Variation of folded polypeptide surface area with probe size

Three types of polypeptide surface area (contact, accessible, and molecular) have been studied as a function of the radius of a probe sphere used to map the surface. The surfaces are: (1) three alpha-helices, the H-helix of myoglobin, the E-helix of leghemoglobin, and an artificial polyalanine helix, each with 26 residues; (2) two globins, myoglobin and leghemoglobin, each with 153 residues; and (3) a two-center model system for which the three types of surface area have been calculated analytically. The two globin helices have almost identical surface areas as a function of probe size as do the two globins. The polyalanine helix surface area is smaller but similar in shape to the globin helix areas. All three helix contact areas tend to the same limit as the probe size increases, and the globin contact areas behave similarly. Fractal dimensions were calculated for the helix and globin contact and molecular surfaces. All fractal dimensions showed strong dependence on probe size. The contact fractal dimension peaks at larger values for both the helices and globins. Most residues do not make contact with large probes (15 A).     

Flexible protein sequence patterns. A sensitive method to detect weak structural similarities

The concept of a flexible protein sequence pattern is defined. In contrast to conventional pattern matching, template or sequence alignment methods, flexible patterns allow residue patterns typical of a complete protein fold to be developed in terms of residue positions (elements), separated by gaps of defined range. An efficient dynamic programming algorithm is presented to enable the best alignment(s) of a pattern with a sequence to be identified. The flexible pattern method is evaluated in detail by reference to the globin protein family, and by comparison to alignment techniques that exploit single sequence, multiple sequence and secondary structural information. A flexible pattern derived from seven globins aligned on structural criteria successfully discriminates all 345 globins from non-globins in the Protein Identification Resource database. Furthermore, a pattern that uses helical regions from just human alpha-haemoglobin identified 337 globins compared to 318 for the best non-pattern global alignment method. Patterns derived from successively fewer, yet more highly conserved positions in a structural alignment of seven globins show that as few as 38 residue positions (25 buried hydrophobic, 4 exposed and 9 others) may be used to uniquely identify the globin fold. The study suggests that flexible patterns gain discriminating power both by discarding regions known to vary within the protein family, and by defining gaps within specific ranges. Flexible patterns therefore provide a convenient and powerful bridge between regular expression pattern matching techniques and more conventional local and global sequence comparison algorithms.     

Thermodynamic and structural studies of cavity formation in proteins suggest that loss of packing interactions rather than the hydrophobic effect dominates the observed energetics

The hydrophobic effect is widely believed to be an important determinant of protein stability. However, it is difficult to obtain unambiguous experimental estimates of the contribution of the hydrophobic driving force to the overall free energy of folding. Thermodynamic and structural studies of large to small substitutions in proteins are the most direct method of measuring this contribution. We have substituted the buried residue Phe8 in RNase S with alanine, methionine, and norleucine. Binding thermodynamics and structures were characterized by titration calorimetry and crystallography, respectively. The crystal structures of the RNase S F8A, F8M, and F8Nle mutants indicate that the protein tolerates the changes without any main chain adjustments. The correlation of structural and thermodynamic parameters associated with large to small substitutions was analyzed for nine mutants of RNase S as well as 32 additional cavity-containing mutants of T4 lysozyme, human lysozyme, and barnase. Such substitutions were typically found to result in negligible changes in DeltaC(p)() and positive values of both DeltaDeltaH degrees and DeltaDeltaS of folding. Enthalpic effects were dominant, and the sign of DeltaDeltaS is the opposite of that expected from the hydrophobic effect. Values of DeltaDeltaG degrees and DeltaDeltaH degrees correlated better with changes in packing parameters such as residue depth or occluded surface than with the change in accessible surface area upon folding. These results suggest that the loss of packing interactions rather than the hydrophobic effect is a dominant contributor to the observed energetics for large to small substitutions. Hence, estimates of the magnitude of the hydrophobic driving force derived from earlier mutational studies are likely to be significantly in excess of the actual value.     

