Location of helical regions in tetrapyrrole-containing proteins by a helical hydrophobic moment analysis. Application to phytochrome

Helical regions in many tetrapyrrole proteins are highly amphiphilic, one side interacting with a hydrophobic core and another side interacting with the polar solvent. The mean helical hydrophobic moment is a measure of amphiphilicity of a helix. Helical regions in myoglobin, the alpha and beta subunits of C-phycocyanin, and cytochrome c can be distinguished from nonhelical regions by use of a hydrophobic moment analysis. 24 of 27 (89%) of the helical regions in these proteins were located by this analysis. Calculations were also performed on chymotrypsin, ribonuclease, and papain, which do not possess as pronounced a hydrophobic core as the tetrapyrrole-containing proteins. Less than 50% of the helical regions were correctly located, indicating a lack of amphiphilicity in the helices of these proteins. The hydrophobic moment analysis was also used to predict helical regions in phytochrome, the ubiquitous photoreceptor in plants. Additionally, this analysis is used to quickly locate internal hydrophilic residues which may be functionally important. The distribution of hydrophobic moments from a random sequence was determined so that qualitative and to some extent quantitative comparisons between different amphiphilic helices may be made.     

Prediction of the burial status of transmembrane residues of helical membrane proteins

Background  

Helical membrane proteins (HMPs) play a crucial role in diverse cellular processes, yet it still remains extremely difficult to determine their structures by experimental techniques. Given this situation, it is highly desirable to develop sequence-based computational methods for predicting structural characteristics of HMPs.     

Empirical lipid propensities of amino acid residues in multispan alpha helical membrane proteins

Characterizing the interactions between amino acid residues and lipid molecules is important for understanding the assembly of transmembrane helices and for studying membrane protein folding. In this study we develop TMLIP (TransMembrane helix-LIPid), an empirically derived propensity of individual residue types to face lipid membrane based on statistical analysis of high-resolution structures of membrane proteins. Lipid accessibilities of amino acid residues within the transmembrane (TM) region of 29 structures of helical membrane proteins are studied with a spherical probe of radius of 1.9 A. Our results show that there are characteristic preferences for residues to face the headgroup region and the hydrocarbon core region of lipid membrane. Amino acid residues Lys, Arg, Trp, Phe, and Leu are often found exposed at the headgroup regions of the membrane, where they have high propensity to face phospholipid headgroups and glycerol backbones. In the hydrocarbon core region, the strongest preference for interacting with lipids is observed for Ile, Leu, Phe and Val. Small and polar amino acid residues are usually buried inside helical bundles and are strongly lipophobic. There is a strong correlation between various hydrophobicity scales and the propensity of a given residue to face the lipids in the hydrocarbon region of the bilayer. Our data suggest a possibly significant contribution of the lipophobic effect to the folding of membrane proteins. This study shows that membrane proteins have exceedingly apolar exteriors rather than highly polar interiors. Prediction of lipid-facing surfaces of boundary helices using TMLIP1 results in a 54% accuracy, which is significantly better than random (25% accuracy). We also compare performance of TMLIP with another lipid propensity scale, kPROT, and with several hydrophobicity scales using hydrophobic moment analysis.     

The role of the charged residues of the GP2 helical regions in Ebola entry

The glycoprotein (GP) of Ebola is the sole structural protein that forms the spikes on the viral envelope. The GP contains two subunits, GP1 and GP2, linked by a disulfide bond, which are responsible for receptor binding and membrane fusion, respectively. In this study, the full length of GP gene of Ebola Zaire species, 2028 base pairs in length, was synthesized using 38 overlapping oligonucleotides by multiple rounds of polymerase chain reaction (PCR). The synthesized GP gene was shown to be efficiently expressed in mammalian cells. Furthermore, an efficient HIV-based pseudotyping system was developed using the synthetic GP gene, providing a safe approach to dissecting the entry mechanism of Ebola viruses. Using this pseudotyping system and mutational analysis, the role of the charged residues in the GP2 helical regions was examined. It was found that substitutions of the most charged residues in the regions did not adversely affect GP expression, processing, or viral incorporation, however, most of the mutations greatly impaired the ability of GP to mediate efficient viral infection. These results demonstrate that these charged residues of GP2 play an important role in GP-mediated Ebola entry into its host cells. We propose that these charged residues are involved in forming the intermediate conformation(s) of GP in membrane fusion and Ebola entry.     

