Evolutionary Aspects of Protein Structure and Folding

Four basic stages of evolution of protein structure are described, basing on recent work of the authors aimed specifically to reconstruct the earliest events in the protein evolution. According to this reconstruction, the initial stage of short peptides comprising, probably, only a few amino acid residues had been followed by formation of closed loops of 25–30 residues, which corresponds to the polymer-statistically optimal ring closure size for mixed polypeptide chains. The next stage involved fusion of relatively small linear genes and formation of protein structures consisting of several closed loops of a nearly standard size, with 4–6 loops (100–200 amino acid residues) in a typical protein fold. The last, modern stage began with combinatorial fusion of the presumably circular 300–600 bp DNA units and, accordingly, formation of multidomain proteins.     

Flowering buds of globular proteins: transpiring simplicity of protein organization

Structural and functional complexity of proteins is dramatically reduced to a simple linear picture when the laws of polymer physics are considered. A basic unit of the protein structure is a nearly standard closed loop of 25-35 amino acid residues, and every globular protein is built of consecutively connected closed loops. The physical necessity of the closed loops had been apparently imposed on the early stages of protein evolution. Indeed, the most frequent prototype sequence motifs in prokaryotic proteins have the same sequence size, and their high match representatives are found as closed loops in crystallized proteins. Thus, the linear organization of the closed loop elements is a quintessence of protein evolution, structure and folding.     

Distinct Stages of Protein Evolution as Suggested by Protein Sequence Analysis

Evolution of proteins encoded in nucleotide sequences began with the advent of the triplet code. The chronological order of the appearance of amino acids on the evolution scene and the steps in the evolution of the triplet code have been recently reconstructed (Trifonov, 2000b) on the basis of 40 different ranking criteria and hypotheses. According to the consensus chronology, the pair of complementary GGC and GCC codons for the amino acids alanine and glycine appeared first. Other codons appeared as complementary pairs as well, which divided their respective amino acids into two alphabets, encoded by triplets with either central purines or central pyrimidines: G, D, S, E, N, R, K, Q, C, H, Y, and W (Glycine alphabet G) and A, V, P, S, L, T, I, F, and M (Alanine alphabet A). It is speculated that the earliest polypeptide chains were very short, presumably of uniform length, belonging to two alphabet types encoded in the two complementary strands of the earliest mRNA duplexes. After the fusion of the minigenes, a mosaic of the alphabets would form. Traces of the predicted mosaic structure have been, indeed, detected in the protein sequences of complete prokaryotic genomes in the form of weak oscillations with the period 12 residues in the form of alteration of two types of 6 residue long units. The next stage of protein evolution corresponded to the closure of the chains in the loops of the size 25–30 residues (Berezovsky et al., 2000). Autocorrelation analysis of proteins of 23 complete archaebacterial and eubacterial genomes revealed that the preferred distances between valine, alanine, glycine, leucine, and isoleucine along the sequences are in the same range of 25–30 residues, indicating that the loops are primarily closed by hydrophobic interactions between the ends of the loops. The loop closure stage is followed by the formation of typical folds of 100–200 amino acids, via end-to-end fusion of the genes encoding the loop-size chains. This size was apparently dictated by the optimal ring closure for DNA. In both cases the closure into the ring (loop) rendered evolutionarily advantageous stability to the respective structures. Further gene fusions lead to the formation of modern multidomain proteins. Recombinational gene splicing is likely to have appeared after the DNA circularization stage. Received: 21 December 2000 / Accepted: 28 February 2001     

Sequence structure of van der Waals locks in proteins

Recent works has suggested that proteins in early evolution have gone through a stage of closed loop elements with a typical contour size of 25-35 residues. These closed loops are still the elementary protein units to these days, and can be used to spell out protein sequence/structure relationship through a relatively small number of protein prototypes. In this study we aimed to identify the sequences that are used to lock the loop ends to one another, and to show how an extensive dictionary of such locking pairs can be created using positional correlation data from a large proteome database, and structural data from PDB databases. Such a dictionary can be used in reconstructing the evolutionary pathway the modern proteins have gone through, and in identifying closed loop elements in modern proteins with yet unknown 3D structure.     

Sequence Structure of van der Waals Locks in Proteins

Abstract

Recent works has suggested that proteins in early evolution have gone through a stage of closed loop elements with a typical contour size of 25–35 residues. These closed loops are still the elementary protein units to these days, and can be used to spell out protein sequence/structure relationship through a relatively small number of protein prototypes. In this study we aimed to identify the sequences that are used to lock the loop ends to one another, and to show how an extensive dictionary of such locking pairs can be created using positional correlation data from a large proteome database, and structural data from PDB databases. Such a dictionary can be used in reconstructing the evolutionary pathway the modern proteins have gone through, and in identifying closed loop elements in modern proteins with yet unknown 3D structure.     