The folding of an enzyme. II. Substructure of barnase and the contribution of different interactions to protein stability.

Barnase is described anatomically in terms of its substructures and their mode of packing. The surface area of hydrophobic residues buried on formation and packing of the structural elements has been calculated. Changes in stability have been measured for 64 mutations, 41 constructed in this study, strategically located over the protein. The purpose is to provide: (1) information on the magnitudes of changes in stabilization energy for mutations of residues that are important in maintaining the structure; and (2) probes for the folding pathway to be used in subsequent studies. The majority of mutations delete functional moieties of side-chains or make isosteric changes. The energetics of the interactions are variable and context-dependent. The following general conclusions may be drawn, however, from this study about the classes of interactions that stabilize the protein. (1) Truncation of buried hydrophobic side-chains has, in general, the greatest effect on stability. For fully buried residues, this averages at 1.5 kcal mol-1 per methylene group with a standard deviation of +/- 0.6 kcal mol-1. Truncation of partly exposed leucine, isoleucine or valine residues that are in the range of 50 to 80 A2 of solvent-accessible area (30 to 50% of the total solvent-accessible area on a Gly-X-Gly tripeptide, i.e. those packed against the surface) has a smaller, but relatively constant effect on stability, at 0.81 kcal mol-1 per methylene group with a statistical standard deviation of +/- 0.18 kcal mol-1. (2) There is a very poor correlation between hydrophobic surface area buried and the free energy change for an extensive data set of hydrophobic mutants. The best correlation is found to be between the free energy change and the number of methylene groups within a 6 A radius of the hydrophobic groups deleted. (3) Burial of the hydroxyl group of threonine in a pocket that is intended for a gamma-methyl group of valine costs 2.5 kcal mol-1, in the range expected for the loss of two hydrogen bonds.(ABSTRACT TRUNCATED AT 400 WORDS)     

The polymeric hemoglobin molecule of Artemia. Interpretation of translated cDNA sequence of nine domains.

Translated cDNA for Artemia hemoglobin provided sequence data for almost nine domains, from the fourth residue of the A helix of one domain through 1405 residues to a stop codon after the ninth domain. The domain sequences were all different (homology between pairs 17-38%) but aligned well with each other and with conventional globins, satisfying the requirements for Phe at CD1, His at F8 and most other highly conserved features of globins including His at E7. Features found to be characteristic of Artemia globin and present in all nine domains were Phe at B10, Tyr at C4, Gly at F5, Phe at G5 and Gly at H22. Approximately 14 residues including a consensus -Val-Asp-Pro-Val-Thr-Gly-Leu- were available to form the linker between each pair of domains. The Artemia sequence data were compared with the crystal structures of Chironomus thummi thummi erythrocruorin III and sperm whale myoglobin in order to identify features of structural similarity and to examine the consequences of the differences. The Artemia sequences were compatible with the main helices and critical features of the globin fold. Possible modifications to the C helix, FG turn, and GH turn were studied in terms of molecular coordinates.     

Non-functional conserved residues in globins and their possible role as a folding nucleus.

Structure-based sequence alignment of 728 sequences of different globin subfamilies shows that in each subfamily there are two clusters of consensually conserved residues. The first is the well-known "functional" cluster which includes six heme-binding conserved residues (Phe CD1, His F8; aliphatic E11, FG5; hydrophobic F4, G5) and seven other conserved residues (Pro C2; aliphatic H19; hydrophobic B10, B13, B14, CD4, E4) that do not bind the heme but belong to its immediate neighborhood. The second cluster revealed here (aliphatic A8, G16, G12; aromatic A12; hydrophobic H8 and possibly H12) is distant from the heme. It is entirely non-polar and includes one turn (i, i+4 positions) from each of helices A, G, and H. It is known that A, G, and H helices formed at the earliest stage of apomyoglobin folding remain relatively stable in the equilibrium molten globule state, and are likely to be tightly packed with each other in this state. We have shown the existence of two similar conserved clusters in c -type cytochromes, heme-binding and distal from the heme. The second cluster in c -cytochromes includes one turn from each of the N and C-terminal alpha-helices. These N and C-terminal helices in cytochrome c are formed at the earliest stage of protein folding, remain relatively stable in the molten globule state, and are tightly packed with each other in this state, similar to the observed behavior of the globins. At least these two large protein families (c -type cytochromes and globins) have a close similarity in the existence and mutual positions of non-functional conserved residues. We assume that non-functional conserved residues are requisite for the fast and correct folding of both of these protein families into their stable 3D structures.     