Stereochemistry of oligomeric proteins

The subunits of many oligomeric proteins are organized into stable arrays with high symmetry. When these proteins interact with small molecules such as enzyme substrates or inhibitors, a variety of non-equivalent forms of the protein may be produced. Some of these forms have the same atomic composition, but differ in the spatial arrangement of the subunits. These species differ in all of their properties (conformation, affinity for substrate, etc.), and the relationships among them and among their subunits may be defined using stereochemical nomenclature which has been developed for small molecules. In many cases, the number of such forms is quite large. Rigorous, group-theoretical methods for enumerating all possible species are described and illustrated for the enzyme aspartate transcarbamoylase.     

Interaction of adjacent amino acid residues in the structured regions of globular proteins

Distribution of the pairs of amino acids i, i + 1 in alpha-helical, beta-sheet and random coil regions from 46 globular proteins comprising 8115 amino acid residues was analyzed. Statistical analysis of the data excludes null hypothesis about random pairing of the amino acid residues i, i + 1 in beta-sheet and random coil configurations. The distribution of the amino acid pairs, i, i + 1 in alpha-helical configurations does not differ from the random pairing.     

Fluorescence and the structure of proteins. XXI. Fluorescence of aminotyrosyl residues in peptides and helical proteins.

1. Five peptides containing tyrosine were converted to the 3-aminotyrosyl peptides by nitration with tetranitromethane and subseuqent reduction of the nitro groups to amino groups. The fluorescence of these aminotyrosyl residues was found to be quite similar to that of 3-aminotyrosine and it is concluded that the fluorescence is not sensitive to incorporation of the amino acid into the peptide chain. 2. Fluorescence of 3-aminotyrosine derivatives was sensitive, however, to the nature of the solvent; as the dielectric constant decreased, fluorescence was enhanced ten fold and the emission maximum shifted from the 350-370 nm value in aqueous solution to 320 nm. It is predicted that similar differences might be expected for exposed and buried aminotyrosyl residues in a protein. 3. Exposed tyrosyl residues on the helical protein tropomyosin and a helical segment of paramyosin were aminated in part (39% and 34% of the total tyrosyl residues, respectively). The fluorescence of the aminated tyrosyl residues on these proteins was similar to that of the aminotyrosyl peptides in an aqueous medium. Although the fluorescence efficiency of an aminotyrosyl residue was much lower than that of a tyrosyl residue, it was easy to distinguish the fluorescence of the aminotyrosyl residues (350-355 nm) on the protein from that arising from unmodified tyrosyl residues (305 nm).     

On the derivation of propensity scales for predicting exposed transmembrane residues of helical membrane proteins

Helical membrane proteins (HMPs) play a crucial role in diverse physiological processes. Given the difficulty in determining their structures by experimental techniques, it is desired to develop computational methods for predicting the burial status of transmembrane residues. Deriving a propensity scale for the 20 amino acids to be exposed to the lipid bilayer from known structures is central to developing such methods. A fundamental problem in this regard is what would be the optimal way of deriving propensity scales. Here, we show that this problem can be reformulated such that an optimal scale is straightforwardly obtained in an analytical fashion. The derived scale favorably compares with others in terms of both algorithmic optimality and practical prediction accuracy. It also allows interesting insights into the structural organization of HMPs. Furthermore, the presented approach can be applied to other bioinformatics problems of HMPs, too. All the data sets and programs used in the study and detailed primary results are available upon request.     

Disposition of polar and nonpolar residues on outer surfaces of transmembrane helical segments of proteins involved in proton translocation

"Helical wheel" projections of transmembrane helical segments of membrane proteins involved in proton translocation were constructed. The particular proteins studied were the uncF protein subunit of the Escherichia coli proton-ATPase, the uncE protein subunit of the E. coli proton-ATPase, and cytochrome oxidase subunit III. Clear demarcation of polar and nonpolar regions on surfaces of transmembrane helical segments was seen in the uncF protein and in uncE protein helical segment two, but not in uncE protein helical segment one. The transmembrane segment of cytochrome oxidase subunit III which includes the dicyclohexylcarbodiimide (DCCD)-reactive residue was very similar to E. coli uncE protein helical segment two. The DCCD-reactive residue in both was clearly located on a nonpolar surface.     

Circular dichroism studies on helical structure preferences of amino acid residues of proteins caused by sodium dodecyl sulfate

The extent of helical structure of 19 intact proteins and of 15 proteins with no disulfide bridges in the absence and presence of 10 mM sodium dodecyl sulfate (SDS) was determined using the curve-fitting method of circular dichroic spectra. The change in helicity caused by the addition of SDS was examined as a function of each amino acid fraction. An increase in the helicity upon the addition of SDS occurred in most of the proteins with no disulfide bridges (C proteins) and containing more than 0.06 Lys fraction. In most of the intact proteins (B proteins), most of which contained disulfide bridges, helicity in SDS decreased with an increase in Lys fraction. The helicity of the C proteins in SDS also tended to increase with an increase in the Leu and Phe fractions, while it decreased with an increase in the Gly fraction. For the helicity of the B proteins in SDS, there was a tendency to increase with increased Asn fraction and decrease with increased His fraction. On the other hand, amino acids were divided into eight groups according to their side-chain properties and the conformational preference for each of the amino acid groups of C proteins was calculated using a simple assumption.     

