Surface beta-strands in proteins: identification using an hydropathy technique

From a representative set of monomeric globular proteins with known three-dimensional structures, beta-strands with lengths > or = 5 amino acids have been identified and catalogued. By ascertaining the accessible surface areas of the constituent residues in these strands, and by checking whether the exposed/buried pattern is 80% or more similar to that in an idealized surface strand, a subset of structures can be delineated in which the beta-strands are all sited on the surface of the protein. The corresponding sequence data show that about 50% of the residues are apolar (Val, Ile, Leu, Phe, Tyr, Ala) and that the common occurrence of valine (14.3%), isoleucine (9.6%), and threonine (8.1%) is a characteristic feature. The frequencies of occurrence of those amino acids in the strands that face the aqueous environment and the interior have also been determined separately and show that most surface strands have a substructure of the form (apolar-X)(n), where X is approximately equally divided between apolar, charged, and hydrophilic residues. Using the frequency data thus obtained, allied with an algorithm to delineate potential surface beta-strands from characteristic hydropathy profiles, it is now possible to search through the sequences of proteins with unknown tertiary structures and make realistic predictions of the presence of this element of structure on the protein surface. In addition, new data are presented on the distribution of the various types of residues on the surface of proteins and in their interior. Significant differences were observed, not all of which have been identified previously. Furthermore, the distribution of the types of residue in a surface beta-strand was compared to that corresponding to the surfaces of all of the proteins in our database. Again, very characteristic differences were observed. These are helpful in recognizing the presence of surface beta-strands.     

Water transfer energetics and solid-like packing of globular proteins

In this article we generalize the use of a relationship based on the occurrence of some characteristic temperatures in protein unfolding, which were originally high-lighted for proteins showing the unfolding enthalpy-entropy convergence. On this basis, we show how to dissect the unfolding Gibbs energy of globular proteins in terms of solid-like and liquid-like contributions, untangling the protein energetics by a scheme which does not suffer from excessive intricacy. We also provide an experimental estimate of unfavorable polar contributions to the protein stability, by which it seems possible to evaluate the number of buried residues in individual proteins. A comparison is assessed with the so-called hard sphere model of globular proteins. Results seem to reconcile the view that the protein interior is liquid-like with the observation that protein organization resembles an assembly of closely packed spheres.     

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.     

Contribution of polar groups in the interior of a protein to the conformational stability

It has been generally believed that polar residues are usually located on the surface of protein structures. However, there are many polar groups in the interior of the structures in reality. To evaluate the contribution of such buried polar groups to the conformational stability of a protein, nonpolar to polar mutations (L8T, A9S, A32S, I56T, I59T, I59S, A92S, V93T, A96S, V99T, and V100T) in the interior of a human lysozyme were examined. The thermodynamic parameters for denaturation were determined using a differential scanning calorimeter, and the crystal structures were analyzed by X-ray crystallography. If a polar group had a heavy energy cost to be buried, a mutant protein would be remarkably destabilized. However, the stability (Delta G) of the Ala to Ser and Val to Thr mutant human lysozymes was comparable to that of the wild-type protein, suggesting a low-energy penalty of buried polar groups. The structural analysis showed that all polar side chains introduced in the mutant proteins were able to find their hydrogen bond partners, which are ubiquitous in protein structures. The empirical structure-based calculation of stability change (Delta Delta G) [Takano et al. (1999) Biochemistry 38, 12698--12708] revealed that the mutant proteins decreased the hydrophobic effect contributing to the stability (Delta G(HP)), but this destabilization was recovered by the hydrogen bonds newly introduced. The present study shows the favorable contribution of polar groups with hydrogen bonds in the interior of protein molecules to the conformational stability.     

Assisted peptide folding by surface pattern recognition

Natively disordered proteins belong to a unique class of biomolecules whose function is related to their flexibility and their ability to adopt desired conformations upon binding to substrates. In some cases these proteins can bind multiple partners, which can lead to distinct structures and promiscuity in functions. In other words, the capacity to recognize molecular patterns on the substrate is often essential for the folding and function of intrinsically disordered proteins. Biomolecular pattern recognition is extremely relevant both in vivo (e.g., for oligomerization, immune response, induced folding, substrate binding, and molecular switches) and in vitro (e.g., for biosensing, catalysis, chromatography, and implantation). Here, we use a minimalist computational model system to investigate how polar/nonpolar patterns on a surface can induce the folding of an otherwise unstructured peptide. We show that a model peptide that exists in the bulk as a molten globular state consisting of many interconverting structures can fold into either a helix-coil-helix or an extended helix structure in the presence of a complementary designed patterned surface at low hydrophobicity (3.7%) or a uniform surface at high hydrophobicity (50%). However, we find that a carefully chosen surface pattern can bind to and catalyze the folding of a natively unfolded protein much more readily or effectively than a surface with a noncomplementary or uniform distribution of hydrophobic residues.     