Folding of Aplysia limacina apomyoglobin involves an intermediate in common with other evolutionarily distant globins

In the globin family, similarities in the folding mechanism have been found among different mammalian apomyoglobins (apoMb). The best-characterized intermediate of sperm whale apoMb, called I(AGH), is mainly stabilized by nativelike contacts among the A, G, and H helices involving a cluster of hydrophobic residues that includes two conserved tryptophans. To verify the hypothesis of a common intermediate in the folding of all members of the globin family, we have extensively studied a site-directed mutant of the myoglobin from Aplysia limacina, distantly related to the mammalian counterpart, in which one of the two tryptophans in the A-G-H cluster [i.e., Trp(H8)130] has been mutated to tyrosine. The results presented here show that this mutation destabilizes both the native state and the acid intermediate I(A) but exerts little or no effect on the thermally stable core of an intermediate species (called I(T)) peculiar to Aplysia apomyoglobin. Dynamic quenching of Trp emission by acrylamide provides information on the accessibility of the chromophores at the native and the intermediate states of wild-type and mutant Aplysia apomyoglobin, consistent with the thermodynamics. Our results agree well with those obtained for the corresponding topological position of apomyoglobin from sperm whale and clearly show that the H8 position is involved in the stabilization of the main intermediate in both apoproteins. This residue thus plays a role which is evolutionarily conserved in the globin family from invertebrates to mammals; our results support the contention that the A-G-H cluster is important in the folding pathway of different globins.     

Axolotl hemoglobin: cDNA-derived amino acid sequences of two alpha globins and a beta globin from an adult Ambystoma mexicanum

Erythrocytes of the adult axolotl, Ambystoma mexicanum, have multiple hemoglobins. We separated and purified two kinds of hemoglobin, termed major hemoglobin (Hb M) and minor hemoglobin (Hb m), from a five-year-old male by hydrophobic interaction column chromatography on Alkyl Superose. The hemoglobins have two distinct alpha type globin polypeptides (alphaM and alpham) and a common beta globin polypeptide, all of which were purified in FPLC on a reversed-phase column after S-pyridylethylation. The complete amino acid sequences of the three globin chains were determined separately using nucleotide sequencing with the assistance of protein sequencing. The mature globin molecules were composed of 141 amino acid residues for alphaM globin, 143 for alpham globin and 146 for beta globin. Comparing primary structures of the five kinds of axolotl globins, including two previously established alpha type globins from the same species, with other known globins of amphibians and representatives of other vertebrates, we constructed phylogenetic trees for amphibian hemoglobins and tetrapod hemoglobins. The molecular trees indicated that alphaM, alpham, beta and the previously known alpha major globin were adult types of globins and the other known alpha globin was a larval type. The existence of two to four more globins in the axolotl erythrocyte is predicted.     

