Raman spectroscopy on blood serum samples of patients with end-stage liver disease.

Raman spectroscopy has shown to be a promising method for the examination of biomedical samples. However, until now, its efficacy has not been established in clinical diagnostics. In this study, Raman spectroscopy's potential application in medical laboratories is evaluated for a large variety (38) of biomarkers. Given 234 serum samples from a cohort of patients with different stages of liver disease, we performed Raman spectroscopy at 780nm excitation wavelength. The Raman spectra were analyzed in combination with the results of routine diagnostics using specifically developed complex mathematical algorithms, including fluorescence filtering, frequency subset selection and several overfitting circumventing strategies, such as independent validation. With the results of this cohort, which were validated in 328 independent samples, a significant proof-of-concept study was completed. This study highlights the need to prevent overfitting and to use independent data for validation. The results reveal that Raman spectroscopy has high potential for use in medical laboratory diagnostics to simultaneously quantify multiple biomarkers.

Voronoia: analyzing packing in protein structures.

The packing of protein atoms is an indicator for their stability and functionality, and applied in determining thermostability, in protein design, ligand binding and to identify flexible regions in proteins. Here, we present Voronoia, a database of atomic-scale packing data for protein 3D structures. It is based on an improved Voronoi Cell algorithm using hyperboloid interfaces to construct atomic volumes, and to resolve solvent-accessible and -inaccessible regions of atoms. The database contains atomic volumes, local packing densities and interior cavities calculated for 61 318 biological units from the PDB. A report for each structure summarizes the packing by residue and atom types, and lists the environment of interior cavities. The packing data are compared to a nonredundant set of structures from SCOP superfamilies. Both packing densities and cavities can be visualized in the 3D structures by the Jmol plugin. Additionally, PDB files can be submitted to the Voronoia server for calculation. This service performs calculations for most full-atomic protein structures within a few minutes. For batch jobs, a standalone version of the program with an optional PyMOL plugin is available for download. The database can be freely accessed at: http://bioinformatics.charite.de/voronoia.

Hydrogen-bonding and packing features of membrane proteins: functional implications.

The recent structural elucidation of about one dozen channels (in which we include transporters) has provided further evidence that these membrane proteins typically undergo large movements during their function. However, it is still not well understood how these proteins achieve the necessary trade-off between stability and mobility. To identify specific structural properties of channels, we compared the helix-packing and hydrogen-bonding patterns of channels with those of membrane coils; the latter is a class of membrane proteins whose structures are expected to be more rigid. We describe in detail how in channels, helix pairs are usually arranged in packing motifs with large crossing angles (|tau| approximately 40 degrees ), where the (small) side chains point away from the packing core and the backbones of the two helices are in close contact. We found that this contributes to a significant enrichment of Calpha-H...O bonds and to a packing geometry where right-handed parallel (tau = -40 degrees +/- 10 degrees ) and antiparallel (tau = +140 degrees +/- 25 degrees ) arrangements are equally preferred. By sharp contrast, the interdigitation and hydrogen bonding of side chains in helix pairs of membrane coils results in narrowly distributed left-handed antiparallel arrangements with crossing angles tau = -160 degrees +/- 10 degrees (|tau| approximately 20 degrees ). In addition, we show that these different helix-packing modes of the two types of membrane proteins correspond to specific hydrogen-bonding patterns. In particular, in channels, three times as many of the hydrogen-bonded helix pairs are found in parallel right-handed motifs than are non-hydrogen-bonded helix pairs. Finally, we discuss how the presence of weak hydrogen bonds, water-containing cavities, and right-handed crossing angles may facilitate the required conformational flexibility between helix pairs of channels while maintaining sufficient structural stability.

Analysis and prediction of helix-helix interactions in membrane channels and transporters.

Membrane proteins span a large variety of different functions such as cell-surface receptors, redox proteins, ion channels, and transporters. Proteins with functional pores show different characteristics of helix-helix packing as other helical membrane proteins. We found that the helix-helix contacts of 13 nonhomologous high-resolution structures of membrane channels and transporters are mainly accomplished by weakly polar amino acids (G > S > T > F) that preferably create contacts every fourth residue, typical for right-handed helix crossings. There is a strong correlation between the now available biological hydrophobicity scale and the propensities of the weakly polar and hydrophobic residues to be buried at helix-helix interfaces or to be exposed to the lipids in membrane channels and transporters. The polar residues, however, make no major contribution towards the packing of their transmembrane helices, and are therefore subsumed to be primarily exposed to the polar milieu during the folding process. The contact formation of membrane channels and transporters is therefore ruled by the solubility of the residues, which we suppose to be the driving force for the assembly of their transmembrane helices. By contrast, in 14 nonhomologous high-resolution structures of other membrane protein coils, also large and polar amino acids (D > S > M > Q) create characteristic contacts every 3.5th residues, which is a signature for left-handed helix crossings. Accordingly, it seems that dependent on the function, different concepts of folding and stabilization are realized for helical membrane proteins. Using a sequence-based matrix prediction method these differences are exploited to improve the prediction of buried and exposed residues of transmembrane helices significantly. When the sequence motifs typical for membrane channels and transporters were applied for the prediction of helix-helix contacts the quality of prediction rises by 16% to an average value of 76%, compared to the same approach when only single amino acid positions are taken into account.

Molecular packing and packing defects in helical membrane proteins.

The packing of helices spanning lipid bilayers is crucial for the stability and function of alpha-helical membrane proteins. Using a modified Voronoi procedure, we calculated packing densities for helix-helix contacts in membrane spanning domains. Our results show that the transmembrane helices of protein channels and transporters are significantly more loosely packed compared with helices in globular proteins. The observed packing deficiencies of these membrane proteins are also reflected by a higher amount of cavities at functionally important sites. The cavities positioned along the gated pores of membrane channels and transporters are noticeably lined by polar amino acids that should be exposed to the aqueous medium when the protein is in the open state. In contrast, nonpolar amino acids surround the cavities in those protein regions where large rearrangements are supposed to take place, as near the hinge regions of transporters or at restriction sites of protein channels. We presume that the observed deficiencies of helix-helix packing are essential for the helical mobility that sustains the function of many membrane protein channels and transporters.

Structural features of transmembrane helices.

A total of 160 transmembrane helices of 15 non-homologous high-resolution X-ray protein structures have been analyzed in respect of their structural features. The dihedral angles and hydrogen bonds of the helical sections that span the hydrophobic interior of the lipid bilayer have been investigated. The Ramachandran plot of protein channels and solute transporters exhibit a significant shift Delta (phi- and psi-angles) of Delta mean (+4.5 degrees and -5.4 degrees ), compared to a reference group of 151 alpha-helices of the same average length derived from water-soluble globular proteins. At the C-termini of transmembrane helices structural motifs equivalent to the Gly-caps of helices in globular proteins have been found, with two third of the transmembrane Gly-caps taking up a primary structure that is typically not found at helix termini exposed to a polar solvent. The structural particularities reported here are relevant for the three-dimensional modelling of membrane protein structures.