Loop fold nature of globular proteins

Protein chains make numerous returns in globules, thus forming loops, closed by tight residue-to-residue contacts-closed loops. Previous statistical analysis of the sizes and locations of the closed loops in all major protein folds revealed that the loops have an almost standard contour length of 25-30 amino acid residues and follow one after another along the chain. In this work the closed loops of the major folds are presented in three dimensions. A special image filtering procedure is introduced that allows one to visualize the standard size closed loops for the first time. The loop positions along the sequences are verified by detection of loop-end clusters.     

Loop folds in proteins and evolutionary conservation of folding nuclei

We show that loops of close contacts involving hydrophobic residues are important in protein folding. Contrary to Berezovsky Berezovsky and Trifonov (J Biomol Struct Dyn 20, 5-6, 2002) the loops important in protein folding usually are much larger in size than 23-31 residues, being instead comparable to the size of the protein for single domain proteins. Additionally what is important are not single loop contacts, but a highly interconnected network of such loop contacts, which provides extra stability to a protein fold and which leads to their conservation in evolution.     

Protein folding: looping from hydrophobic nuclei.

Protein structure can be viewed as a compact linear array of nearly standard size closed loops of 25-30 amino acid residues (Berezovsky et al., FEBS Letters 2000; 466: 283-286) irrespective of details of secondary structure. The end-to-end contacts in the loops are likely to be hydrophobic, which is a testable hypothesis. This notion could be verified by direct comparison of the loop maps with Kyte and Doolittle hydropathicity plots. This analysis reveals that most of the ends of the loops are hydrophobic, indeed. The same conclusion is reached on the basis of positional autocorrelation analysis of protein sequences of 23 fully sequenced bacterial genomes. Hydrophobic residues valine, alanine, glycine, leucine, and isoleucine appear preferentially at the 25-30 residues distance one from another. These observations open a new perspective in the understanding of protein structure and folding: a consecutive looping of the polypeptide chain with the loops ending primarily at hydrophobic nuclei.     

Protein Modules Conserved Since LUCA

Universal scale of the sequence conservation has been recently introduced based on omnipresence of the protein sequence motifs across species. A large spectrum of short sequences, up to eight residues has been found to reside in all or almost all prokaryotic organisms. By this discovery a principally novel quantitative approach is introduced to the problem of reconstruction of the last universal common ancestor (LUCA). The most conserved elements (protein modules) with defined structures and sequences harboring the omnipresent motifs are outlined in this work, by combining the sequence and protein crystal structure data. The structurally conserved modules involve 25–30 amino acid residues and have appearance of closed loops, loop-n-lock structures. This confirms earlier conclusions on the loop-fold structure of globular proteins. Many of the topmost conserved modules represent the primary closed loop prototypes, that have been derived by whole genome sequence searches. The data presented, thus, make a basis for further developments toward the earliest stages of protein evolution. [Reviewing Editor: Dr. Martin Kreitman]     

Closed loops of TIM barrel protein fold

The closed loops within the proteins of the TIM-barrel fold family are analyzed and compared sequence- and structure-wise. The size distribution of the closed loops of the TIM-barrels confirms universal preference to the standard size of 25-30 residues. 3D structural RMSD comparisons of the closed loops and presentation of their sequences in binary form suggest that the TIM-barrel proteins are built from descendants of several types of basic closed loop prototypes. Comparison of these prototypes points to a likely common ancestor--the alpha helix containing closed loops of 28 amino acids. The presumed ancestor is characterized by specific binary consensus sequence.     

Protein structure and folding: a new start.

Standard building blocks of proteins--closed loops of 25-30 amino acid residues--have been recently discovered and further characterized by combined efforts of several laboratories. New challenging views on the protein structure, folding, and evolution are introduced by these studies. In particular, the role of van der Waals contacts in protein stability is better understood. They can be considered as locks closing the polypeptide chain returns and forming the loop-n-lock elements. The linearity of the arrangement of the standard loops in the proteins has important evolutionary implications. Selection pressure to maintain the loops of nearly standard size is reflected in the protein sequences as characteristic distance between hydrophobic residues, equal to the loop end-to-end distance. Further characterization of the loop-n-lock units reveals several sequence/structure prototypes, which suggests a new basis for protein classification. The following is a review of these studies.     

Closed Loops of TIM Barrel Protein Fold

Abstract

The closed loops within the proteins of the TIM-barrel fold family are analyzed and compared sequence- and structure-wise. The size distribution of the closed loops of the TIM-barrels confirms universal preference to the standard size of 25–30 residues. 3D structural RMSD comparisons of the closed loops and presentation of their sequences in binary form suggest that the TIM-barrel proteins are built from descendants of several types of basic closed loop prototypes. Comparison of these prototypes points to a likely common ancestor—the alpha helix containing closed loops of 28 amino acids. The presumed ancestor is characterized by specific binary consensus sequence.     

