Lysine carboxylation: unveiling a spontaneous post-translational modification.

The carboxylation of lysine residues is a post-translational modification (PTM) that plays a critical role in the catalytic mechanisms of several important enzymes. It occurs spontaneously under certain physicochemical conditions, but is difficult to detect experimentally. Its full impact is unknown.

Computational studies of membrane proteins: models and predictions for biological understanding.

We discuss recent progresses in computational studies of membrane proteins based on physical models with parameters derived from bioinformatics analysis. We describe computational identification of membrane proteins and prediction of their topology from sequence, discovery of sequence and spatial motifs, and implications of these discoveries. The detection of evolutionary signal for understanding the substitution pattern of residues in the TM segments and for sequence alignment is also discussed.

Lipid-binding surfaces of membrane proteins: evidence from evolutionary and structural analysis.

Membrane proteins function in the diverse environment of the lipid bilayer. Experimental evidence suggests that some lipid molecules bind tightly to specific sites on the membrane protein surface. These lipid molecules often act as co-factors and play important functional roles.

Structural signatures of enzyme binding pockets from order-independent surface alignment: a study of metalloendopeptidase and NAD binding proteins.

Detecting similarities between local binding surfaces can facilitate identification of enzyme binding sites and prediction of enzyme functions, and aid in our understanding of enzyme mechanisms. Constructing a template of local surface characteristics for a specific enzyme function or binding activity is a challenging task, as the size and shape of the binding surfaces of a biochemical function often vary. Here we introduce the concept of signature binding pockets, which captures information on preserved and varied atomic positions at multiresolution levels.

Cloning and characterization of a single-chain fragment of monoclonal antibody to ACE suitable for lung endothelial targeting.

Monoclonal antibody (mAb) 9B9 to angiotensin-converting enzyme (ACE) demonstrates selective accumulation in lung tissue of the rat, hamster, cat, monkey and human after systemic injection. It has also been demonstrated that mAb 9B9 is the useful tool for targeting therapeutic agents or genes to lung endothelium. In this study, we describe the generation and characterization of a single-chain derivative (scFv) of mAb9B9 (scFv 9B9).

Structural model of rho1 GABAC receptor based on evolutionary analysis: Testing of predicted protein-protein interactions involved in receptor assembly and function.

The homopentameric rho1 GABA(C) receptor is a ligand-gated ion channel with a binding pocket for gamma-aminobutyric acid (GABA) at the interfaces of N-terminal extracellular domains. We combined evolutionary analysis, structural modeling, and experimental testing to study determinants of GABA(C) receptor assembly and channel gating. We estimated the posterior probability of selection pressure at amino acid residue sites measured as omega-values and built a comparative structural model, which identified several polar residues under strong selection pressure at the subunit interfaces that may form intersubunit hydrogen bonds or salt bridges.

Detecting remote homologues using scoring matrices calculated from the estimation of amino acid substitution rates of beta-barrel membrane proteins.

Beta-barrel membrane proteins (MP) are found in Gram-negative bacteria, mitochondria and chloroplasts. They play important roles in metabolism of bacteria, where they are involved in transport of solutes in and out of the cell. Beta-barrel proteins may also act as proteases, lipases and may be important for cell-cell adhesion.

Chapter 4. Predicting and characterizing protein functions through matching geometric and evolutionary patterns of binding surfaces.

Predicting protein functions from structures is an important and challenging task. Although proteins are often thought to be packed as tightly as solids, closer examination based on geometric computation reveals that they contain numerous voids and pockets. Most of them are of random nature, but some are binding sites providing surfaces to interact with other molecules.

Evolutionary patterns of retinal-binding pockets of type I rhodopsins and their functions.

Genome sequencing projects resulted in the identification of a large number of new sequence homologs of archaeal rhodopsins in marine bacteria, fungi, and unicellular algae. It is an important task to unambiguously predict the functions of these new rhodopsins, as it is difficult to perform individual experiments on every newly discovered sequence. The transmembrane segments of rhodopsins have similar three-dimensional structures where the seven transmembrane helices form a tightly packed scaffold to accommodate a covalently bound retinal.

