Influence of medium- and long-range interactions in different folding types of globular proteins

Recognition of protein fold from amino acid sequence is a challenging task. The structure and stability of proteins from different fold are mainly dictated by inter-residue interactions. In our earlier work, we have successfully used the medium- and long-range contacts for predicting the protein folding rates, discriminating globular and membrane proteins and for distinguishing protein structural classes. In this work, we analyze the role of inter-residue interactions in commonly occurring folds of globular proteins in order to understand their folding mechanisms. In the medium-range contacts, the globin fold and four-helical bundle proteins have more contacts than that of DNA-RNA fold although they all belong to all-alpha class. In long-range contacts, only the ribonuclease fold prefers 4-10 range and the other folding types prefer the range 21-30 in alpha/beta class proteins. Further, the preferred residues and residue pairs influenced by these different folds are discussed. The information about the preference of medium- and long-range contacts exhibited by the 20 amino acid residues can be effectively used to predict the folding type of each protein.     

Helix-coil transition theory including long-range electrostatic interactions: application to globular proteins

An extension of the Zimm–Bragg two-state theory for the helix–coil transition in polypeptides, which takes into account the effect of peptide charge–dipole interactions on helix stability, is presented. This new theory incorporates these interactions in an expression that is parameterized on recently obtained experimental data on polypeptides for which electrostatic effects are known to influence helix content. Unlike previous two-state or multistate models, which are parameterized on protein x-ray data, the present theoretical treatment in independent of such protein data. The theoretical model is applied to a series of peptides derived from the C-peptide of ribonuclease A, which have been the object of recent spectroscopic studies. The new theoretical approach can account for most of the structural information derived from studies of these C-peptides, and for overall average helix probabilities that are close in magnitude to those observed for these polypeptides in solution. An application of this new formulation for the prediction of the locations of α-helices in globular proteins from their amino acid sequence is also presented.     

Local and nonlocal interactions in globular proteins and mechanisms of alcohol denaturation.

How important are helical propensities in determining the conformations of globular proteins? Using the two-dimensional lattice model and two monomer types, H (hydrophobic) and P (polar), we explore both nonlocal interactions, through an HH contact energy, as developed in earlier work, and local interactions, through a helix energy, σ. By computer enumeration, the partition functions for short chains are obtained without approximation for the full range of both types of energy. When nonlocal interactions dominate, some sequences undergo coil-globule collapse to a unique native structure. When local interactions dominate, all sequences undergo helix–coil transitions. For two different conformational properties, the closest correspondence between the lattice model and proteins in the Protein Data Bank is obtained if the model local interactions are made small compared to the HH contact interaction, suggesting that helical propensities may be only weak determinants of globular protein structures in water. For some HP sequences, varying σ/ leads to additional sharp transitions (sometimes several) and to “conformational switching” between unique conformations. This behavior resembles the transitions of globular proteins in water to helical states in alcohols. In particular, comparison with experiments shows that whereas urea as a denaturant is best modeled as weakening both local and nonlocal interactions, trifluoroethanol is best modeled as mainly weakening HH interactions and slightly enhancing local helical interactions.     

G-protein-coupled receptor domain overexpression in Halobacterium salinarum: long-range transmembrane interactions in heptahelical membrane proteins