Geometric cooperativity and anticooperativity of three-body interactions in native proteins

Characterizing multibody interactions of hydrophobic, polar, and ionizable residues in protein is important for understanding the stability of protein structures. We introduce a geometric model for quantifying 3-body interactions in native proteins. With this model, empirical propensity values for many types of 3-body interactions can be reliably estimated from a database of native protein structures, despite the overwhelming presence of pairwise contacts. In addition, we define a nonadditive coefficient that characterizes cooperativity and anticooperativity of residue interactions in native proteins by measuring the deviation of 3-body interactions from 3 independent pairwise interactions. It compares the 3-body propensity value from what would be expected if only pairwise interactions were considered, and highlights the distinction of propensity and cooperativity of 3-body interaction. Based on the geometric model, and what can be inferred from statistical analysis of such a model, we find that hydrophobic interactions and hydrogen-bonding interactions make nonadditive contributions to protein stability, but the nonadditive nature depends on whether such interactions are located in the protein interior or on the protein surface. When located in the interior, many hydrophobic interactions such as those involving alkyl residues are anticooperative. Salt-bridge and regular hydrogen-bonding interactions, such as those involving ionizable residues and polar residues, are cooperative. When located on the protein surface, these salt-bridge and regular hydrogen-bonding interactions are anticooperative, and hydrophobic interactions involving alkyl residues become cooperative. We show with examples that incorporating 3-body interactions improves discrimination of protein native structures against decoy conformations. In addition, analysis of cooperative 3-body interaction may reveal spatial motifs that can suggest specific protein functions.     

Origins of globular structure in proteins

Thermodynamic incompatibility of polymers in a common solvent is possibly a driving force for formation and evolution of globular protein structures. Folding of polypeptide chains leads to a decrease in both excluded volume of molecules and chemical differences between surfaces of globular molecules with chemical information hidden in the hydrophobic interior. Folding of polypeptide chains results in 'molecular or thermodynamic mimicry' of globular proteins and in at least more than 10-fold higher phase separation threshold values of mixed protein solutions compared to those of classical polymers. Unusually high co-solubility might be necessary for efficient biological functioning of proteins, e.g. enzymes, enzyme inhibitors, etc.     

Remote homology detection of integral membrane proteins using conserved sequence features

Compared with globular proteins, transmembrane proteins are surrounded by a more intricate environment and, consequently, amino acid composition varies between the different compartments. Existing algorithms for homology detection are generally developed with globular proteins in mind and may not be optimal to detect distant homology between transmembrane proteins. Here, we introduce a new profile-profile based alignment method for remote homology detection of transmembrane proteins in a hidden Markov model framework that takes advantage of the sequence constraints placed by the hydrophobic interior of the membrane. We expect that, for distant membrane protein homologs, even if the sequences have diverged too far to be recognized, the hydrophobicity pattern and the transmembrane topology are better conserved. By using this information in parallel with sequence information, we show that both sensitivity and specificity can be substantially improved for remote homology detection in two independent test sets. In addition, we show that alignment quality can be improved for the most distant homologs in a public dataset of membrane protein structures. Applying the method to the Pfam domain database, we are able to suggest new putative evolutionary relationships for a few relatively uncharacterized protein domain families, of which several are confirmed by other methods. The method is called Searcher for Homology Relationships of Integral Membrane Proteins (SHRIMP) and is available for download at http://www.sbc.su.se/shrimp/.     