Prediction of transmembrane helix orientation in polytopic membrane proteins.

Background: Membrane proteins compose up to 30% of coding sequences within genomes. However, their structure determination is lagging behind compared with soluble proteins due to the experimental difficulties. Therefore, it is important to develop reliable computational methods to predict structures of membrane proteins.

Prediction of buried helices in multispan alpha helical membrane proteins.

Analysis of a database of structures of membrane proteins shows that membrane proteins composed of 10 or more transmembrane (TM) helices often contain buried helices that are inaccessible to phospholipids. We introduce a method for identifying TM helices that are least phospholipid accessible and for prediction of fully buried TM helices in membrane proteins from sequence information alone. Our method is based on the calculation of residue lipophilicity and evolutionary conservation.

The membrane-water interface region of membrane proteins: structural bias and the anti-snorkeling effect.

Membrane proteins have important roles in many cellular processes. Computational analysis of their sequences and structures has provided much insight into the organizing principles of transmembrane helices. In a recent study, the membrane-water interface region was examined in detail for the first time.

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.

Inferring functional relationships of proteins from local sequence and spatial surface patterns.

We describe a novel approach for inferring functional relationship of proteins by detecting sequence and spatial patterns of protein surfaces. Well-formed concave surface regions in the form of pockets and voids are examined to identify similarity relationship that might be directly related to protein function. We first exhaustively identify and measure analytically all 910,379 surface pockets and interior voids on 12,177 protein structures from the Protein Data Bank.

Interhelical hydrogen bonds in transmembrane region are important for function and stability of Ca2+-transporting ATPase.

Ca2+-transporting adenosine triphosphatase (ATPase) of sarcoplasmic reticulum couples ATP hydrolysis with ion transport. Phosphorylation of the cytosolic region of the calcium-bound conformation (E1) of the protein leads to drastic conformational rearrangements of the transmembrane helices and the release of Ca2+. The resulting calcium-free conformation (E2) is less stable than the E1 form.

Position-dependence of stabilizing polar interactions of asparagine in transmembrane helical bundles.

Recent studies with model peptides and statistical analyses of the crystal structures of membrane proteins have shown that buried polar interactions contribute significantly to the stabilization of the three-dimensional structures of membrane proteins. Here, we probe how the location of these polar groups along the transmembrane helices affect their free energies of interaction. Asn residues were placed singly and in pairs at three positions within a model transmembrane helix, which had previously been shown to support the formation of trimers in micelles.

Higher-order interhelical spatial interactions in membrane proteins.

Higher-order interactions are important for protein folding and assembly. We introduce the concept of interhelical three-body interactions as derived from Delaunay triangulation and alpha shapes of protein structures. In addition to glycophorin A, where triplets are strongly correlated with protein stability, we found that tight interhelical triplet interactions exist extensively in other membrane proteins, where many types of triplets occur far more frequently than in soluble proteins.

Automated assignment and 3D structure calculations using combinations of 2D homonuclear and 3D heteronuclear NOESY spectra.

The NOAH/DIAMOD suite uses feedback filtering and self-correcting distance geometry to generate 3D structures from unassigned NOESY spectra. In this study we determined the minimum set of experiments needed to generate a high quality structure bundle. Different combinations of 3D 15N-edited, 13C-edited HSQC-NOESY and 2D homonuclear 1H-1H NOESY spectra of the 77 amino acid protein, myeloid progenitor inhibitory factor-1 (MPIF-1) were used as input for NOAH/DIAMOD calculations.

Interhelical hydrogen bonds and spatial motifs in membrane proteins: polar clamps and serine zippers.

Polar and ionizable amino acid residues are frequently found in the transmembrane (TM) regions of membrane proteins. In this study, we show that they help to form extensive hydrogen bond connections between TM helices. We find that almost all TM helices have interhelical hydrogen bonding.