strong N-H to C R -H bend coupling. In contrast, for R-helix-like Ψ and Φ Ramachandran angles the N-H and C R -H bonds are approximately trans The physical origin of this Ψ angle AmIII 3 frequency dependence is that the hydrogen van der Waals radii in the C R -H and N-H bonds are in contact for positive Ψ angles Relative Impact of the Ψ and Φ Ramachandran Angles on the AmIII 3 Frequency. Although the projections of the N-H and C R -H bending motions on each other (and as a result the degree of coupling between them) depend on both the Ψ and Φ Ramachandran angles, an examination of a model of a peptide bond Asher et al. Mirkin and Krimm 73 theoretically examined the Ψ and Φ frequency dependence of the AmIII band of "alanine dipeptide" (N-acetyl-L-alanine-N-methylamide). They concentrated on peptide bond 2, whose frequencies were close to those measured experimentally. Although Mirkin and Krimm claim in their conclusions, that AmIII frequency shows strong dependence on both Ψ and Φ Ramachandran angles, we note that the impact of changes in the Φ angle is relatively modest if we only include the allowed regions of the Ramachandran plot In the allowed regions of Ramachandran plot, Mirkin and Krimm 73 calculated a 25-40 cm -1 AmIII frequency span over the allowed Ψ angles for fixed Φ angles In addition, the largest 16 cm -1 span in the AmIII frequency with Φ angle occurs in an almost forbidden region of the Ramachandran plot between the -sheet and R-helical regions (at Φ angles of -134°and -90°and Ψ angle of 60°, Figures 47 for details). Black line (s): Fit of calculated points using the eq 3 (see text for detail). Note: Grey regions show the forbidden and/or nearly forbidden Ψ Ramachandran angles based on recent Ramachandran plots. 1930 J. Phys. Chem. B, Vol. 110, No. 4, 2006 Mikhonin et al. 3 and 4). In contrast, in the R-helical region of the Ramachandran plot the AmIII frequency of alanine dipeptide shows no more than 8 cm -1 Φ angular span, while in the -strand region of the Ramachandran plot the AmIII frequency shows no more than 6 cm -1 Φ dependence Ianoul et al.'s 59 combined experimental and theoretical studies of Ac-X-OCH 3 (X ) Val, Ile, Leu, Lys, Ala) revealed a 9 cm -1 AmIII 3 frequency shift upon an 18°increase of the Φ Ramachandran angle from -96 to -78°. In addition, Ianoul et al. also performed theoretical calculations for Ala-Ala at a fixed R-helix-like Ψ angle of -21°and calculated only a 3 cm -1 AmIII 3 frequency upshift upon the 20°increase of Φ angle from -95°to -75°. Thus, Ianoul et al. never observed more than a 9 cm -1 shift of AmIII 3 frequency due to variation of the Φ Ramachandran angle. In addition, we recently 60 measured the UVRR AmIII 3 frequencies of two different secondary structure conformations in aqueous solutions with very similar Φ angles, but very different Ψ angles. Specifically, an equimolar mixture of PLL and PGA forms an antiparallel -sheet 60 (Ψ ≈ 135°, Φ ≈ -139°), which shows an AmIII 3 frequency at 1227 cm -1 . In contrast individual PLL and PGA samples form extended 2.5 1 -helices 60 (Ψ ≈ 170°, Φ ≈ -130°), which show AmIII 3 frequencies at ∼1271 cm -1 . To summarize, the total Φ angular span of the AmIII 3 frequencies appears experimentally 59 to be no more than 9 cm -1 and no more than 16 cm -1 in the allowed regions of the Ramachandran plot from theoretical calculations. Thus, we conclude that Ψ Ramachandran angular dependence of the AmIII 3 frequency dominates the Φ angular dependence in the allowed regions of Ramachandran plot. If we totally neglect the Φ angular dependence of AmIII 3 frequency, this could enable an error in the Ψ-dependent AmIII 3 frequency of no more than (8 cm -1 (since the total Φ angular span of AmIII 3 frequencies no higher than 16 cm -1 , Formation of PB-water and PB-PB HBs upshift the AmIII 3 frequency, in part, due to the resulting increased C(O)dN double ν III3 (ψ,φ,HB P-P ,HB P-W ,T) = ν III3 (ψ,HB P-P ,HB P-W ,T) (2) ν III3 (ψ,HB P-P ,HB P-W ,T) = {ν 0 -A sin(ψ -R 0 )} + Δν III3 (HB P-P ,HB P-W ,T) (4) Peptide Secondary Structure Folding Reaction Coordinate J. Phys. Chem. B, Vol. 110, No. 4, 2006 1931 bond character. 75, The relationships given below by eqs 5 (for non-HB PB in a vacuum), 6A-D (PB in aqueous solutions), and 7A-C (PB in the absence of water) are shown in Correlation between AmIII 3 Frequency and Ψ Ramachandran Angle in the Absence of HB. We measured the UVR AmIII 3 frequencies for the AP R-helix 42,89 (∼1263 cm -1 , 0°C), XAO PPII 42,43 (1247 cm -1 , 0°C), PLL and PGA 2.5 1 -helix 60 (∼1271 cm -1 , 0°C), and PLL-PGA mixture antiparallel -sheet 60 (∼1227 cm -1 , 0°C) conformations of different polypeptides in aqueous solutions. Each of these conformations has known Ramachandran angles We can calculate the AmIII 3 frequencies that would result from the above peptide conformations in the fictitious case where the PB did not partake in any HB at all. This would