Probing protein structure by solvent perturbation of NMR spectra: the surface accessibility of bovine pancreatic trypsin inhibitor.

In the absence of specific interactions, the relative attenuation of protein NMR signals due to added stable free radicals such as TEMPOL should reflect the solvent accessibility of the molecular surface. The quantitative correlation between observed attenuation and surface accessibility was investigated with a model system, i.e., the small protein bovine pancreatic trypsin inhibitor. A detailed discussion is presented on the reliability and limits of the approach, and guidelines are provided for data acquisition, treatment, and interpretation. The NMR-derived accessibilities are compared with those obtained from x-ray diffraction and molecular dynamics data. Although the time-averaged accessibilities from molecular dynamics are ideally suited to fit the NMR data, better agreement was observed between the paramagnetic attenuations of the fingerprint cross-peaks of homonuclear proton spectra and the total NH and H alpha accessibilities calculated from x-ray coordinates, than from time-averaged molecular dynamics simulations. In addition, the solvent perturbation response appears to be a promising approach for detecting the thermal conformational evolution of secondary structure elements in proteins.     

Probing protein structure by solvent perturbation of nuclear magnetic resonance spectra. Nuclear magnetic resonance spectral editing and topological mapping in proteins by paramagnetic relaxation filtering.

Soluble spin labels, which "bleach" the surface proton resonances of a protein to n.m.r. measurements, can provide useful information about protein conformation and dynamics. The use of the soluble nitroxide, TEMPOL, has been explored to show the correlation of the paramagnetic perturbations of protein two-dimensional n.m.r. data with proton exposure to the free radical in hen egg-white lysozyme. The results demonstrate that the nitroxide approaches the protein randomly, and that the extent of the observed paramagnetic effects reflects the native folding pattern of the protein. A correlation of spectral simplification with the known tertiary structure establishes the feasibility of new strategies for topological mapping of surface and buried protons of the protein. Application to the elucidation of protein structure and to the study of dynamical processes is discussed.     

Location of chromophoric residues in ribonuclease T1 by solvent perturbation difference spectroscopy.

When beef liver microsomes are labeled with UDP-[³H]N-acetyl glucosamine, three different lipid-bound saccharides are labeled: dolichol pyrophosphoryl-GlcNAc, dolichol pyrophosphoryl-(GlcNAc)₂ and a previously uncharacterized compound (component III). Incubation with UDP leads to the disappearance of dolichol pyrophosphoryl-(GlcNac)₂ from microsomes prelabeled with UDP-³H-N-acetyl glucosamine. This result provides further support for the suggestion of Leloir $\text{et}\text{al.}$ (6) that dolichol pyrophosphoryl-(GlcNAc)₂ is synthesized from dolichol pyrophosphoryl-GlcNAc and UDP-N-acetyl glucosamine. Component III cannot be discharged with UMP or UDP. It elutes from DEAE-cellulose exactly as the other components. The saccharide portion of component III has a molecular weight of 800–900 and contains glucose in addition to N-acetyl glucosamine.     

Interpretation of thermal perturbation spectra of proteins.

The thermal perturbation difference spectrum of reduced lysozyme has a long wave length extremum at 304 nm at pH 6.15 and a very small extremum at 306 nm at pH 1.5. These results differ from those of Leach & Smith (1972), which showed an extremum at 293 nm, the same as for model tryptophyl compounds. Our result may arise from a conformational difference between the two sample temperatures. The interpretation of thermal perturbation spectra of proteins is discussed. Contributions from thermally induced concentration differences, buried chromophores, and chromophores in crevices are considered in the interpretation of the thermal perturbation spectrum of bovine serum albumin. It is suggested that chromophores in pauci-aqueous crevices may appear buried toward thermal perturbation spectroscopy but accessible toward solvent perturbation and chemical reagents.     

Determination of the secondary structure of proteins from circular dichroism spectra. IV. Contribution of aromatic amino acid residues into circular dichroism spectra of proteins in the peptide region

A new method for determination of the secondary protein structure from the CD spectra taking into account the contribution of aromatic amino acid residues is proposed. New proteins reference CD spectra for five secondary structures (alpha-helices, antiparallel and parallel beta-structures, beta-bends and irregular form) without contribution of aromatic residues are obtained. By means of this new method the secondary structure of sixteen different proteins was analysed. There is a good correlation of these results with the X-ray data.     

Determination of the primary structure of homologous proteins by sequence analysis of peptide mixtures

We propose a rapid method to determine the primary structure of a protein knowing the sequence of a homologous protein. The method consists in submitting both the reduced and alkylated proteins to an enzymatic or chemical hydrolysis and performing the sequence analysis of the peptide mixtures. The assessment of the unknown sequence and the degree of identity of the two proteins are reached by comparing the two sequence analyses. The sequences of all the possible peptides present in the two mixtures are reconstructed and the differences in the two sequences are determined. If necessary, the differences can be confirmed by performing a mass spectrometric analysis of the two mixtures. We used this procedure on two homologous proteins of known sequence to furnish an application example of the method.     

Probing stability and dynamics of proteins by protease digestion. II: Identification of the initial chymotryptic cleavage sites of homologous cytochromes c

Three homologous cytochromes c from horse, rabbit and tuna were subjected to chymotryptic digestion and their initial cleavage sites were identified. The sites in oxidized cytochromes c are the COOH-terminal sides of Tyr-48, Phe-46 and Tyr-46 for horse, rabbit and tuna cytochromes c, respectively. The results show that the chymotrypsin attacks a single site in each protein; the sites are located at the almost identical position on the polypeptide chain. Through the time-course studies of digestion, it was found that the three cytochromes c have different chymotrypsin-susceptibility at the initial cleavage site in the order of horse less than rabbit less than tuna. Studies on chymotryptic digestion of tuna cytochrome c in the reduced form revealed that the haem-reduction does not alter the initial cleavage site but increases the resistance to the proteolysis at the site. The uniqueness of the initial cleavage site in each cytochrome c species suggests that the protease susceptibility reflects some overall properties of the protein. At the same time, it was clarified that the initial cleavage site is also affected by a neighboring region by the fact that another potential cleavage site is located near the site in question. In order to elucidate the initial cleavage site, several physical properties of tuna cytochrome c molecule deduced from the X-ray 3D structure, accessible surface area, temperature factor, effective hydrophobicity and electrostatic potential, were compared with the experimental results and it was concluded that these properties given by a residue have no direct relationship with the chymotrypsin susceptibility.     

Determination of secondary structure of globular proteins using circular dichroism spectra

A ridge regression method is presented for prediction of the secondary structure of proteins by the circular dichroism spectra (CD) from 190 to 236 nm. Eight types of the secondary structure were calculated on a microcalculator. The method is based on the X-ray data of Kabsh and Sander. The teaching rule is constructed on CD spectra of 30 proteins of all structural classes of the globular proteins (alpha, alpha/beta, alpha + beta and beta-proteins). The errors of the methods are analysed by removing each protein from the reference set and analyzing its structure in terms of the remaining proteins. Correlation coefficients and root-mean square deviations between CD and X-ray data were: 0.99 and 0.03 for alpha-helix, 0.86 and 0.02 for 3(10)-helix, 0.92 and 0.06 for antiparallel beta-sheet, 0.86 and 0.03 for parallel beta-sheet, 0.94 and 0.01 for T3 beta-turn, 0.85 and 0.02 for other beta-turn, 0.84 and 0.03 for S-bends, 0.83 and 0.04 for "random" structure.