Loop Folds in Proteins and Evolutionary Conservation of Folding Nuclei

Abstract

We show that loops of close contacts involving hydrophobic residues are important in protein folding. Contrary to Berezovsky and Trifonov (J. Biomol. Struct. Dyn. 20, 5–6, 2002) the loops important in protein folding usually are much larger in size than 23–31 residues, being instead comparable to the size of the protein for single domain proteins. Additionally what is important are not single loop contacts, but a highly interconnected network of such loop contacts, which provides extra stability to a protein fold and which leads to their conservation in evolution.     

The chemistry of the reaction determines the invariant amino acids during the evolution and divergence of orotidine 5'-monophosphate decarboxylase

Orotidine 5'-phosphate (OMP) decarboxylase has the largest rate enhancement for any known enzyme. For an average protein of 270 amino acids from more than 80 species, only 8 amino acids are invariant, and 7 of these correspond to ligand-binding residues in the crystal structures of the enzyme from four species. It appears that the chemistry required for catalysis determines the invariant residues for this enzyme structure. A motif of three invariant amino acids at the catalytic site (DXKXXD) is also found in the enzyme hexulose-phosphate synthase. Although the core of OMP decarboxylase is conserved, it has undergone a variety of changes in subunit size or fusion to other protein domains, such as orotate phosphoribosyltransferase, during evolution in different kingdoms. The phylogeny of OMP decarboxylase shows a unique subgroup distinct from the three kingdoms of life. The enzyme subunit size almost doubles from Archaea (average mass of 24.5 kDa) to certain fungi (average mass of 41.7 kDa). These observed changes in subunit size are produced by insertions at 12 sites, largely in loops and on the exterior of the core protein. The consensus for all sequences has a minimal size of <20 kDa.     

Closed loops: persistence of the protein chain returns

It has recently been discovered that globular proteins are universally built from standard loop-n-lock units of about 30 amino acid residues. The hypothesis has been put forward on the loop stage in the protein evolution when the units were autonomous. Later they joined together making longer chains. One would expect that the early individual loop-n-lock elements might still be detected in modern protein sequences as remnants of the hypothetical 30-residue sequence prototypes. Among several strong sequence motifs, extracted from protein sequences of 23 complete bacterial proteomes, one 32-residue prototype was studied here in detail. Numerous sequence segments related to the prototype are identified in the crystal structures of proteins of a PDB_SELECT database. Analysis of the respective chain trajectories for the cases with different degrees of sequence conservation confirms that the majority of the segments correspond to the closed loops. In the evolutionary diversification of the prototypes the secondary structure yields first, while the sequence is still moderately conserved. The last feature to go is the chain return property. Apparently, the opening of the loops would severely destabilize the protein fold, which explains their conservation.     

The fusion kinetics of influenza hemagglutinin expressing cells to planar bilayer membranes is affected by HA density and host cell surface

Time-resolved admittance measurements were used to follow formation of individual fusion pores connecting influenza virus hemagglutinin (HA)- expressing cells to planar bilayer membranes. By measuring in-phase, out-of-phase, and dc components of currents, pore conductances were resolved with millisecond time resolution. Fusion pores developed in stages, from small pores flickering open and closed, to small successful pores that remained open until enlarging their lumens to sizes greater than those of viral nucleocapsids. The kinetics of fusion and the properties of fusion pores were studied as functions of density of the fusion protein HA. The consequences of treating cell surfaces with proteases that do not affect HA were also investigated. Fusion kinetics were described by waiting time distributions from triggering fusion, by lowering pH, to the moment of pore formation. The kinetics of pore formation became faster as the density of active HA was made greater or when cell surface proteins were extensively cleaved with proteases. In accord with this faster kinetics, the intervals between transient pore openings within the flickering stage were shorter for higher HA density and more extensive cell surface treatment. Whereas the kinetics of fusion depended on HA density, the lifetimes of open fusion pores were independent of HA density. However, the lifetimes of open pores were affected by the proteolytic treatment of the cells. Faster fusion kinetics correlated with shorter pore openings. We conclude that the density of fusion protein strongly affects the kinetics of fusion pore formation, but that once formed, pore evolution is not under control of fusion proteins but rather under the influence of mechanical forces, such as membrane bending and tension.     