Co-evolving residues in membrane proteins

MOTIVATION: The analysis of co-evolving residues has been exhaustively evaluated for the prediction of intramolecular amino acid contacts in soluble proteins. Although a variety of different methods for the detection of these co-evolving residues have been developed, the fraction of correctly predicted contacts remained insufficient for their reliable application in the construction of structural models. Membrane proteins, which constitute between one-fourth and one-third of all proteins in an organism, were only considered in few individual case studies. RESULTS: We present the first general study of correlated mutations in alpha-helical membrane proteins. Using seven different prediction algorithms, we extracted co-evolving residues for 14 membrane proteins having a solved 3D structure. On average, distances between correlated pairs of residues lying on different transmembrane segments were found to be significantly smaller compared to a random prediction. Covariation of residues was frequently found in direct sequence neighborhood to helix-helix contacts. Based on the results obtained from individual prediction methods, we constructed a consensus prediction for every protein in the dataset that combines obtained correlations from different prediction algorithms and simultaneously removes likely false positives. Using this consensus prediction, 53% of all predicted residue pairs were found within one helix turn of an observed helix-helix contact. Based on the combination of co-evolving residues detected with the four best prediction algorithms, interacting helices could be predicted with a specificity of 83% and sensitivity of 42%. AVAILABILITY: http://webclu.bio.wzw.tum.de/helixcorr/     

Buried water molecules in helical transmembrane proteins

Buried water molecules (having no contact with bulk solvent) in 30 helical transmembrane (TM) protein structures were identified. The average amount of buried water in helical TM proteins is about the same as for all water-soluble (WS) proteins, but it is greater than the average for helical WS proteins. Buried waters in TM proteins make more polar contacts, and are more frequently found contacting helices than in WS proteins. The distribution of the buried water binding sites across the membrane profile shows that the sites to some extent reflect protein function. There is also evidence for asymmetry of the sites, with more in the extracellular half of the membrane. Many of the buried water contact sites are conserved across families of proteins, including family members having different functions. This suggests that at least some buried waters play a role in structural stabilization. Disease-causing mutations, which are known to result in misfolded TM proteins, occur at buried water contact sites at a higher than random frequency, which also supports a stabilizing role for buried water molecules.     

Inputting information about amino acid residues in protein bioinformatics: a case study on predicting helical regions with a neural network

The best way to introduce information about amino acid residues into calculations of protein bioinformatics was examined. That was done for predicting helical regions with a neural network. Several fundamental and instructive ways for information processing were developed and are described.     

Sugar residues on proteins

Glycoproteins have become increasingly important in the structure and function of many different mammalian systems; for example, membrane glycoproteins and glycoprotein hormones. It is, therefore, important to understand their chemistry, which would include an understanding of both the carbohydrate and protein parts of the molecule. Since the chemical characterization of the protein moiety has been extensively examined and the techniques for its characterization are well worked out, only the carbohydrate portion of glycoproteins will be reviewed in this article. The chemical nature of the carbohydrate moiety of glycoproteins will be examined. First, the types of monosaccharides present in animal systems, especially those in the mammalian systems, will be described. Next, various types of simple and complex carbohydrate chains will be discussed to establish the diversity, size, and number of chains present in the carbohydrate units in different glycoproteins. Then, the type of linkages of the carbohydrate to the protein will be examined to determine if the primary sequence of protein is important in determining the size and type of carbohydrate chains present in glycoproteins. Finally, the current methods of structural elucidation such as monosaccharide sequence, intersugar bonds, and anomeric linkages in the carbohydrate moiety of glycoproteins will be reviewed. These methods include the techniques of periodate oxidation, methylation, partial acid hydrolysis, and specific glycosidase digestion of glycoproteins, as well as the latest techniques using micromethods of carbohydrate quantitation and characterization involving gas chromatography and mass spectrometry. The function of the carbohydrate in glycoproteins will also be considered. First, hormone glycoproteins will be discussed in their relationship to the immunological and biological function of the glycoprotein when the carbohydrate is sequentially removed. Next, the function of the carbohydrate in the turnover of glycoproteins will be discussed. These topics will be considered in order to develop an understanding of a specific function(s) of the carbohydrate in glycoproteins.