Sites of modification of hemospan, a poly(ethylene glycol)-modified human hemoglobin for use as an oxygen therapeutic

Hemospan is an acellular hemoglobin-based oxygen therapeutic in clinical trials in Europe and the United States. The product is prepared by site-specific conjugation of maleimide-activated poly(ethylene) glycol (PEG, MW approximately 5500) to human oxyhemoglobin through maleimidation reactions either (1) directly to reactive Cys thiols or (2) at surface Lys groups following thiolation using 2-iminothiolane. The thiolation/maleimidation reactions lead to the addition of approximately 8 PEGs per hemoglobin tetramer. Identification of PEG modified globins by SDS-PAGE and MALDI-TOF reveals a small percentage of protein migrating at the position for unmodified globin chains and the remaining as separate bands representing globin chains conjugated with 1 to 4 PEGs per chain. Identification of PEG modification sites on individual alpha and beta globins was made using reverse-phase HPLC, showing a series of alpha globins conjugated with 0 to 3 PEGs and a series of beta globins conjugated with 0 to 4 PEGs per globin. Mass analysis of tryptic peptides from hemoglobin thiolated and maleimidated with N-ethyl maleimide showed the same potential sites of modification regardless of thiolation reaction ratio, with seven sites identified on beta globins at beta8, beta17, beta59, beta66, beta93, beta95, and beta132 and three sites identified on alpha globins at alpha7, alpha16, and alpha40.     

Redesign of artificial globins: effects of residue replacements at hydrophobic sites on the structural properties

Artificial sequences of the 153 amino acids have been designed to fit the main-chain framework of the sperm whale myoglobin (Mb) structure based on a knowledge-based 3D-1D compatibility method. The previously designed artificial globin (DG1) folded into a monomeric, compact, highly helical and globular form with overall dimensions similar to those of the target structure, but it lacked structural uniqueness at the side-chain level [Isogai, Y., Ota, M., Fujisawa, T. , Izuno, H., Mukai, M., Nakamura, H., Iizuka, T., and Nishikawa, K. (1999) Biochemistry 38, 7431-7443]. In this study, we redesigned hydrophobic sites of DG1 to improve the structural specificity. Several Leu and Met residues in DG1 were replaced with beta-branched amino acids, Ile and Val, referring to the 3D profile of DG1 to produce three redesigned globins, DG2-4. These residue replacements resulted in no significant changes of their compactness and alpha-helical contents in the absence of denaturant, whereas they significantly affected the dependence of the secondary structure on the concentration of guanidine hydrochloride. The analyses of the denaturation curves revealed higher global stabilities of the designed globins than that of natural apoMb. Among DG1-4, DG3, in which 11 Leu residues of DG1 are replaced with seven Ile and four Val residues, and one Met residue is replaced with Val, displayed the lowest stability but the most cooperative folding-unfolding transition and the most dispersed NMR spectrum with the smallest line width. The present results indicate that the replacements of Leu (Met) with the beta-branched amino acids at appropriate sites reduce the freedom of side-chain conformation and improve the structural specificity at the expense of stability.     

A phylogenomic profile of globins

Background

Globins occur in all three kingdoms of life: they can be classified into single-domain globins and chimeric globins. The latter comprise the flavohemoglobins with a C-terminal FAD-binding domain and the gene-regulating globin coupled sensors, with variable C-terminal domains. The single-domain globins encompass sequences related to chimeric globins and «truncated» hemoglobins with a 2-over-2 instead of the canonical 3-over-3 α-helical fold.

Results

A census of globins in 26 archaeal, 245 bacterial and 49 eukaryote genomes was carried out. Only ~25% of archaea have globins, including globin coupled sensors, related single domain globins and 2-over-2 globins. From one to seven globins per genome were found in ~65% of the bacterial genomes: the presence and number of globins are positively correlated with genome size. Globins appear to be mostly absent in Bacteroidetes/Chlorobi, Chlamydia, Lactobacillales, Mollicutes, Rickettsiales, Pastorellales and Spirochaetes. Single domain globins occur in metazoans and flavohemoglobins are found in fungi, diplomonads and mycetozoans. Although red algae have single domain globins, including 2-over-2 globins, the green algae and ciliates have only 2-over-2 globins. Plants have symbiotic and nonsymbiotic single domain hemoglobins and 2-over-2 hemoglobins. Over 90% of eukaryotes have globins: the nematode Caenorhabditis has the most putative globins, ~33. No globins occur in the parasitic, unicellular eukaryotes such as Encephalitozoon, Entamoeba, Plasmodium and Trypanosoma.

