Measures of residue density in protein structures

A hierarchy of residue density assessments and packing properties in protein structures are contrasted, including a regular density, a variety of charge densities, a hydrophobic density, a polar density, and an aromatic density. These densities are investigated by alternative distance measures and also at the interface of multiunit structures. Amino acids are divided into nine structural categories according to three secondary structure states and three solvent accessibility levels. To take account of amino acid abundance differences across protein structures, we normalize the observed density by the expected density defining a density index. Solvent accessibility levels exert the predominant influence in determinations of the regular residue density. Explicitly, the regular density values vary approximately linearly with respect to solvent accessibility levels, the linearity parameters depending on the amino acid. The charge index reveals pronounced inequalities between lysine and arginine in their interactions with acidic residues. The aromatic density calculations in all structural categories parallel the regular density calculations, indicating that the aromatic residues are distributed as a random sample of all residues. Moreover, aromatic residues are found to be over-represented in the neighborhood of all amino acids. This result might be attributed to nucleation sites and protein stability being substantially associated with aromatic residues.

Atom density in protein structures

The residue environment in protein structures is studied with respect to the density of carbon (C), oxygen (O), and nitrogen (N) atoms within a certain distance (say 5 Å) of each residue. Two types of environments are evaluated: one based on side-chain atom contacts (abbreviated S-S) and the other based on all atom (side-chain + backbone) contacts (abbreviated A-A). Different atom counts are observed about nine-residue structural categories defined by three solvent accessibility levels and three secondary structure states. Among the structural categories, the S-S atom count ratios generally vary more than the A-A atom count ratios because of the fact that the backbone (O) and (N) atoms contribute equal counts. Secondary structure affects the (C) density for the A-A contacts whereas secondary structure has little influence on the (C) density for the S-S contacts. For S-S contacts, a greater density of (O) over (N) atom neighbors stands out in the environment of most amino acid types. By contrast, for A-A contacts, independent of the solvent accessibility levels, the ratio (O)/(N) is ≈1 in helical states, consistent with the geometry of α-helical residues whose side-chains tilt oppositely to the amino to carboxy α-helical axis. The highest ratio of neighbor (O)/(N) is achieved under solvent exposed conditions. This (O) vs. (N) prevalence is advantageous at the protein surface that generally exhibits an acid excess that helps to enhance protein solubility in the cell and to avoid nonspecific interactions with phosphate groups of DNA, RNA, and other plasma constituents.

Isolation and characterization of an amino acid-selective channel protein present in the chloroplastic outer envelope membrane

The reconstituted pea chloroplastic outer envelope protein of 16 kDa (OEP16) forms a slightly cation-selective, high-conductance channel with a conductance of Λ = 1,2 nS (in 1 M KCl). The open probability of OEP16 channel is highest at 0 mV (Popen = 0.8), decreasing exponentially with higher potentials. Transport studies using reconstituted recombinant OEP16 protein show that the OEP16 channel is selective for amino acids but excludes triosephosphates or uncharged sugars. Crosslinking indicates that OEP16 forms a homodimer in the membrane. According to its primary sequence and predicted secondary structure, OEP16 shows neither sequence nor structural homologies to classical porins. The results indicate that the intermembrane space between the two envelope membranes might not be as freely accessible as previously thought.

Exploring the folding free energy surface of a three-helix bundle protein

The multidimensional free energy surface for a small fast folding helical protein is explored based on first-principle calculations. The model represents the 46-residue segment from fragment B of staphylococcal protein A. The relationship between collapse and tertiary structure formation, and the order of collapse and secondary structure formation, are investigated. We find that the initial collapse process gives rise to a transition state with about 30% of the native tertiary structure and 50–70% of the native helix content. We also observe two distinct distributions of native helix in this collapsed state (Rg ≈ 12 Å), one with about 20% of the native helical hydrogen bonds, the other with near 70%. The former corresponds to a local minimum. The barrier from this metastable state to the native state is about 2 kBT. In the latter case, folding is essentially a downhill process involving topological assembly. In addition, the order of formation of secondary structure among the three helices is examined. We observe cooperative formation of the secondary structure in helix I and helix II. Secondary structure in helix III starts to form following the formation of certain secondary structure in both helix I and helix II. Comparisons of our results with those from theory and experiment are made.