Statistical analysis of protein structures suggests that buried ionizable residues in proteins are hydrogen bonded or form salt bridges

It is well known that nonpolar residues are largely buried in the interior of proteins, whereas polar and ionizable residues tend to be more localized on the protein surface where they are solvent exposed. Such a distribution of residues between surface and interior is well understood from a thermodynamic point: nonpolar side chains are excluded from the contact with the solvent water, whereas polar and ionizable groups have favorable interactions with the water and thus are preferred at the protein surface. However, there is an increasing amount of information suggesting that polar and ionizable residues do occur in the protein core, including at positions that have no known functional importance. This is inconsistent with the observations that dehydration of polar and in particular ionizable groups is very energetically unfavorable. To resolve this, we performed a detailed analysis of the distribution of fractional burial of polar and ionizable residues using a large set of ?2600 nonhomologous protein structures. We show that when ionizable residues are fully buried, the vast majority of them form hydrogen bonds and/or salt bridges with other polar/ionizable groups. This observation resolves an apparent contradiction: the energetic penalty of dehydration of polar/ionizable groups is paid off by favorable energy of hydrogen bonding and/or salt bridge formation in the protein interior. Our conclusion agrees well with the previous findings based on the continuum models for electrostatic interactions in proteins. Proteins 2011; © 2011 Wiley‐Liss, Inc.     

Correlations between internal mobility and stability of globular proteins.

The recent work is surveyed which leads to the suggestions that the conformation of globular proteins in solution corresponds to a dynamic ensemble of rapidly interconverting spatial structures, that clusters of hydrophobic amino acid side chains have an important role in the architecture of protein molecules, and that mechanistic aspects of protein denaturation can be correlated with internal mobility seen in the native conformation. These conclusions resulted originally from high resolution 1H nuclear magnetic resonance (NMR) studies of aromatic ring mobility, exchange of interior amide protons and thermal denaturation of the basic pancreatic trypsin inhibitor and a group of related proteins. Various new approaches to further characterize proteins in solution have now been taken and preliminary data are presented. These include computer graphics to outline hydrophobic clusters in globular protein structures, high resolution 1H-NMR experiments at variable hydrostatic pressure and 13C-NMR relaxation measurements. At the present early stage of these new investigations it appears that the hydrophobic cluster model for globular proteins is compatible with the data obtained.     

On the significance of alternating patterns of polar and non-polar residues in beta-strands

A common assumption about protein sequences in beta-strands is that they have alternating patterns of polar and non-polar residues. It is thought that such patterns reflect the interior/exterior geometry of amino acid residue side-chains on a beta-sheet. Here we study the prevalence of simple hydrophobicity patterns in parallel and antiparallel beta-sheets in proteins of known structure and in the sequences of amyloidogenic proteins. The occurrence of 32 possible pentapeptide binary patterns (polar (P)/non-polar (N)) is computed in 1911 non-homologous protein structures. Despite their tendency to aggregate in experimentally designed proteins, the purely alternating hydrophobic/polar patterns (PNPNP and NPNPN) are most frequent in beta-sheets, typically occurring in antiparallel strands. The overall distribution of the pentapeptide binary patterns is significantly different in strands within parallel and antiparallel sheets. In both types of sheets, complementary patterns (where the hydrophobic and polar residues pair with one another) associate preferentially. We do not find alternating patterns to be common in amyloidogenic proteins or in short fragments involved directly in amyloid formation. However, we do note some similarities between patterns present in amyloidogenic sequences and those in parallel strands.     

Spatial sign-alternating charge clusters in globular proteins

Large sign-alternating charge clusters formed by the charged side groups of amino acid residues and N- and C-terminal groups were found in the majority of considered globular proteins, namely 235 in a total of 274 protein structures, i.e. 85.8%. The clusters were determined by the criteria proposed earlier: charged groups were included in the cluster if their charged N and O atoms were located at distances between 2.4 and 7.0 A. The set of selected proteins consisted of known non-homologous protein structures from the Protein Data Bank with a resolution less than or equal to 2.5 A and pair sequence similarity less than 25%. Molecular masses of the proteins were from 5.5 to 91.5 kDa and protein chain length from 50 to 830 residues. The distribution of charged groups on the protein surface between isolated charged groups, small clusters with two and three groups, and large clusters with four or more groups were found to be approximately similar making 33, 35 and 32% of the total amount of protein charged groups, respectively. The large sign-alternating charge clusters with four or more charged groups were studied in greater detail. The amount of such clusters depends on the protein chain length. The small proteins contain 1-3 clusters while the large proteins display 4-6 or more clusters. On average, 1.5 clusters per each 100 residues were observed. In contrast with this, the size of a cluster, i.e. the number of charged groups inside a cluster, does not depend on the protein molecular mass, and large clusters are observed for proteins from a range of molecular masses. Clusters consisting of four to six charged groups occur most frequently, although extra large clusters are also often revealed. We can conclude that sign-alternating charge clusters are a common feature of the protein surface of globular protein. They are suggested to play a general functional role as a local polar factor of protein surface.     

