β-Strand-mediated interactions of protein domains

Protein domains exist by themselves or in combination with other domains to form complex multidomain proteins. Defining domain boundaries in proteins is essential for understanding their evolution and function but is not trivial. More specifically, partitioning domains that interact by forming a single β-sheet is known to be particularly troublesome for automatic structure-based domain decomposition pipelines. Here, we study edge-to-edge β-strand interactions between domains in a protein chain, to help define the boundaries for some more difficult cases where a single β-sheet spanning over two domains gives an appearance of one. We give a number of examples where β-strands belonging to a single β-sheet do not belong to a single domain and highlight the difficulties of automatic domain parsers on these examples. This work can be used as a baseline for defining domain boundaries in homologous proteins or proteins with similar domain interactions in the future.     

The surface of β-sheet proteins contains amphiphilic regions which may provide clues about protein folding

A major bottleneck in the field of biochemistry is our limited understanding of the processes by which a protein folds into its native conformation. Much of the work on this issue has focused on the conserved core of the folded protein. However, one might imagine that a ubiquitous motif for unaided folding or for the recognition of chaperones may involve regions on the surface of the native structure. We explore this possibility by an analysis of the spatial distribution of regions with amphiphilic α-helical potential on the surface of β-sheet proteins. All proteins, Including β-sheet proteins, contain regions with amphiphilic α-helical potential. That is, any α-helix formed by that region would be amphiphilic, having both hydrophobic and hydrophilic surfaces. In the three-dimensional structure of all β-sheet proteins analyzed, we have found a distinct pattern in the spatial distribution of sequences with amphiphilic α-helical potential. The amphiphilic regions occur in ring shaped clusters approximately 20 to 30 Å in diameter on the surface of the protein. In addition, these regions have a strong preference for positively charged amino acids and a lower preference for residues not favorable to α-helix formation. Although the purpose of these amphiphilic regions which are not associated with naturally occurring α-helix is unknown, they may play a critical role in highly conserved processes such as protein folding. © 1996 Wiley-Liss, Inc.     

Multiscale simulations of protein folding: application to formation of secondary structures

A multiscale simulation method of protein folding is proposed, using atomic representation of protein and solvent, combing genetic algorithms to determine the key protein structures from a global view, with molecular dynamic simulations to reveal the local folding pathways, thus providing an integrated landscape of protein folding. The method is found to be superior to previously investigated global search algorithms or dynamic simulations alone. For secondary structure formation of a selected peptide, RN24, the structures and dynamics produced by this method agree well with corresponding experimental results. Three most populated conformations are observed, including hairpin, β-sheet and α-helix. The energetic barriers separating these three structures are comparable to the kinetic energy of the atoms of the peptide, implying that the transition between these states can be easily triggered by kinetic perturbations, mainly through electrostatic interactions between charged atoms. Transitions between α-helix and β-sheet should jump over at least two energy barriers and may stay in the energetic trap of hairpin. It is proposed that the structure of proteins should be jointly governed by thermodynamic and dynamic factors; free energy is not the exclusive dominant for stability of proteins.     

An Overlapping Region between the Two Terminal Folding Units of the Outer Surface Protein A (OspA) Controls Its Folding Behavior

Although many naturally occurring proteins consist of multiple domains, most studies on protein folding to date deal with single-domain proteins or isolated domains of multi-domain proteins. Studies of multi-domain protein folding are required for further advancing our understanding of protein folding mechanisms. Borrelia outer surface protein A (OspA) is a β-rich two-domain protein, in which two globular domains are connected by a rigid and stable single-layer β-sheet. Thus, OspA is particularly suited as a model system for studying the interplays of domains in protein folding. Here, we studied the equilibria and kinetics of the urea-induced folding–unfolding reactions of OspA probed with tryptophan fluorescence and ultraviolet circular dichroism. Global analysis of the experimental data revealed compelling lines of evidence for accumulation of an on-pathway intermediate during kinetic refolding and for the identity between the kinetic intermediate and a previously described equilibrium unfolding intermediate. The results suggest that the intermediate has the fully native structure in the N-terminal domain and the single layer β-sheet, with the C-terminal domain still unfolded. The observation of the productive on-pathway folding intermediate clearly indicates substantial interactions between the two domains mediated by the single-layer β-sheet. We propose that a rigid and stable intervening region between two domains creates an overlap between two folding units and can energetically couple their folding reactions.     