A structural census of the current population of protein sequences

We examine the occurrence of the ≈300 known protein folds in different groups of organisms. To do this, we characterize a large fraction of the currently known protein sequences (≈140,000) in structural terms, by matching them to known structures via sequence comparison (or by secondary-structure class prediction for those without structural homologues). Overall, we find that an appreciable fraction of the known folds are present in each of the major groups of organisms (e.g., bacteria and eukaryotes share 156 of 275 folds), and most of the common folds are associated with many families of nonhomologous sequences (i.e., >10 sequence families for each common fold). However, different groups of organisms have characteristically distinct distributions of folds. So, for instance, some of the most common folds in vertebrates, such as globins or zinc fingers, are rare or absent in bacteria. Many of these differences in fold usage are biologically reasonable, such as the folds of metabolic enzymes being common in bacteria and those associated with extracellular transport and communication being common in animals. They also have important implications for database-based methods for fold recognition, suggesting that an unknown sequence from a plant is more likely to have a certain fold (e.g., a TIM barrel) than an unknown sequence from an animal.

Two-dimensional IR correlation spectroscopy: Sequential events in the unfolding process of the λ Cro-V55C repressor protein

A question often posed in protein folding/unfolding studies is whether the process is fully cooperative or whether it contains sequential elements. To address this question, one needs tools capable of resolving different events. It seems that, at least in certain cases, two-dimensional (2D) IR correlation spectroscopy can provide answers to this question. To illustrate this point, we have turned to the Cro-V55C dimer of the λ Cro repressor, a protein known to undergo thermal unfolding in two discrete steps through a stable equilibrium intermediate. The secondary structure of this intermediate is compatible with that of a partially unfolded protein and involves a reorganization of the N terminus, whereas the antiparallel β-ribbon formed by the C-terminal part of each subunit remains largely intact. To establish whether the unfolding process involves sequential events, we have performed a 2D correlation analysis of IR spectra recorded over the temperature range of 20–95°C. The 2D IR correlation analysis indeed provides evidence for a sequential formation of the stable intermediate, which is created in three (closely related) steps. A first step entails the unfolding of the short N-terminal β-strand, followed by the unfolding of the α-helices in a second step, and the third step comprises the reorganization of the remaining β-sheet and of some unordered segments in the protein. The complete unfolding of the stable intermediate at higher temperatures also undergoes sequential events that ultimately end with the breaking of the H bonds between the two β-strands at the dimer interface.

Conformational influences of glycosylation of a peptide: A possible model for the effect of glycosylation on the rate of protein folding

Improved strategies for synthesis make it possible to expand the range of glycopeptides available for detailed conformational studies. The glycopeptide 1 was synthesized using a new solid phase synthesis of carbohydrates and a convergent coupling to peptide followed by deprotection. Its conformational properties were subjected to NMR analysis and compared with a control peptide 2 prepared by conventional solid phase methods. Whereas peptide 2 fails to manifest any appreciable secondary structure, the glycopeptide 1 does show considerable conformational bias suggestive of an equilibrium between an ordered and a random state. The implications of this ordering effect for the larger issue of protein folding are considered.

Localization of the N terminus of hepatitis B virus capsid protein by peptide-based difference mapping from cryoelectron microscopy

Recently, cryoelectron microscopy of isolated macromolecular complexes has advanced to resolutions below 10 Å, enabling direct visualization of α-helical secondary structure. To help correlate such density maps with the amino acid sequences of the component proteins, we advocate peptide-based difference mapping, i.e., insertion of peptides, ≈10 residues long, at targeted points in the sequence and visualization of these peptides as bulk labels in cryoelectron microscopy-derived difference maps. As proof of principle, we have appended an extraneous octapeptide at the N terminus of hepatitis B virus capsid protein and determined its location on the capsid surface by difference imaging at 11 Å resolution. Hepatitis B virus capsids are icosahedral particles, ≈300 Å in diameter, made up of T-shaped dimers (subunit Mr, 16–21 kDa, depending on construct). The stems of the Ts protrude outward as spikes, whereas the crosspieces pack to form the contiguous shell. The two N termini per dimer reside on either side of the spike-stem, at the level at which it enters the shell. This location is consistent with formation of the known intramolecular disulfide bond between the cysteines at positions 61 and −7 (in the residual propeptide) in the “e-antigen” form of the capsid protein and has implications for why this clinically important antigen remains unassembled in vivo.