Conclusion

Although Bacteria have all three types of globins, Archaeado not have flavohemoglobins and Eukaryotes lack globin coupled sensors. Since the hemoglobins in organisms other than animals are enzymes or sensors, it is likely that the evolution of an oxygen transport function accompanied the emergence of multicellular animals.     

Evolution of the sulfide-binding function within the globin multigenic family of the deep-sea hydrothermal vent tubeworm Riftia pachyptila

The giant extracellular hexagonal bilayer hemoglobin (HBL-Hb) of the deep-sea hydrothermal vent tube worm Riftia pachyptila is able to transport simultaneously O(2) and H(2)S in the blood from the gills to a specific organ: the trophosome that harbors sulfide-oxidizing endosymbionts. This vascular HBL-Hb is made of 144 globins from which four globin types (A1, A2, B1, and B2) coevolve. The H(2)S is bound at a specific location (not on the heme site) onto two of these globin types. In order to understand how such a function emerged and evolved in vestimentiferans and other related annelids, six partial cDNAs corresponding to the six globins known to compose the multigenic family of R. pachyptila have been identified and sequenced. These partial sequences (ca. 120 amino acids, i.e., 80% of the entire protein) were used to reconstruct molecular phylogenies in order to trace duplication events that have led to the family organization of these globins and to locate the position of the free cysteine residues known to bind H(2)S. From these sequences, only two free cysteine residues have been found to occur, at positions Cys + 1 (i.e., 1 a.a. from the well-conserved distal histidine) and Cys + 11 (i.e., 11 a.a. from the same histidine) in globins B2 and A2, respectively. These two positions are well conserved in annelids, vestimentiferans, and pogonophorans, which live in sulfidic environments. The structural comparison of the hydrophobic environment that surrounds these cysteine residues (the sulfide-binding domain) using hydrophobic cluster analysis plots, together with the cysteine positions in paralogous strains, suggests that the sulfide-binding function might have emerged before the annelid radiation in order to detoxify this toxic compound. Moreover, globin evolutionary rates are highly different between paralogous strains. This suggests that either the two globin subfamilies involved in the sulfide-binding function (A2 and B2) have evolved under strong directional selective constraints (negative selection) and that the two other globins (A1 and B1) have accumulated more substitutions through positive selection or have evolved neutrally after a relaxation of selection pressures. A likely scenario on the evolution of this multigenic family is proposed and discussed from this data set.     

Thermodynamic and kinetic characterization of apoHmpH, a fast-folding bacterial globin

Despite the widespread presence of the globin fold in most living organisms, only eukaryotic globins have been employed as model proteins in folding/stability studies so far. This work introduces the first thermodynamic and kinetic characterization of a prokaryotic globin, that is, the apo form of the heme-binding domain of flavohemoglobin (apoHmpH) from Escherichia coli. This bacterial globin has a widely different sequence but nearly identical structure to its eukaryotic analogues. We show that apoHmpH is a well-folded monomeric protein with moderate stability at room temperature [apparent ΔG°_UN(w) = − 3.1 ± 0.3 kcal mol^− 1; m_UN = − 1.7 kcal mol^− 1 M^− 1] and predominant α-helical structure. Remarkably, apoHmpH is the fastest-folding globin known to date, as it refolds about 4- to 16-fold more rapidly than its eukaryotic analogues (e.g., sperm whale apomyoglobin and soybean apoleghemoglobin), populating a compact kinetic intermediate (β_I = 0.9 ± 0.2) with significant helical content. Additionally, the single Trp120 (located in the native H helix) becomes locked into a fully native-like environment within 6 ms, suggesting that this residue and its closest spatial neighbors complete their folding at ultrafast (submillisecond) speed. In summary, apoHmpH is a bacterial globin that shares the general folding scheme (i.e., a rapid burst phase followed by slower rate-determining phases) of its eukaryotic analogues but displays an overall faster folding and a kinetic intermediate with some fully native-like traits. This study supports the view that the general folding features of bacterial and eukaryotic globins are preserved through evolution while kinetic details differ.     