Accounting for protein-solvent contacts facilitates design of nonaggregating lattice proteins

The folding specificity of proteins can be simulated using simplified structural models and knowledge-based pair-potentials. However, when the same models are used to simulate systems that contain many proteins, large aggregates tend to form. In other words, these models cannot account for the fact that folded, globular proteins are soluble. Here we show that knowledge-based pair-potentials, which include explicitly calculated energy terms between the solvent and each amino acid, enable the simulation of proteins that are much less aggregation-prone in the folded state. Our analysis clarifies why including a solvent term improves the foldability. The aggregation for potentials without water is due to the unrealistically attractive interactions between polar residues, causing artificial clustering. When a water-based potential is used instead, polar residues prefer to interact with water; this leads to designed protein surfaces rich in polar residues and well-defined hydrophobic cores, as observed in real protein structures. We developed a simple knowledge-based method to calculate interactions between the solvent and amino acids. The method provides a starting point for modeling the folding and aggregation of soluble proteins. Analysis of our simple model suggests that inclusion of these solvent terms may also improve off-lattice potentials for protein simulation, design, and structure prediction.     

A novel measure characterized by a polar energy surface approximation for recognition and classification of transmembrane protein structures

A new theoretical method has been developed for recognition and classification of membrane proteins. The method is based on computation of a polar energy surface that can reveal characteristic interaction patterns for individual helices even if crystal or NMR structure coordinates are not available. A protein with N transmembrane helices is described as a set of N vectors that are derived from a Fourier analysis of this polar energy surface computed for each helix. We then derive a polarity difference score (PDS) for any two proteins computed as the root mean square deviation between the respective vector coordinate sets. The score was found to correlate with the degree of structural similarity between the following three protein families for which tertiary structures have been determined: bacteriorhodopsin, rhodopsin, and the cytochrome c oxidase III subunit.     

Mapping the binding sites of challenging drug targets

An increasing number of medically important proteins are challenging drug targets because their binding sites are too shallow or too polar, are cryptic and thus not detectable without a bound ligand or located in a protein–protein interface. While such proteins may not bind druglike small molecules with sufficiently high affinity, they are frequently druggable using novel therapeutic modalities. The need for such modalities can be determined by experimental or computational fragment based methods. Computational mapping by mixed solvent molecular dynamics simulations or the FTMap server can be used to determine binding hot spots. The strength and location of the hot spots provide very useful information for selecting potentially successful approaches to drug discovery.     

Molecular structure of an apolipoprotein determined at 2.5-A resolution

The three-dimensional structure of an apolipoprotein isolated from the African migratory locust Locusta migratoria has been determined by X-ray analysis to a resolution of 2.5 A. The overall molecular architecture of this protein consists of five long alpha-helices connected by short loops. As predicted from amino acid sequence analyses, these helices are distinctly amphiphilic with the hydrophobic residues pointing in toward the interior of the protein and the hydrophilic side chains facing outward. The molecule falls into the general category of up-and-down alpha-helical bundles as previously observed, for example, in cytochrome c'. Although the structure shows the presence of five long amphiphilic alpha-helices, the alpha-helical moment and hydrophobicity of the entire molecule fall into the range found for normal globular proteins. Thus, in order for the amphiphilic helices to play a role in the binding of the protein to a lipid surface, there must be a structural reorganization of the protein which exposes the hydrophobic interior to the lipid surface. The three-dimensional motif of this apolipoprotein is compatible with a model in which the molecule binds to the lipid surface via a relatively nonpolar end and then spreads on the surface in such a way as to cause the hydrophobic side chains of the helices to come in contact with the lipid surface, the charged and polar residues to remain in contact with water, and the overall helical motif of the protein to be maintained.     