A transcriptional activating region with two contrasting modes of protein interaction

A C-terminal segment of the yeast activator Gal4 manifests two functions: When tethered to DNA, it elicits gene activation, and it binds the inhibitor Gal80. Here we examine the effects on these two functions of cysteine and proline substitutions. We find that, although certain cysteine substitutions diminish interaction with Gal80, those substitutions have little effect on the activating function in vivo and interaction with TATA box-binding protein (TBP) in vitro. Proline substitutions introduced near residues critical for Gal80 binding abolish that interaction but once again have no effect on the activating function. Crosslinking experiments show that a defined position in the activating peptide is in close proximity to TBP and Gal80 in the two separate reactions and show that binding of the inhibitor blocks binding to TBP. Thus, the same stretch of amino acids are involved in two quite different protein–protein interactions: binding to Gal80, which depends on a precise sequence and the formation of a defined secondary structure, or interactions with the transcriptional machinery in vivo, which are not impaired by perturbations of either sequence or structure.

A T42A Ran Mutation: Differential Interactions with Effectors and Regulators, and Defect in Nuclear Protein Import

Ran, the small, predominantly nuclear GTPase, has been implicated in the regulation of a variety of cellular processes including cell cycle progression, nuclear-cytoplasmic trafficking of RNA and protein, nuclear structure, and DNA synthesis. It is not known whether Ran functions directly in each process or whether many of its roles may be secondary to a direct role in only one, for example, nuclear protein import. To identify biochemical links between Ran and its functional target(s), we have generated and examined the properties of a putative Ran effector mutation, T42A-Ran. T42A-Ran binds guanine nucleotides as well as wild-type Ran and responds as well as wild-type Ran to GTP or GDP exchange stimulated by the Ran-specific guanine nucleotide exchange factor, RCC1. T42A-Ran·GDP also retains the ability to bind p10/NTF2, a component of the nuclear import pathway. In contrast to wild-type Ran, T42A-Ran·GTP binds very weakly or not detectably to three proposed Ran effectors, Ran-binding protein 1 (RanBP1), Ran-binding protein 2 (RanBP2, a nucleoporin), and karyopherin β (a component of the nuclear protein import pathway), and is not stimulated to hydrolyze bound GTP by Ran GTPase-activating protein, RanGAP1. Also in contrast to wild-type Ran, T42A-Ran does not stimulate nuclear protein import in a digitonin permeabilized cell assay and also inhibits wild-type Ran function in this system. However, the T42A mutation does not block the docking of karyophilic substrates at the nuclear pore. These properties of T42A-Ran are consistent with its classification as an effector mutant and define the exposed region of Ran containing the mutation as a probable effector loop.

Recombinant scrapie-like prion protein of 106 amino acids is soluble

The N terminus of the scrapie isoform of prion protein (PrPSc) can be truncated without loss of scrapie infectivity and, correspondingly, the truncation of the N terminus of the cellular isoform, PrPC, still permits conversion into PrPSc. To assess whether additional segments of the PrP molecule can be deleted, we previously removed regions of putative secondary structure in PrPC; in the present study we found that deletion of each of the four predicted helices prevented PrPSc formation, as did deletion of the stop transfer effector region and the C178A mutation. Removal of a 36-residue loop between helices 2 and 3 did not prevent formation of protease-resistant PrP; the resulting scrapie-like protein, designated PrPSc106, contained 106 residues after cleavage of an N-terminal signal peptide and a C-terminal sequence for glycolipid anchor addition. Addition of the detergent Sarkosyl to cell lysates solubilized PrPSc106, which retained resistance to digestion by proteinase K. These results suggest that all the regions of proposed secondary structure in PrP are required for PrPSc formation, as is the disulfide bond stabilizing helices 3 and 4. The discovery of PrPSc106 should facilitate structural studies of PrPSc, investigations of the mechanism of PrPSc formation, and the production of PrPSc-specific antibodies.

Energy-based de novo protein folding by conformational space annealing and an off-lattice united-residue force field: Application to the 10-55 fragment of staphylococcal protein A and to apo calbindin D9K

The conformational space annealing (CSA) method for global optimization has been applied to the 10-55 fragment of the B-domain of staphylococcal protein A (protein A) and to a 75-residue protein, apo calbindin D9K (PDB ID code 1CLB), by using the UNRES off-lattice united-residue force field. Although the potential was not calibrated with these two proteins, the native-like structures were found among the low-energy conformations, without the use of threading or secondary-structure predictions. This is because the CSA method can find many distinct families of low-energy conformations. Starting from random conformations, the CSA method found that there are two families of low-energy conformations for each of the two proteins, the native-like fold and its mirror image. The CSA method converged to the same low-energy folds in all cases studied, as opposed to other optimization methods. It appears that the CSA method with the UNRES force field, which is based on the thermodynamic hypothesis, can be used in prediction of protein structures in real time.

