Infrared spectroscopy of proteins.

Distance distributions between paramagnetic centers in the range of 1.8 to 6 nm in membrane proteins and up to 10 nm in deuterated soluble proteins can be measured by the DEER technique. The number of paramagnetic centers and their relative orientation can be characterized. DEER does not require crystallization and is not limited with respect to the size of the protein or protein complex. Diamagnetic proteins are accessible by site-directed spin labeling. To characterize structure or structural changes, experimental protocols were optimized and techniques for artifact suppression were introduced. Data analysis programs were developed, and it was realized that interpretation of the distance distributions must take into account the conformational distribution of spin labels. First methods have appeared for deriving structural models from a small number of distance constraints. The present scope and limitations of the technique are illustrated.

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DEER distance measurements on proteins

Infrared spectroscopy is a useful technique for the determination of conformation and orientation of membrane-associated proteins and lipids. The technique is especially powerful for detecting conformational changes by recording spectral differences before and after perturbations in physiological solution. Polarized infrared measurements on oriented membrane samples have revealed valuable information on the orientation of chemical groupings and substructures within membrane molecules which is difficult to obtain by other methods. The application of infrared spectroscopy to the static and dynamic structure of proteins and peptides in lipid bilayers is reviewed with some emphasis on the importance of sample preparation. Limitations of the technique with regard to the absolute determination of secondary structure and orientation and new strategies for structural assignments are also discussed.

http://shaker.umh.es/docencia/doctorado_IR/FTIR_review3.pdf

Infrared spectroscopy of proteins and peptides in lipid bilayers

The helix is a common secondary structural motif found in proteins, and the mechanism of helix-coil interconversion is key to understanding the protein-folding problem. We report the observation of the fast kinetics (nanosecond to millisecond) of helix melting in a small 21-residue alanine-based peptide. The unfolding reaction is initiated using a laser-induced temperature jump and probed using time-resolved infrared spectroscopy. The model peptide exhibits fast unfolding kinetics with a time constant of 160 +/- 60 ns at 28 degrees C in response to a laser-induced temperature jump of 18 degrees C which is completed within 20 ns. Using the unfolding time and the measured helix-coil equilibrium constant of the model peptide, a folding rate constant of approximately 6 x 10(7) s-1 (t1/2 = 16 ns) can be inferred for the helix formation reaction at 28 degrees C. These results demonstrate that secondary structure formation is fast enough to be a key event at early times in the protein-folding process and that helices are capable of forming before long range tertiary contacts are made.

Fast events in protein folding: helix melting and formation in a small peptide.

A quantum mechanical/molecular mechanical (QM/MM) approach based on an approximate density functional theory, the so-called self-consistent charge density functional tight binding (SCC-DFTB) method, has been implemented in the CHARMM program and tested on a number of systems of biological interest. In the gas phase, SCC-DFTB gives reliable energetics for models of the triosephosphate isomerase (TIM) catalyzed reactions. The rms errors in the energetics compared to B3LYP/6-31+G(d,p) are about 2−4 kcal/mol; this is to be contrasted with AM1, where the corresponding errors are 9−11 kcal/mol. The method also gives accurate vibrational frequencies. For the TIM reactions in the presence of the enzyme, the overall SCC-DFTB/CHARMM results are in somewhat worse agreement with the B3LYP/6-31+G(d,p)/CHARMM values; the rms error in the energies is 5.4 kcal/mol. Single-point B3LYP/CHARMM energies at the SCC-DFTB/CHARMM optimized structures were found to be very similar to the full B3LYP/CHARMM values. The relative sta...

A QM/MM Implementation of the Self-Consistent Charge Density Functional Tight Binding (SCC-DFTB) Method

SHORT alanine peptides, containing 16 or 17 residues, appear to form α-helices in aqueous solution1–4. But the main spectroscopic analyses used on helical peptides (circular dichroism5 and nuclear magnetic resonance6–8) cannot distinguish between an α-helix (in which the ith residue is hydrogen-bonded to residue i+4; ref. 9) and the next most common peptide helix, the 310-helix10 (i → i + 3 hydrogen-bonding). To address this problem we have designed single and doubly spin-labelled analogues of alanine-based peptides in which the nitroxide spin label forms an unbranched side chain extending from the sulphur atom of a cysteine residue. Here we report the circular dichroism, Fourier-transform infrared and electron-spin resonance spectra of these peptides under helix-forming conditions. The infrared absorbance gives an amide I' band with a frequency that is substantially different from that observed for α-helices. The electron-spin resonance spectra of doubly labelled helices show that the ranking of distances between side chains, around a single turn (residues 4–8), is inconsistent with an α-helical structure. Our experiments suggest that the more likely peptide geometry is a 310-helix.

Short alanine-based peptides may form 310-helices and not α-helices in aqueous solution

A variety of host L-alanine homo-peptides (to the pentamer) containing one or two spin-labelled TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) residues were synthesized by solution methods and fully characterized. The conformational features of the terminally blocked, doubly spin-labelled TOAC-(Ala)2-TOAC-Ala-pentapeptide were examined in the crystal state by X-ray diffraction and in solution using a combination of techniques (Fourier transform infrared, circular dichroism, cyclic voltammetry and electron spin resonance) in comparison with singly labelled shorter peptides. The 3(10)-helical structure of the pentapeptide, promoted by the two C alpha, alpha-disubstituted glycines under favourable experimental conditions, allows an interaction to take place between the two nitroxide TOAC side chains spaced by one turn of the helix. Taken together, these results suggest that TOAC is an excellent probe for exploring bends and helices in doubly labelled peptides.

Synthesis and conformational studies of peptides containing TOAC, a spin-labelled Cα,α-disubstituted glycine

FTIR spectroscopy of alanine-based peptides: assignment of the amide I' modes for random coil and helix

Distinguishing helix conformations in alanine-rich peptides using the unnatural amino acid TOAC and electron spin resonance

The unnatural, conformationally constrained nitroxide amino acid TOAC, a Cα,α-disubstituted glycine, stabilizes helical structure and provides a means for studying rigidly spin labelled peptides by electron spin resonance. Water soluble, alanine-rich sixteen and twenty residue sequences are examined with single and double TOAC labelling. Lineshape analysis of the singly labelled peptide reveals evidence of substantial anisotropic rotational diffusion. Combined circular dichroism and electron spin resonance of the double labelled sequences demonstrates that both the 16-residue and 20-residue peptides are completely folded and adopt predominantly the a-helical conformation. However, lineshape fitting of the 16-residue sequences cooled in MeOH revealed a surprising result. Instead of the typical α-helix geometry of 3.6 residues per turn found in peptide and protein crystal structures, these doubly TOAC labelled peptides adopt a more open geometry of approximately 3.8-3.9 residues per turn. Although this conf...

Gary V. Martinez

Papers

Short alanine-based peptides may form 310-helices and not α-helices in aqueous solution

Synthesis and conformational studies of peptides containing TOAC, a spin-labelled Cα,α-disubstituted glycine

FTIR spectroscopy of alanine-based peptides: assignment of the amide I' modes for random coil and helix

Distinguishing helix conformations in alanine-rich peptides using the unnatural amino acid TOAC and electron spin resonance

Electron spin resonance and structural analysis of water soluble, alanine-rich peptides incorporating TOAC