Polar residues in the protein core of Escherichia coli thioredoxin are important for fold specificity

Most globular proteins contain a core of hydrophobic residues that are inaccessible to solvent in the folded state. In general, polar residues in the core are thermodynamically unfavorable except when they are able to form intramolecular hydrogen bonds. Compared to hydrophobic interactions, polar interactions are more directional in character and may aid in fold specificity. In a survey of 263 globular protein structures, we found a strong positive correlation between the number of polar residues at core positions and protein size. To probe the importance of buried polar residues, we experimentally tested the effects of hydrophobic mutations at the five polar core residues in Escherichia coli thioredoxin. Proteins with single hydrophobic mutations (D26I, C32A, C35A, T66L, and T77V) all have cooperative unfolding transitions like the wild type (wt), as determined by chemical denaturation. Relative to wt, D26I is more stable while the other point mutants are less stable. The combined 5-fold mutant protein (IAALV) is less stable than wt and has an unfolding transition that is substantially less cooperative than that of wt. NMR spectra as well as amide deuterium exchange indicate that IAALV is likely sampling a number of low-energy structures in the folded state, suggesting that polar residues in the core are important for specifying a well-folded native structure.     

Receptor-dependent compartmentalization of PPIP5K1, a kinase with a cryptic polyphosphoinositide binding domain

Amyloid-related diseases are a group of illnesses in which an abnormal accumulation of proteins into fibrillar structures is evident. Results from a wide range of studies, ranging from identification of amyloid-β dimers in the brain to biophysical characterization of the interactions between amyloidogenic peptides and lipid membranes during fibril growth shed light on the initial events which take place during amyloid aggregation. Accounts of fibril disaggregation and formation of globular aggregates due to interactions with lipids or fatty acids further demonstrate the complexity of the aggregation process and the difficulty to treat amyloid-related diseases. There is an inherent difficulty in generalizing from studies of aggregation in vitro, but the involvement of too many cellular components limits the ability to follow amyloid aggregation in a cellular (or extracellular) context. Fortunately, the development of experimental methods to generate stable globular aggregates suggests new means of studying the molecular events associated with amyloid aggregation. Furthermore, simulation studies enable deeper understanding of the experimental results and provide useful predictions that can be tested in the laboratory. Computer simulations can nowadays provide molecular or even atomistic details that are experimentally not available or very difficult to obtain. In the present review, recent developments on modelling and experiments of amyloid aggregation are reviewed, and an integrative account on how isolated interactions (as observed in vitro and in silico) combine during the course of amyloid-related diseases is presented. Finally, it is argued that an integrative approach is necessary to get a better understanding of the protein aggregation process.     

Spatial profiling of protein hydrophobicity: native vs. decoy structures

A recent study of 30 soluble globular protein structures revealed a quasi-invariant called the hydrophobic ratio. This invariant, which is the ratio of the distance at which the second order hydrophobic moment vanished to the distance at which the zero order moment vanished, was found to be 0.75 +/- 0.05 for 30 protein structures. This report first describes the results of the hydrophobic profiling of 5,387 non-redundant globular protein domains of the Protein Data Bank, which yields a hydrophobic ratio of 0.71 +/- 0.08. Then, a new hydrophobic score is defined based on the hydrophobic profiling to discriminate native-like proteins from decoy structures. This is tested on three widely used decoy sets, namely the Holm and Sander decoys, Park and Levitt decoys, and Baker decoys. Since the hydrophobic moment profiling characterizes a global feature and requires reasonably good statistics, this imposes a constraint upon the size of the protein structures in order to yield relatively smooth moment profiles. We show that even subject to the limitations of protein size (both Park & Levitt and Baker sets are small protein decoys), the hydrophobic moment profiling and hydrophobic score can provide useful information that should be complementary to the information provided by force field calculations.     

Identifying foldable regions in protein sequence from the hydrophobic signal

Structural genomics initiatives aim to elucidate representative 3D structures for the majority of protein families over the next decade, but many obstacles must be overcome. The correct design of constructs is extremely important since many proteins will be too large or contain unstructured regions and will not be amenable to crystallization. It is therefore essential to identify regions in protein sequences that are likely to be suitable for structural study. Scooby-Domain is a fast and simple method to identify globular domains in protein sequences. Domains are compact units of protein structure and their correct delineation will aid structural elucidation through a divide-and-conquer approach. Scooby-Domain predictions are based on the observed lengths and hydrophobicities of domains from proteins with known tertiary structure. The prediction method employs an A*-search to identify sequence regions that form a globular structure and those that are unstructured. On a test set of 173 proteins with consensus CATH and SCOP domain definitions, Scooby-Domain has a sensitivity of 50% and an accuracy of 29%, which is better than current state-of-the-art methods. The method does not rely on homology searches and, therefore, can identify previously unknown domains.