be done by subtracting the HB-induced AmIII 3 frequency shifts By fitting the above four "non-HB" data points to eq 3, we obtain the following semiempirical relationship, which relates 1932 J. Phys. Chem. B, Vol. 110, No. 4, 2006 Mikhonin et al. the AmIII 3 frequency to the Ψ Ramachandran angle dependent coupling between N-H and C R -H bending motions The blue curve in Correlation of AmIII 3 Frequency and Ψ Ramachandran Angle for Two-End-On PB-PB HBs: Infinite r-Helix, Interior Strands of -Sheet in Water. Each PB in infinitely long R-helices and in interior strands of multistranded -sheets in aqueous solutions (Appendix, The green curve in The magenta curve in Peptide Secondary Structure Folding Reaction Coordinate J. Phys. Chem. B, Vol. 110, No. 4, 2006 1933 cm -1 HB-induced upshift as well as the temperature-dependent term to eq 5 and write The black curve in Correlation of AmIII 3 Frequency and Ψ Angle for a PB in Water If Its HB State Is Unknown. If the HB state of a PB in aqueous solution is unknown, we suggest the use of eq 6E, which is the "average" of eqs 6A-D. This will minimize the error in determination of the Ψ Ramachandran angle and will allow the estimation of the Ψ angle with the error bounds discussed below. Correlation between AmIII 3 Frequency and Ψ Ramachandran Angle in Peptide Crystals. Figures 6 and 7 show that the crystal data appear to roughly follow the sinusoidal relationship between the AmIII 3 frequency and the Ψ Ramachandran angle (see red dashed curve in Anhydrous r-Helical and -Sheet Conformations. If we dehydrate a two-end-on PB-PB HB R-helical conformation, we will see a 5 cm -1 AmIII 3 frequency downshift due to the loss of hydrogen bonding to the normally present sheath of water. The In the case of a PB where only the CdO group is involved in PB-PB HB, we estimate that the AmIII 3 frequency is 12 cm -1 upshifted with respect to non-HB PB (Appendix, The In the case of PB, where only the NH group is PB-PB HB, we estimate the AmIII 3 frequency to be 35 cm -1 upshifted with respect to non-HB PB (Appendix, The black curve in Thus, the families of eqs 6A-D and 7A-C predict the correlation between the AmIII 3 frequency and the Ψ angle for the common conformations of peptides and proteins. If the HB is known for a particular PB, the appropriate equation can be used to determine its Ψ angle from the observed AmIII 3 frequency. In the case where the HB state of a PB in aqueous solution is unknown, one can use eq 6E. These relationships will become less accurate if the PB has an unusual Φ angle or unusual HB pattern (see below). Prediction of UVRR AmIII 3 Frequencies of Other Secondary Structures. On the basis of the known Ψ Ramachandran angle and HB patterns, we can predict the AmIII 3 frequencies of other secondary structures such as the π-helix, 3 10 -helix UVRR spectra of HEWL amyloid fibrils, 100 which are dominated by -sheet conformations contain three spectroscopic features in the AmIII 3 region: ∼1210, ∼1230, and ∼1255 cm -1 . The dominating ∼1230 cm -1 feature certainly derives from antiparallel -sheet, though a minor contribution of several turn conformations is also possible The error associated with neglecting the Φ angle also gives rise to the uncertainty in the Ψ angle determination. Ianoul et al. Additional bias can occur if we do not know the HB state of a PB in water. This could give rise to a bias of the AmIII 3 frequency of (6 cm -1 , which would lead to a Ψ angle bias of (6°in eq 6E. Thus, a typical UV Raman measurement of a typical sample would find a random error of e(8°in the Ψ angle, assuming a known HB state. However, extreme alterations in the unknown HB state of a PB in water could additionally bias the Ψ angle by (6°. Conclusions We used UV resonance Raman spectroscopy to investigate the dependence of the AmIII 3 frequency on the Ψ Ramachandran angle and on the nature of PB HBs. These results allow us to formulate relationships that allow us to estimate the Ψ Ramachandran angles from observed AmIII 3 frequencies for both aqueous solutions of peptides and proteins as well as for the anhydrous states of peptides and proteins. A typical Raman measurement of a typical sample would find a random error of e(8°in the Ψ angle, assuming a known HB state. However, if the HB state of a PB in water is unknown, extreme alterations in such a state could additionally bias the Ψ angle by (6°. We are optimistic that these relationships will be very useful for protein conformational studies, especially in the field of protein folding. This is because any attempt to understand reaction mechanisms, such as protein folding, requires elucidation of the relevant reaction coordinate(s). The Ψ angle is precisely the reaction coordinate that determines secondary structure changes. As shown elsewhere, 60 the correlation we propose can be used to determine features of the energy landscape along this Ψ reaction coordinate