Mutation R120G in αB-crystallin, which is linked to a desmin-related myopathy, results in an irregular structure and defective chaperone-like function

αB-crystallin, a member of the small heat shock protein family, possesses chaperone-like function. Recently, it has been shown that a missense mutation in αB-crystallin, R120G, is genetically linked to a desmin-related myopathy as well as to cataracts [Vicart, P., Caron, A., Guicheney, P., Li, A., Prevost, M.-C., Faure, A., Chateau, D., Chapon, F., Tome, F., Dupret, J.-M., et al. (1998) Nat. Genet. 20, 92–95]. By using α-lactalbumin, alcohol dehydrogenase, and insulin as target proteins, in vitro assays indicated that R120G αB-crystallin had reduced or completely lost chaperone-like function. The addition of R120G αB-crystallin to unfolding α-lactalbumin enhanced the kinetics and extent of its aggregation. R120G αB-crystallin became entangled with unfolding α-lactalbumin and was a major portion of the resulting insoluble pellet. Similarly, incubation of R120G αB-crystallin with alcohol dehydrogenase and insulin also resulted in the presence of R120G αB-crystallin in the insoluble pellets. Far and near UV CD indicate that R120G αB-crystallin has decreased β-sheet secondary structure and an altered aromatic residue environment compared with wild-type αB-crystallin. The apparent molecular mass of R120G αB-crystallin, as determined by gel filtration chromatography, is 1.4 MDa, which is more than twice the molecular mass of wild-type αB-crystallin (650 kDa). Images obtained from cryoelectron microscopy indicate that R120G αB-crystallin possesses an irregular quaternary structure with an absence of a clear central cavity. The results of this study show, through biochemical analysis, that an altered structure and defective chaperone-like function of αB-crystallin are associated with a point mutation that leads to a desmin-related myopathy and cataracts.

DNA annealing by Rad52 Protein is stimulated by specific interaction with the complex of replication protein A and single-stranded DNA

Homologous recombination in Saccharomyces cerevisiae depends critically on RAD52 function. In vitro, Rad52 protein preferentially binds single-stranded DNA (ssDNA), mediates annealing of complementary ssDNA, and stimulates Rad51 protein-mediated DNA strand exchange. Replication protein A (RPA) is a ssDNA-binding protein that is also crucial to the recombination process. Herein we report that Rad52 protein effects the annealing of RPA–ssDNA complexes, complexes that are otherwise unable to anneal. The ability of Rad52 protein to promote annealing depends on both the type of ssDNA substrate and ssDNA binding protein. RPA allows, but slows, Rad52 protein-mediated annealing of oligonucleotides. In contrast, RPA is almost essential for annealing of longer plasmid-sized DNA but has little effect on the annealing of poly(dT) and poly(dA), which are relatively long DNA molecules free of secondary structure. These results suggest that one role of RPA in Rad52 protein-mediated annealing is the elimination of DNA secondary structure. However, neither Escherichia coli ssDNA binding protein nor human RPA can substitute in this reaction, indicating that RPA has a second role in this process, a role that requires specific RPA–Rad52 protein interactions. This idea is confirmed by the finding that RPA, which is complexed with nonhomologous ssDNA, inhibits annealing but the human RPA–ssDNA complex does not. Finally, we present a model for the early steps of the repair of double-strand DNA breaks in yeast.

Overexpression of a glutamate receptor (GluR2) ligand binding domain in Escherichia coli: Application of a novel protein folding screen

Expression of the S1S2 ligand binding domain [Kuusinen, A., Arvola, M. & Keinänen, K. (1995) EMBO J. 14, 6327–6332] of the rat α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-selective glutamate receptor GluR2 in Escherichia coli under control of a T7 promoter leads to production of >100 mg/liter of histidine-tagged S1S2 protein (HS1S2) in the form of inclusion bodies. Using a novel fractional factorial folding screen and a rational, step-by-step approach, multiple conditions were determined for the folding of the HS1S2 α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid binding domain. Characterization of the HS1S2 ligand binding domain showed that it is water-soluble, monomeric, has significant secondary structure, and is sensitive to trypsinolysis at sites close to the beginning of the putative transmembrane regions. Application of a fractional factorial folding screen to other proteins may provide a useful means to evaluate E. coli as an economical and convenient expression host.