Conformational properties of the iso-1-cytochrome C denatured state: dependence on guanidine hydrochloride concentration

Production of seven single surface histidine variants of yeast iso-1-cytochrome c allowed measurement of the apparent pK(a), pK(a)(obs), for histidine-heme loop formation for loops of nine to 83 amino acid residues under varying denaturing conditions (2 M to 6 M guanidine hydrochloride, gdnHCl). A linear correlation between pK(a)(obs) and the log of the loop size is expected for a random coil, pK(a)(obs) proportional to k log(n), where k is a scaling factor and n is the number of monomers in the loop. For small loops of nine, 16, and 22 monomers, no dependence of pK(a)(obs) on loop size was observed at any denaturant concentration indicating effects from chain stiffness. For larger loops of 37, 56, 72, and 83 monomers, the dependence of pK(a)(obs) on log(n) was linear and the slope of that dependence decreased with increasing concentration of denaturant. The scaling factor obtained at 5 M and 6 M gdnHCl for the larger loop sizes was approximately -2.0, close to the value of -2.2 expected for a random coil with excluded volume. However, scaling factors obtained under less harsh denaturing conditions (2 M to 4.5 M gdnHCl) deviated strongly from that expected for a random coil, being in the range -3 to -4. The gdnHCl dependence of pK(a)(obs) at each loop size was also evaluated to obtain denaturant m-values. Short loops where chain stiffness dominates had similar m-values of approximately 0.25 kcal/mol M. For larger loops m-values decrease with increasing loop size indicating that less hydrophobic area is sequestered when larger loops form. It is known that the earliest events in protein folding involve the formation of simple loops. The data from these studies provide direct insight into the relative probability with which loops of different sizes will form, as well as the factors which affect loop formation.     

Evolution of CuZn superoxide dismutase and the Greek key beta-barrel structural motif

Detailed analysis of the CuZn superoxide dismutase (SOD) structure provides new results concerning the significance and molecular basis for sequence conservation, intron-exon boundary locations, gene duplication, and Greek key beta-barrel evolution. Using 15 aligned sequences, including a new mouse sequence, specific roles have been assigned to all 23 invariant residues and additional residues exhibiting functional equivalence. Sequence invariance is dominated by 15 residues that form the active site stereochemistry, supporting a primary biological function of superoxide dismutation. The beta-strands have no sequence insertions and deletions, whereas insertions occur within the loops connecting the beta-strands and at both termini. Thus, the beta-barrel with only four invariant residues is apparently over-determined, but dependent on multiple cooperative side chain interactions. The regions encoded by exon I, a proposed nucleation site for protein folding, and exon III, the Zn loop involved in stability and catalysis, are the major structural subdomains not included in the internal twofold axis of symmetry passing near the catalytic Cu ion. This provides strong confirmatory evidence for gene evolution by duplication and fusion followed by the addition of these two exons. The proposed evolutionary pathway explains the structural versatility of the Greek key beta-barrel through functional specialization and subdomain insertions in new loop connections, and provides a rationale for the size of the present day enzyme.     

Protein sequences yield a proteomic code

Analysis of crystallized protein structures suggests that globular proteins are organized as consecutively connected units of 25-35 residues. These units are closed loops, that is returns of the polypeptide chain trajectory to a close contact with itself. This universal feature of apparently polymer-statistical nature is a basis for a principally novel view on the globular proteins as loop fold structures. The same unit size has been detected in protein sequences translated from complete prokaryotic genomes by positional autocorrelation analysis, which strongly indicates the evolutionary connection of the units. The units are further characterized by prototype sequences matching to their numerous derivatives in the translated genomes. The matches to five strongest prokaryotic prototypes and three prototypes of C. elegans are identified in the sequences of crystallized proteins, and their structures analyzed. Corresponding segments of the polypeptide chains in majority of cases form closed loops, though evolutionary fate of every prototype element is shown to be rather diverse. Then loop ends can be separated by a sequence-wise distant segments and stabilized by the spatial interactions in the context of the overall globular structure. The units belong to a presumably limited spectrum of the sequence prototypes, full repertoire of which would constitute a proteomic code.     

Role of interstrand loops in the formation of intramolecular cross-beta-sheets by homopolyamino acids

A matrix treatment of the formation of intramolecular anti-parallel β-sheets from a statistical coil has been extended to incorporate interstrand loops of arbitrary size. The behavior of the model is compared with a simpler version in which all pairs of contiguous strands were connected by β-bends. When large interstrand loops are allowed, there are many more types of sheets than is the case when all contiguous strands must be connected by tight or β-bends. For this reason, the larger interstrand loops make it easier to introduce the initial sheet into a statistical coil, and the sheet content is enhanced in the early stages of stages of sheet formation (i.e., at small values of the growth parameter t). As the transition continues (i.e., as t increases), a stage will be reached where occupancy of the statistical coil state is negligible because nearly all residues are in sheets or interstrand loops. Now, additional sheet formation can be accomplished only at the expense of residues in the interior of interstrand loops. For this reason, the larger interstrand loops make it more difficult to complete the final stages of sheet formation. These effects are especially dramatic in the formation of cross-β-sheets.