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Pre-mRNA processing protein 40 (Prp40) is a nuclear protein that has a role in pre-mRNA splicing. Prp40 possesses two leucine-rich nuclear export signals, but little is known about the function of Prp40 in the export process. Another protein that has a role in protein export is centrin, a member of the EF-hand superfamily of Ca2+-binding proteins. Prp40 was found to be a centrin target by yeast-two-hybrid screening using both Homo sapiens centrin 2 (Hscen2) and Chlamydomonas reinhardtii centrin (Crcen). We identified a centrin-binding site within H. sapiens Prp40 homolog A (HsPrp40A), which contains a hydrophobic triad W1L4L8 that is known to be important in the interaction with centrin. This centrin-binding site is highly conserved within the first nuclear export signal consensus sequence identified in Saccharomyces cerevisiae Prp40. Here, we examine the interaction of HsPrp40A peptide (HsPrp40Ap) with both Hscen2 and Crcen by isothermal titration calorimetry. We employed the thermodynamic parameterization to estimate the polar and apolar surface area of the interface. In addition, we have defined the molecular mechanism of thermally induced unfolding and dissociation of the Crcen-HsPrp40Ap complex using two-dimensional infrared correlation spectroscopy. These complementary techniques showed for the first time, to our knowledge, that HsPrp40Ap interacts with centrin in vitro, supporting a coupled functional role for these proteins in pre-mRNA splicing.

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Trifluoroacetate (TFA) is a strong anion byproduct of solid-phase peptide synthesis. Fourier transform infrared (FT-IR) spectroscopy can be used to ascertain the presence of this excipient in peptide samples for quality assessment. TFA absorbs as a strong sharp peak (1675 cm-1) within the amide I' band of the spectral region. A peptide sample and the TFA excipient can be studied simultaneously by FT-IR and 2D IR correlation spectroscopies. In addition, these techniques are able to determine the effect of TFA on the stability of the peptide. Herein, we describe the spectroscopic characterization of the GXXG loop peptide (GXXGlp), which is present in KH domain containing proteins. The sequence of the Homo sapiens Krr1 GXXGlp is evolutionarily conserved (165KRRQRLIGPKGSTLKALELLTNCY189) and has been associated with ssDNA interaction and ribosome biogenesis. Our goal was to determine the structural elements present in this peptide and evaluate whether TFA affects the stability of GXXGlp during thermal stress. We observed differences in the molecular behavior of the synthetic peptide in the presence and absence of TFA at various peptide concentrations. Finally, 2D IR correlation spectroscopy was used for the determination of the unfolding process, mechanism and extent of peptide aggregation, and the effect of TFA on the stability of the peptide. This spectroscopic method can be applied to the characterization of any synthetic peptide.

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Trifluoroacetic acid (TFA) may be the cause of the bottleneck in high resolution structure determination for protein-peptide complexes. Fragment based drug design often involves the use of synthetic peptides which contain TFA (excipient). Our goal was to explore the effects of this excipient on a model complex: centrin-melittin-TFA. We performed Fourier transform infrared, two-dimensional infrared correlation spectroscopies and spectral simulations to analyze the amide I'/I'* band for the components and the ternary complex. Melittin (MLT) was observed to have increased helicity upon its interaction with centrin, followed by the thermally induced aggregation of MLT within the ternary complex in the TFA presence.

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Centrins are calcium binding proteins that belong to the EF-hand superfamily with diverse biological functions. Herein we present the first systematic study that establishes the relative stability of related centrins via complementary biophysical techniques. Our results define the stepwise molecular behavior of human centrins by two-dimensional infrared (2D IR) correlation spectroscopy, the change in heat capacity and enthalpy of denaturation by differential scanning calorimetry, and the relative stability of the helical regions of centrins by circular dichroism. More importantly, 2D IR correlation spectroscopy provides unique information about the similarities and differences in dynamics between these related proteins. The thermally induced molecular behavior of human centrins can be used to predict biological target interactions that have a relative dependence on calcium affinity. This information is essential for understanding why certain isoforms may be used to rescue a phenotype and therefore also for explaining the different functions these proteins may have in vivo. Furthermore, this comparative approach can be applied to the study of recombinant therapeutic protein candidates for the treatment of disease states.