The aminergic alpha(2b)-adrenergic receptor (alpha(2b)-AR) third intracellular loop (alpha(2b)-AR 3i) mediates receptor subcellular compartmentalization and signal transduction processes via ligand-dependent interaction with G(i)- and G(o)- proteins. To understand the structural origins of these processes we engineered several lengths of alpha(2b)-AR 3i into the third intracellular loop of the proton pump bacteriorhodopsin (bR) and produced the fusion proteins in quantities suitable for physical studies. The fusion proteins were expressed in the Archaeon Halobacterium salinarum and purified. A highly expressed fusion protein was crystallized from bicelles and diffracted to low resolution on an in-house diffractometer. The bR-alpha(2b)-AR 3i(203-292) protein possessed a photocycle slightly perturbed from that of the wild-type bR. The first half of the fusion protein photocycle, correlated with proton release, is accelerated by a factor of 3, whereas the second half, correlated with proton uptake, is slightly slower than wild-type bR. In addition, there is a large decrease in the pK(a), (from 9.6 to 8.3) of the terminal proton release group in the unphotolyzed state of bR-alpha(2b)-AR 3i as deduced from the pH-dependence of the M-formation. Perturbation of a cytoplasmic loop has thus resulted in the perturbation of proton release at the extracellular surface. The current work indicates that long-range and highly coupled intramolecular interactions exist that are capable of "transducing" structural perturbations (e.g., signals) across the cellular membrane. This gene fusion approach may have general applicability for physical studies of G-protein-coupled receptor domains in the context of the bR structural scaffold.     

Repulsive interactions between polar and apolar atoms in globular proteins

A significant number of tetrahedral carbon-backbone nitrogen pairs in ferrocytochrome c have interatomic distances that are more than 0.4 Å shorter than the sum of the atomic van der Waals' radii. The non-bonded repulsions of these pairs destabilize the native structure by as much as 50 to 60 kcal/mol. A detailed examination of the close contacts suggests that these result from packing forces associated with the formation of secondary and tertiary structure in the protein.     

Curvature-mediated interactions between membrane proteins.

Membrane proteins can deform the lipid bilayer in which they are embedded. If the bilayer is treated as an elastic medium, then these deformations will generate elastic interactions between the proteins. The interaction between a single pair is repulsive. However, for three or more proteins, we show that there are nonpairwise forces whose magnitude is similar to the pairwise forces. When there are five or more proteins, we show that the nonpairwise forces permit the existence of stable protein aggregates, despite their pairwise repulsions.     

Solvent effects and polar interactions in the structural stability and dynamics of globular proteins.

Using detailed hydrogen bonding, surface exposure, internal environment, and solvent interaction calculations on several proteins, in conjunction wit data from quantum mechanical hydrogen-bonding studies, various contributions to the free energy of globular estimated and their likely relative significance discussed. A picture emerges of globular proteins as extremely well-fitting jigsaw-puzzles, in which no single driving force dominates the marginal stability of the native conformation. Rather, the folded structure is seen as the result of a complex global maximization of several strongly-interacting driving forces. In particular, the necessity to maintain very efficient internal hydrogen-bonding, and the role of the solvent as a hydrogen-bond sink, are stressed as strong constraints on the (incomplete) maximization of hydrophobic effects. The possible significance of internal dipole-induced dipole interactions is discussed tentatively. Although quantitative estimates of the various contributions remain uncertain, consideration of effective force constants suggests that polar, including solvent, interactions may largely determine the overall curvatures of the native conformation's potential well, and be important in controlling the flexibility of local regions which are important for the exact positioning of groups during enzyme catalysis, as well as the molecule's overall dynamics. In contrast, hydrophobic interactions change less for small geometrical perturbations, and seem more relevant to directing the folding protein. along a path to a region in configurational space where the polar interactions can switch on for the final "docking".     

Role of laminin terminal globular domains in basement membrane assembly

Laminins contribute to basement membrane assembly through interactions of their N- and C-terminal globular domains. To further analyze this process, recombinant laminin-111 heterotrimers with deletions and point mutations were generated by recombinant expression and evaluated for their ability to self-assemble, interact with nidogen-1 and type IV collagen, and form extracellular matrices on cultured Schwann cells by immunofluorescence and electron microscopy. Wild-type laminin and laminin without LG domains polymerized in contrast to laminins with deleted alpha1-, beta1-, or gamma1-LN domains or with duplicated beta1- or alpha1-LN domains. Laminins with a full complement of LN and LG domains accumulated on cell surfaces substantially above those lacking either LN or LG domains and formed a lamina densa. Accumulation of type IV collagen onto the cell surface was found to require laminin with separate contributions arising from the presence of laminin LN domains, nidogen-1, and the nidogen-binding site in laminin. Collectively, the data support the hypothesis that basement membrane assembly depends on laminin self-assembly through formation of alpha-, beta-, and gamma-LN domain complexes and LG-mediated cell surface anchorage. Furthermore, type IV collagen recruitment into the laminin extracellular matrices appears to be mediated through a nidogen bridge with a lesser contribution arising from a direct interaction with laminin.     

