Aggregated β-sheet structures are associated with amyloid and prion diseases. Techniques capable of revealing detailed structural and dynamical information on β-sheet structure are thus of great biomedical and biophysical interest. In this work, the infrared (IR) amide I spectral characteristics of stacked β-sheets were modeled using the transition dipole coupling model. For a test set of β-sheet stacks, the simulated amide I spectrum was analyzed with respect to the following parameters; intersheet distance, relative rotation of the sheets with respect to each other and the effect of number of sheets stacked. The amide I maximum shifts about 5 cm(-1) to higher wavenumbers when the intersheet distance between two identical β-sheets decreases from 20 to 5 Å. Rotation around the normal of one of the sheets relative to the other results in maximum intersheet coupling near 0° and 180°. Upon of rotation from 0° to 90° at an intersheet distance of 9 Å, the amide I maximum shifts about 3 cm(-1). Tilting of one of the sheets by 30° from the normal results in a shift of the amide I maximum by less than 1 cm(-1). When stacking several β-sheets along the normal, the amide I maximum shifts to higher wavenumbers with increasing stack size. The amide I maximum shifts about 6 cm(-1) when stacking four sheets with an intersheet distance of 9 Å. The study provides an aid in the interpretation of the IR amide I region for experiments involving β-sheets and creates awareness of the many effects that determine the spectrum of β-sheet structures.

A new simulation protocol for the prediction of the infrared absorption of the amide I vibration of proteins was developed. The method incorporates known effects on the intrinsic frequencies (backbone conformation, inter-peptide and peptide-solvent hydrogen bonding) and couplings (nearest neighbor coupling, transition dipole coupling) of amide I oscillators in a parametrized manner. Model parameters for the simulation of amide I spectra were determined through fitting and optimization of simulated spectra to experimentally measured infrared spectra of 44 proteins that represent maximum structural variation in terms of different folds and secondary structure contents. Prediction of protein spectra using the optimized parameters resulted in good agreement with experimental spectra and in a considerable improvement compared to a description involving only transition dipole coupling.

Protonation of the Ca(2+) ligands of the SR Ca(2+)-ATPase (SERCA1a) was studied by a combination of rapid scan FTIR spectroscopy and electrostatic calculations. With FTIR spectroscopy, we investigated the pH dependence of C=O bands of the Ca(2+)-free phosphoenzyme (E2P) and obtained direct experimental evidence for the protonation of carboxyl groups upon Ca(2+) release. At least three of the infrared signals from protonated carboxyl groups of E2P are pH dependent with pK(a) values near 8.3: a band at 1758 cm(-1) characteristic of nonhydrogen-bonded carbonyl groups, a shoulder at 1720 cm(-1), and part of a band at 1710 cm(-1), both characteristic of hydrogen-bonded carbonyl groups. The bands are thus assigned to H(+) binding residues, some of which are involved in H(+) countertransport. At pH 9, bands at 1743 and 1710 cm(-1) remain which we do not attribute to Ca(2+)/H(+) exchange. We also obtained evidence for a pH-dependent conformational change in beta-sheet or turn structures of the ATPase. With MCCE on the E2P analog E2(TG+MgF(4)(2-)), we assigned infrared bands to specific residues and analyzed whether or not the carbonyl groups of the acidic Ca(2+) ligands are hydrogen bonded. The carbonyl groups of Glu(771), Asp(800), and Glu(908) were found to be hydrogen bonded and will thus contribute to the lower wave number bands. The carbonyl group of some side-chain conformations of Asp(800) is left without a hydrogen-bonding partner; they will therefore contribute to the higher wave number band.

The sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) pumps Ca(2+) and countertransport protons. Proton pathways in the Ca(2+) bound and Ca(2+)-free states are suggested based on an analysis of crystal structures to which water molecules were added. The pathways are indicated by chains of water molecules that interact favorably with the protein. In the Ca(2+) bound state Ca(2)E1, one of the proposed Ca(2+) entry paths is suggested to operate additionally or alternatively as proton pathway. In analogs of the ADP-insensitive phosphoenzyme E2P and in the Ca(2+)-free state E2, the proton path leads between transmembrane helices M5 to M8 from the lumenal side of the protein to the Ca(2+) binding residues Glu-771, Asp-800 and Glu-908. The proton path is different from suggested Ca(2+) dissociation pathways. We suggest that separate proton and Ca(2+) pathways enable rapid (partial) neutralization of the empty cation binding sites. For this reason, transient protonation of empty cation binding sites and separate pathways for different ions are advantageous for P-type ATPases in general.

The amide I spectrum of multimers of helical protein segments was simulated using transition dipole coupling (TDC) for long-range interactions between individual amide oscillators and DFT data from dipeptides (la Cour Jansen et al. 2006, J. Chem. Phys. 125, 44312) for nearest neighbor interactions. Vibrational coupling between amide groups on different helices shift the helix absorption to higher wavenumbers. This effect is small for helix dimers (1 cm −1 ) at 10Å distance and only moderately affected by changes in the relative orientation between the helices. However, the effect becomes considerable when several helices are bundled in membrane proteins. Particular examples are the 7-helix membrane proteins bacteriorhodopsin (BR) and rhodopsin, where the upshift is 4.3 and 5.3 cm −1 , respectively, due to inter-helical coupling within a BR monomer. A further upshift of 4.0 cm −1 occurs when BR monomers associate to trimers. We propose that inter-helical vibrational coupling explains the experimentally observed unusually high wavenumber of the amide I band of BR.

Adenylate kinase (AdK) and apyrase were employed as helper enzymes to remove ADP in infrared spectroscopic experiments that study the sarcoplasmic reticulum Ca(2+)-ATPase. The infrared absorbance changes of their enzymatic reactions were characterized and used to monitor enzyme activity. AdK transforms ADP to ATP and AMP, whereas apyrase consumes ATP and ADP to generate AMP and inorganic phosphate. The benefits of using them as helper enzymes are severalfold: i), both remove ADP generated after ATP hydrolysis by ATPase, which enables repeat of ATP-release experiments several times with the same sample without interference by ADP; ii), AdK helps maintain the presence of ATP for a longer time by regenerating 50% of the initial ATP; iii), apyrase generates free P(i), which can help stabilize the ADP-insensitive phosphoenzyme (E2P); and iv), apyrase can be used to monitor ADP dissociation from transient enzyme intermediates with relatively high affinity to ADP, as shown here for ADP dissociation from the ADP-sensitive phosphoenzyme intermediate (Ca(2)E1P). The respective infrared spectra indicate that ADP dissociation relaxes the closed conformation immediately after phosphorylation partially back toward the open conformation of Ca(2)E1 but does not trigger the transition to E2P. The helper enzyme approach can be extended to study other nucleotide-dependent proteins.

A general method to study the phosphate group of phosphoenzymes with infrared difference spectroscopy by helper enzyme-induced isotope exchange was developed. This allows the selective monitoring of the phosphate P-O vibrations in large proteins, which provides detailed information on several band parameters. Here, isotopic exchange was achieved at the oxygen atoms of the catalytically important phosphate group that transiently binds to the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a). [gamma-(18)O(3)]ATP phosphorylated the ATPase, which produced phosphoenzyme that was initially isotopically labeled. The helper enzyme adenylate kinase regenerated the substrate ATP from ADP (added or generated upon ATP hydrolysis) with different isotopic composition than used initially. With time this produced the unlabeled phosphoenzyme. The method was tested on the ADP-insensitive phosphoenzyme state of the Ca(2+)-ATPase for which the vibrational frequencies of the phosphate group are known, and it was established that the helper enzyme is effective in mediating the isotope exchange process.