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Centrin is a calcium binding protein (CaBP) belonging to the EF-hand superfamily. As with other proteins within this family, centrin is a calcium sensor with multiple biological target proteins. We chose to study Chlamydomonas reinhardtii centrin (Crcen) and its interaction with melittin (MLT) as a model for CaBP complexes due to its amphipathic properties. Our goal was to determine the molecular interactions that lead to centrin-MLT complex formation, their relative stability, and the conformational changes associated with the interaction, when compared to the single components. For this, we determined the thermodynamic parameters that define Crcen-MLT complex formation. Two-dimensional infrared (2D IR) correlation spectroscopy were used to study the amide I', I'*, and side chain bands for (13)C-Crcen, MLT, and the (13)C-Crcen-MLT complex. This approach resulted in the determination of MLT's increased helicity, while centrin was stabilized within the complex. Herein we provide the first complete molecular description of centrin-MLT complex formation and the dissociation process. Also, discussed is the first structure of a CaBP-MLT complex by X-ray crystallography, which shows that MLT has a different binding orientation than previously characterized centrin-bound peptides. Finally, all of the experimental results presented herein are consistent with centrin maintaining an extended conformation while interacting with MLT. The molecular implications of these results are: (1) the recognition of hydrophobic contacts as requirements for initial binding, (2) minimum electrostatic interactions within the C-terminal end of the peptide, and (3) van der Waals interactions within MLTs N-terminal end are required for complex formation.

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Chlamydomonas reinhardtii centrin is a member of the EF-hand calcium-binding superfamily. It is found in the basal body complex and is important for flagellar motility. Like other members of the EF-hand family, centrin interacts with and modulates the function of other proteins in a calcium-dependent manner. To understand how C. reinhardtii centrin interacts with its protein targets, it has been crystallized in the presence of the model peptide melittin and X-ray diffraction data have been collected to 2.2 A resolution. The crystals are orthorhombic, with unit-cell parameters a = 52.1, b = 114.4, c = 34.8 A, and are likely to belong to space group P2(1)2(1)2.

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Five highly homologous epidermal growth factor receptor ligands were studied by mass spectral analysis, hydrogen/deuterium (H/D) exchange via attenuated total reflectance Fourier transform-infrared spectroscopy, and two-dimensional correlation analysis. These studies were performed to determine the order of events during the exchange process, the extent of H/D exchange, and associated kinetics of exchange for a comparative analysis of these ligands. Furthermore, the secondary structure composition of amphiregulin (AR) and heparin-binding-epidermal growth factor (HB-EGF) was determined. All ligands were found to have similar contributions of 3(10)-helix and random coil with varying contributions of beta-sheets and beta-turns. The extent of exchange was 40%, 65%, 55%, 65%, and 98% for EGF, transforming growth factor-alpha (TGF-alpha), AR, HB-EGF, and epiregulin (ER), respectively. The rate constants were determined and classified as fast, intermediate, and slow: for EGF the 0.20 min(-1) (Tyr), 0.09 min(-1) (Arg, beta-turns), and 1.88 x 10(-3) min(-1) (beta-sheets and 3(10)-helix); and for TGF-alpha 0.91 min(-1) (Tyr), 0.27 min(-1) (Arg, beta-turns), and 1.41 x 10(-4) min(-1) (beta-sheets). The time constants for AR 0.47 min(-1) (Tyr), 0.04 min(-1) (Arg), and 1.00 x 10(-4) min(-1) (buried 3(10)-helix, beta-turns, and beta-sheets); for HB-EGF 0.89 min(-1) (Tyr), 0.14 min(-1) (Arg and 3(10)-helix), and 1.00 x 10(-3) min(-1) (buried 3(10)-helix, beta-sheets, and beta-turns); and for epiregulin 0.16 min(-1) (Tyr), 0.03 min(-1) (Arg), and 1.00 x 10(-4) min(-1) (3(10)-helix and beta-sheets). These results provide essential information toward understanding secondary structure, H/D exchange kinetics, and solvation of these epidermal growth factor receptor ligands in their unbound state.

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Centrin is an EF-hand calcium-binding protein found in microtubule organizing centers of organisms ranging from algae and yeast to man. Phosphorylation in the centrin C-terminal domain occurs in mitosis and is associated with alterations in contractile fibers. To obtain insight into the structural basis for the functional effect of phosphorylation, Chlamydomonas reinhardtii centrin C-terminal domain phosphorylated at Ser167 (pCRC-C) has been produced and characterized. The structure of pCRC-C was compared to the unmodified protein by NMR spectroscopy. The effect of phosphorylation on target binding was examined for the complex of pCRC-C and a 19 residue centrin-binding fragment of Kar1. Remarkably, the efficient and selective phosphorylation by PKA was suppressed in the complex. Moreover, comparisons of NMR chemical shift differences induced by phosphorylation reveal a greater effect from phosphorylation in the context of the Kar1 complex than for the free protein. These results directly demonstrate that phosphorylation modulates the structure and biochemical activities of centrin.