Hydrogen bonding in globular proteins.

A global census of the hydrogen bonds in 42 X-ray-elucidated proteins was taken and the following demographic trends identified: (1) Most hydrogen bonds are local, i.e. between partners that are close in sequence, the primary exception being hydrogen-bonded ion pairs. (2) Most hydrogen bonds are between backbone atoms in the protein, an average of 68%. (3) All proteins studied have extensive hydrogen-bonded secondary structure, an average of 82%. (4) Almost all backbone hydrogen bonds are within single elements of secondary structure. An approximate rule of thirds applies: slightly more than one-third (37%) form i----i--3 hydrogen bonds, almost one-third (32%) form i----i--4 hydrogen bonds, and slightly less than one-third (26%) reside in paired strands of beta-sheet. The remaining 5% are not wholly within an individual helix, turn or sheet. (5) Side-chain to backbone hydrogen bonds are clustered at helix-capping positions. (6) An extensive network of hydrogen bonds is present in helices. (7) To a close approximation, the total number of hydrogen bonds is a simple function of a protein's helix and sheet content. (8) A unique quantity, termed the reduced number of hydrogen bonds, is defined as the maximum number of hydrogen bonds possible when every donor:acceptor pair is constrained to be 1:1. This quantity scales linearly with chain length, with 0.71 reduced hydrogen bond per residue. Implications of these results for pathways of protein folding are discussed.     

Analysing the origin of long-range interactions in proteins using lattice models

Background  

Long-range communication is very common in proteins but the physical basis of this phenomenon remains unclear. In order to gain insight into this problem, we decided to explore whether long-range interactions exist in lattice models of proteins. Lattice models of proteins have proven to capture some of the basic properties of real proteins and, thus, can be used for elucidating general principles of protein stability and folding.     

A sequence-profile-based HMM for predicting and discriminating beta barrel membrane proteins

MOTIVATION: Membrane proteins are an abundant and functionally relevant subset of proteins that putatively include from about 15 up to 30% of the proteome of organisms fully sequenced. These estimates are mainly computed on the basis of sequence comparison and membrane protein prediction. It is therefore urgent to develop methods capable of selecting membrane proteins especially in the case of outer membrane proteins, barely taken into consideration when proteome wide analysis is performed. This will also help protein annotation when no homologous sequence is found in the database. Outer membrane proteins solved so far at atomic resolution interact with the external membrane of bacteria with a characteristic beta barrel structure comprising different even numbers of beta strands (beta barrel membrane proteins). In this they differ from the membrane proteins of the cytoplasmic membrane endowed with alpha helix bundles (all alpha membrane proteins) and need specialised predictors. RESULTS: We develop a HMM model, which can predict the topology of beta barrel membrane proteins using, as input, evolutionary information. The model is cyclic with 6 types of states: two for the beta strand transmembrane core, one for the beta strand cap on either side of the membrane, one for the inner loop, one for the outer loop and one for the globular domain state in the middle of each loop. The development of a specific input for HMM based on multiple sequence alignment is novel. The accuracy per residue of the model is 83% when a jack knife procedure is adopted. With a model optimisation method using a dynamic programming algorithm seven topological models out of the twelve proteins included in the testing set are also correctly predicted. When used as a discriminator, the model is rather selective. At a fixed probability value, it retains 84% of a non-redundant set comprising 145 sequences of well-annotated outer membrane proteins. Concomitantly, it correctly rejects 90% of a set of globular proteins including about 1200 chains with low sequence identity (<30%) and 90% of a set of all alpha membrane proteins, including 188 chains.     