Aqueous alteration processes in Jezero crater, Mars—implications for organic geochemistry

The Perseverance rover landed in Jezero crater, Mars, in February 2021. We used the Scanning Habitable Environments with Raman and Luminescence for Organics and Chemicals (SHERLOC) instrument to perform deep-ultraviolet Raman and fluorescence spectroscopy of three rocks within the crater. We identify evidence for two distinct ancient aqueous environments at different times. Reactions with liquid water formed carbonates in an olivine-rich igneous rock. A sulfate-perchlorate mixture is present in the rocks, which probably formed by later modifications of the rocks by brine. Fluorescence signatures consistent with aromatic organic compounds occur throughout these rocks and are preserved in minerals related to both aqueous environments

Development of Novel, Simple, Multianalyte Sensors for Remote Environmental Analysis

We will develop simple, inexpensive new chemical sensing materials which can be used as visual color test strips to sensitively and selectively report on the concentration and identity of environmental pollutants such as cations of Pb, U, Pu, Sr, Hg, Cs, Co as well as other species. We will develop inexpensive chemical test strips which can be immersed in water to determine these analytes in the field. We will also develop arrays of these chemical sensing materials which will be attached to fiber optic bundles to be used as rugged multichannel optrodes to simultaneously monitor numerous analytes remotely in hostile environments. These sensing materials are based on the intelligent polymerized crystalline colloidal array (PCCA) technology we recently developed. This sensing motif utilizes a mesoscopically periodic array of colloidal particles polymerized into an acrylamide hydrogel. This array Bragg diffracts light in the visible spectral region due to the periodic array of colloidal particles. This material also contains chelating agents for the analytes of interest. When an analyte binds, its charge is immobilized within the acrylamide hydrogel. The resulting Donnan potential causes an osmotic pressure which swells the array proportional to the concentration of analyte bound. The diffracted wavelength shifts and the color changes. The change in the wavelength diffracted reports on the identity and concentration of the target analyte. Our successful development of these simple, inexpensive highly sensitive chemical sensing optrodes, which are easily coupled to simple optical instrumentation, could revolutionize environmental monitoring. In addition, we will develop highly rugged versions, which can be attached to core penetrometers and which can be used to determine analytes in buried core samples. Research Progress and Implications This report summarizes work after 21 months of a three year project. We have developed a new method to crosslink our PCCA sensing materials with disulfide bridges. We cleave these bridges to expose thiols which complex with heavy metals. In the presence of appropriate analytes, two or more of the thiols will complex the analyte and form a crosslink. As these crosslinks are formed, the gel will shrink. We have demonstrated this sensing motif with arsenous acid, including the reversibility of the sensing response. We are now investigating the utility of this sensing material for other heavy metals. Further work is planned to increase the sensor specificity by attempting to tailor the binding sites for specific analytes. Ion screening and templating are just two of the possible routes to increase selectivity

Development of Novel, Simple Multianalyte Sensors for Remote Environmental Analysis

Advancement of our polymerized crystalline colloidal array chemical sensing technology. They have dramatically advanced their polymerized crystalline colloidal array chemical sensing technology. They fabricated nonselective sensors for determining pH and ionic strength. They also developed selective sensors for glucose and organophosphorus mimics of nerve gas agents. They developed a trace sensor for cations in water which utilized a novel crosslinking sensing motif. In all of these cases they have been able to theoretically model their sensor response by extending hydrogel volume phase transition theory. They also developed transient sampling methods to allow their ion sensing methods to operate at high ionic strengths. They also developed a novel optrode to provide for simple sampling