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Centrin is a conserved calcium binding protein belonging to the EF-hand superfamily with two independent structural domains. This protein is found to be phosphorylated near the carboxyl terminal end. Our goal was to perform a novel comparative study of phosphorylated and unphosphorylated centrin by Fourier transform infrared (FT-IR) spectroscopy, two-dimensional correlation spectroscopy (2D-COS) analysis and differential scanning calorimetry (DSC). To achieve this goal, we have bacterially expressed, isolated, purified and phosphorylated centrin. We verified the extent of phosphorylation to be >97% for centrin by MALDI MS analysis and determined the absence of aggregated protein. The thermal denaturation temperature and ΔCp were determined to be T(m) = 112.1 °C (ΔCp = 7.8 Kcal/mole/ΔC) and T(m) = 111.0°C (ΔCp = 5.0 Kcal/mole/°C) for holo-centrin and phosphorylated centrin, respectively. We have also described the molecular dynamics leading up to the thermal denaturation of the protein: for holo-centrin the vibrational modes associated with the calcium binding sites aspartates and glutamates, loops then the arginines, followed by the structured backbone vibrational modes the α-helix at 1635 cm(-1) then ß-sheet and finally the more exposed α-helix at 1650 cm(-1); while for phosphorylated centrin aspartate, glutamate and arginine, followed by the backbone associated vibrational modes α-helix (1650 cm(-1)), loop then the ß-sheet (1633 cm(-1)) and finally the α-helix (1637 cm(-1)). Therefore, the effect on domain stability due to phosphorylation at Ser(167) was observed in the loops as well as the α-helix at 1650 cm(-1).

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The liquid states and the liquid-liquid equilibrium of surfactant molecules forming an interphase between air and water have been considered using Monte Carlo computer simulations. Specifically, the expanded and compressed liquid phases observed for surfactant molecules were characterized as a function of pressure and temperature. Simple modified beadlike potentials were implemented in order to describe the interparticle forces between the hydrophobic and hydrophilic portions of surfactant molecules at the air/water interface. A simulation box was defined such that the monolayer was exposed to an externally applied lateral pressure in a modified isothermal-isobaric ensemble, whereas the water bath was modeled in a canonical ensemble. The simulation resembles the experimental setup used to measure lateral pressure (Pi) versus area isotherms obtained with Langmuir troughs. The applied lateral pressure-surface area phase diagram clearly showed the coexistence of the expanded and compressed liquid phases within certain temperature and pressure ranges. Distribution functions of distances and enthalpies for the monolayer were computed to clearly identify each liquid phase and the coexistence region.

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Chlamydomonas reinhardtii centrin is a 169-amino acid residue calcium binding protein belonging to the EF-hand protein superfamily. Centrin is associated with the microtubule organizing center (MTOC) in all eukaryotes, and in Chlamydomonas, centrin is a component of the flagellar basal body apparatus. Recombinant full-length centrin, calmodulin, and terminal domain fragments [Ccen-N (residues 1-94) and Ccen-C (residues 99-169)] were used to examine hydrogen-deuterium (H --> D) exchange dynamics using combined attenuated total reflectance (ATR) Fourier transform-infrared (FT-IR) spectroscopy, curve fit, and two-dimensional correlation analysis. Analysis of the Ccen-N and Ccen-C fragments allowed separation of domain specific solvent exchange events and together with analysis of the full-length proteins provides novel insight into domain accessibility to the aqueous environment and the internal dynamics of the protein.

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Centrin is a low molecular mass (20 kDa) protein that belongs to the EF-hand superfamily of calcium-binding proteins. Local and overall changes were investigated for interactions between cations and Chlamydomonas centrin using Fourier transform infrared (FT-IR) and circular dichroic (CD) spectroscopies. FT-IR spectral features studied included the amide I' band and the side-chain absorbances for aspartate residues located almost exclusively at the calcium-binding sites in the spectral region of 1700-1500 cm(-1). The amide I' band is exquisitely sensitive to changes in protein secondary structure and is observed to shift from 1626.5 to 1642.7 cm(-1) in the presence and absence of calcium. These spectral bands are complex and were further studied using two-dimensional Fourier transform infrared (2D-FT-IR) correlation along with curve-fitting routines. Using these methods the secondary structure contributions were determined for holocentrin and apocentrin. The alpha-helical content in centrin was determined to be 60%-53% in the presence and absence of cations, respectively. Furthermore, the beta-strand content was determined to be 12%-36%, while the random coil component remained almost constant at 7%-13.5% in the presence and absence of cations, respectively. Changes in the side-chain band are mostly due to the monodentate coordination of aspartate to the cation. A shift of approximately 4 cm(-1) (for the COO- antisymmetric stretch in Asp) from 1565 to 1569 cm(-1) is observed for apocentrin and holocentrin, respectively. Thermal dependence revealed reversible conformational transition temperatures for apocentrin at 37 degrees C and holocentrin at 45 degrees C, suggesting greater stability for holocentrin.

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