Spatial optimization of electrostatic interactions between the ionized groups in globular proteins

A model approach is suggested to estimate the degree of spatial optimization of the electrostatic interactions in protein molecules. The method is tested on a set of 44 globular proteins, representative of the available crystallographic data. The theoretical model is based on macroscopic computation of the contribution of charge–charge interactions to the electrostatic term of the free energy for the native proteins and for a big number of virtual structures with randomly distributed on protein surface charge consetellations (generated by a Monte-Carlo technique). The statistical probability of occurrence of random structures with electrostatic energies lower than the energy of the native protein is suggested as a criterion for spatial optimization of the electrostatic interactions. The results support the hypothesis that the folding process optimizes the stabilizing effect of electrostatic interactions, but to very different degree for different proteins. A parallel analysis of ion pairs shows that the optimization of the electrostatic term in globular proteins has increasingly gone in the direction of rejecting the repulsive short contacts between charges of equal sign than of creating of more salt bridges (in comparison with the statistically expected number of shortrange ion pairs in the simulated random structures). It is observed that the decrease in the spatial optimization of the electrostatic interactions is usually compensated for by an appearance of disulfide bridges in the covalent structure of the examined proteins. © 1994 Wiley-Liss, Inc.     

A simple method for discriminating between cell membrane and cytosolic proteins

* Transgenic plants expressing either green fluorescent protein (GFP)-genomic DNA or GFP-cDNA fusions have been used as powerful tools to define the subcellular localization of many proteins. Because most plant cells are highly vacuolated, the cytosol is confined to a thin layer at the periphery of the cells, making it very difficult to distinguish among cell wall, cell membrane and cytosolic GFP-fusion proteins. * Plasmolysis tests inform about cell-wall localization of GFP-tagged proteins, but they do not discriminate between its cell membrane and/or cytoplasmic localization. By observing the GFP signal in transgenic protoplasts placed at a hypotonic solution, it was possible to distinguish between cell membrane and cytosolic GFP-tagged proteins. * The osmotic disruption of the protoplast vacuole in the hypotonic solution allows the diffusion of the GFP signal from the cell periphery to the central part of the cell volume when the GFP is fused to a soluble protein. By contrast, such diffusion does not occur when the protein under study is attached to the cell membrane. * The present method is easier, faster and cheaper than subcellular fractionating studies and/or immunoelectron microscopy, which have been traditionally used to discern between cell membrane and cytosolic proteins.     

Topology of globular proteins. II.

This paper continues the inquiry into the likelihood of occurrence of protein structures which are “knotted”. The probability of knotted con formations is calculated by a simple Monte Carlo method for the proteins lysozyme and ribonuclease and for an elementary bicyclic polymer. Ribonuclease shows no measurable inclination towards tangling, but some 10%. of the lysozyme conformations are knotted. The implications of this result for the role of disulfide bridge formation in protein renaturation are discussed.     

Hydrogen bonding stabilizes globular proteins.

It is clear that intramolecular hydrogen bonds are essential to the structure and stability of globular proteins. It is not clear, however, whether they make a net favorable contribution to this stability. Experimental and theoretical studies are at odds over this important question. Measurements of the change in conformational stability, delta (delta G), for the mutation of a hydrogen bonded residue to one incapable of hydrogen bonding suggest a stabilization of 1.0 kcal/mol per hydrogen bond. If the delta (delta G) values are corrected for differences in side-chain hydrophobicity and conformational entropy, then the estimated stabilization becomes 2.2 kcal/mol per hydrogen bond. These and other experimental studies discussed here are consistent and compelling: hydrogen bonding stabilizes globular proteins.