A conserved interdomain interaction is a determinant of folding cooperativity in the GST fold

The canonical glutathione transferase (GST) fold found in many monomeric and dimeric proteins consists of two domains that differ in structure and conformational dynamics. However, no evidence exists that the two domains unfold/fold independently at equilibrium, indicating the significance of interdomain interactions in governing cooperativity between domains. Bioinformatics analyses indicate the interdomain interface of the GST fold is large, predominantly hydrophobic with a high packing density explaining cooperative interdomain behavior. Structural alignments reveal a topologically conserved lock-and-key interaction across the domain interface in which a bulky hydrophobic residue ("key") protrudes from the surface of the N-domain and inserts into a pocket ("lock") in the C-domain. To better understand the molecular basis for the contribution of interdomain interactions toward cooperativity within the GST fold in the absence of any influence from quaternary interactions, studies were done with two monomeric GST proteins: Escherichia coli Grx2 (EcGrx2) and human CLIC1 (hCLIC1). Replacing the methionine "key" residue with alanine is structurally nondisruptive, whereas it significantly diminishes the folding cooperativity of both proteins. The loss in cooperativity between domains in the mutants is reflected by a change in the equilibrium folding mechanism from a wild-type two-state process to a three-state process, populating a stable folding intermediate.     

Solution 1H NMR study of the heme cavity and folding topology of the abbreviated chain 118-residue globin from the cyanobacterium Nostoc commune

The globin from the cyanobacterium Nostoc commune, abbreviated GlbN, which appears to serve as a part of a terminal oxidase rather than as a respiratory pigment, displays relatively normal O2 binding properties, despite the highly abbreviated polypeptide chain, (118 residues) relative to more conventional globins [Thorsteinsson, M. V. , Bevan, D. R., Potts, M., Dou, Y., Eich, R. F., Hargrove, M. S., Gibson, Q. H., and Olson, J. S. (1999) Biochemistry 38, 2117-2126]. The nature of the heme cavity and the general folding topology of this cyanoglobin were investigated by solution 1H NMR to establish the extent to which, and the manner in which, this compact globin adheres to the standard globin fold. This represents by far the smallest globin subjected to structural analysis. The paramagnetic cyanomet derivative was selected because its characteristically large magnetic anisotropy imparts significant dipolar shifts which both improve resolution to greatly facilitate assignments and serve as indicators of the folding topology of the globin. Identification of the axial His 70 and highly conserved Phe 35 (CD1) determined the absolute orientation of the heme and proximal His. Sequential assignments of four helical and one loop segments, which exhibit dipolar contacts to the heme and among each other, confirm the presence of well-conserved F, G, and H helices and the FG corner. The majority of the abbreviation of the chain relative to the more conventional length globins is accommodated in the A-D helices, of which the last is completely missing. The distal residue which provides a H-bond to bound ligand is identified as Gln 43, but the expected helical position E7 could not be confirmed. His 46, placed at position E10, is found to adopt alternate orientations into, and out of, the heme cavity depending on protonation state, suggesting the presence of a Bohr effect at low pH. It is shown that the dipolar shifts exhibited by backbone protons for the assigned residues conform well to those observed for other cyanomet globins and further support a conserved Mb fold. Perturbed medium-range dipolar contacts and the pH-independent backbone proton lability of the F helix are interpreted in terms of a holoprotein which is less stable than a conventional length globin.     

The complete amino acid sequences of four globins from the land leech Haemadipsa zeylanica var. japonica

The amino acid sequences of four globins from the land leech, Haemadipsa zeylanica var. japonica, were determined using nucleotide sequencing and protein sequencing. The mature globin-molecules were composed of 146 amino acid residues for M-1 globin, 156 for M-2 globin, 143 for D-1 globin, and 149 for D-2 globin. Alignment of the four kinds of globins by Clustal X revealed 22 invariant amino acids. The four globins were 26–33% identical. A striking feature of amino acid alteration was: the replacement of the E7 distal-His of D-1 globin by phenylalanine because histidine is conserved among the rest of the globins of H. zeylanica, those of other representative species (Lumbricus and Tylorrhynchus) of Annelida and most other hemoglobins. A phylogenetic tree constructed of 18 globin structures including two species of leeches, H. zeylanica (a land leech) and Macrobdella decora (a freshwater leech), T. heterochaetus (a representative species of polychaetes), L. terrestris (a representative species of oligochaetes), and human α and β globins strongly indicated that the leech globins first separated from globin lineage of annelids.     

Genome-wide analysis of the globin gene family of C. elegans

The aim of our study was to annotate sequences for 35 putative globins from the nematode Caenorhabditis elegans. All these proteins are expressed, but seven of these differ from the gene predictions in Wormbase. The entire polypeptide sequences for 31 genes and the core globin domain of four proteins were confirmed or corrected. All core globin domains were aligned manually following a procedure that was designed to fit the putative sequences to the crystal structure based alignment of 56 known globin crystal structures. Neighbor-joining analysis of the resulting alignment showed that the majority of these globins are very divergent from each other, possibly suggesting a long evolutionary divergence. The surprisingly high number and low sequence conservation of putative globins in this small organism urges a detailed functional analysis.     

The role of atomic level steric effects and attractive forces in protein folding

Protein folding into tertiary structures is controlled by an interplay of attractive contact interactions and steric effects. We investigate the balance between these contributions using structure‐based models using an all‐atom representation of the structure combined with a coarse‐grained contact potential. Tertiary contact interactions between atoms are collected into a single broad attractive well between the C^β atoms between each residue pair in a native contact. Through the width of these contact potentials we control their tolerance for deviations from the ideal structure and the spatial range of attractive interactions. In the compact native state dominant packing constraints limit the effects of a coarse‐grained contact potential. During folding, however, the broad attractive potentials allow an early collapse that starts before the native local structure is completely adopted. As a consequence the folding transition is broadened and the free energy barrier is decreased. Eventually two‐state folding behavior is lost completely for systems with very broad attractive potentials. The stabilization of native‐like residue interactions in non‐perfect geometries early in the folding process frequently leads to structural traps. Global mirror images are a notable example. These traps are penalized by the details of the repulsive interactions only after further collapse. Successful folding to the native state requires simultaneous guidance from both attractive and repulsive interactions. Proteins 2012. © 2011 Wiley Periodicals, Inc.     

A model of globin evolution

Putative globins have been identified in 426 bacterial, 32 Archaeal and 67 eukaryote genomes. Among these sequences are the hitherto unsuspected presence of single domain sensor globins within Bacteria, Fungi, and a Euryarchaeote. Bayesian phylogenetic trees suggest that their occurrence in the latter two groups could be the result of lateral gene transfer from Bacteria. Iterated psiblast searches based on groups of globin sequences indicate that bacterial flavohemoglobins are closer to metazoan globins than to the other two lineages, the 2-over-2 globins and the globin-coupled sensors. Since Bacteria is the only kingdom to have all the subgroups of the three globin lineages, we propose a working model of globin evolution based on the assumption that all three lineages originated and evolved only in Bacteria. Although the 2-over-2 globins and the globin-coupled sensors recognize flavohemoglobins, there is little recognition between them. Thus, in the first stage of globin evolution, we favor a flavohemoglobin-like single domain protein as the ancestral globin. The next stage comprised the splitting off to single domain 2-over-2 and sensor-like globins, followed by the covalent addition of C-terminal domains resulting in the chimeric flavohemoglobins and globin-coupled sensors. The last stage encompassed the lateral gene transfers of some members of the three globin lineages to specific groups of Archaea and Eukaryotes.