Insect juvenile hormone binding protein shows ancestral fold present in human lipid-binding proteins

Low molecular weight juvenile hormone binding proteins (JHBPs) are specific carriers of juvenile hormone (JH) in the hemolymph of butterflies and moths. As hormonal signal transmitters, these proteins exert a profound effect on insect development. The crystal structure of JHBP from Galleria mellonella shows an unusual fold consisting of a long α-helix wrapped in a highly curved antiparallel β-sheet. JHBP structurally resembles the folding pattern found in tandem repeats in some mammalian lipid-binding proteins, with similar organization of one cavity and a disulfide bond between the long helix and the β-sheet. JHBP reveals, therefore, an archetypal fold used by nature for hydrophobic ligand binding. The JHBP molecule possesses two hydrophobic cavities. Several lines of experimental evidence conclusively indicate that JHBP binds JH in only one cavity, close to the N- and C-termini, and that this binding induces a structural change. The second cavity, located at the opposite end of the molecule, could bind another ligand.     

Secondary structure perturbations in salt-induced protein precipitates

The secondary structure implications of precipitation induced by a chaotropic salt, KSCN, and a structure stabilizing salt, Na₂SO₄, were studied for twelve different proteins. α-helix and β-sheet content of precipitate and native structures were estimated from the analysis of amide I band Raman spectra. A statistical analysis of the estimated perturbations in the secondary structure contents indicated that the most significant event is the formation of β-sheet structures with a concomitant loss of α-helix on precipitation with KSCN. The conformational changes for each protein were also analyzed with respect to elements of primary, secondary and tertiary structure existing in the native protein; primary structure was quantified by the fractions of hydrophobic and charged amino acids, secondary structure by x-ray estimates of α-helix and β-sheet contents of native proteins and tertiary structure by the dipole moment and solvent-accessible surface area. For the KSCN precipitates, factors affecting β-sheet content included the fraction of charged amino acids in the primary sequence and the surface area. Changes in α-helix content were influenced by the initial helical content and the dipole moment. The enhanced β-sheet contents of precipitates observed in this work parallel protein structural changes occurring in other aggregative phenomena.     

Structure determination of archaea-specific ribosomal protein L46a reveals a novel protein fold

Three archaea-specific ribosomal proteins recently identified show no sequence homology with other known proteins. Here we determined the structure of L46a, the most conserved one among the three proteins, from Sulfolobus solfataricus P2 using NMR spectroscopy. The structure presents a twisted β-sheet formed by the N-terminal part and two helices at the C-terminus. The L46a structure has a positively charged surface which is conserved in the L46a protein family and is the potential rRNA-binding site. Searching homologous structures in Protein Data Bank revealed that the structure of L46a represents a novel protein fold. The backbone dynamics identified by NMR relaxation experiments reveal significant flexibility at the rRNA binding surface. The potential position of L46a on the ribosome was proposed by fitting the structure into a previous electron microscopy map of the ribosomal 50S subunit, which indicated that L46a contacts to domain I of 23S rRNA near a multifunctional ribosomal protein L7ae.     

Understanding the folding and stability of a designed WW domain protein with replica exchange molecular dynamics simulations

WW domain proteins are usually regarded as simple models for understanding the folding mechanism of β-sheet. CC45 is an artificial protein that is capable of folding into the same structure as WW domain. In this article, the replica exchange molecular dynamics simulations are performed to investigate the folding mechanism of CC45. The analysis of thermal stability shows that β-hairpin 1 is more stable than β-hairpin 2 during the unfolding process. Free energy analysis shows that the unfolding of this protein substantially proceeds through solvating the smaller β-hairpin 2, followed by the unfolding of β-hairpin 1. We further propose the unfolding process of CC45 and the folding mechanism of two β-hairpins. These results are similar to the previous folding studies of formin binding protein 28 (FBP28). Compared with FBP28, it is found that CC45 has more aromatic residues in N-terminal loop, and these residues contact with C-terminal loop to form the outer hydrophobic core, which increases the stability of CC45. Knowledge about the stability and folding behaviour of CC45 may help in understanding the folding mechanisms of the β-sheet and in designing new WW domains.     

β-sheet topology prediction with high precision and recall for β and mixed α/β proteins

The prediction of the correct β-sheet topology for pure β and mixed α/β proteins is a critical intermediate step toward the three dimensional protein structure prediction. The predicted beta sheet topology provides distance constraints between sequentially separated residues, which reduces the three dimensional search space for a protein structure prediction algorithm. Here, we present a novel mixed integer linear optimization based framework for the prediction of β-sheet topology in β and mixed α/β proteins. The objective is to maximize the total strand-to-strand contact potential of the protein. A large number of physical constraints are applied to provide biologically meaningful topology results. The formulation permits the creation of a rank-ordered list of preferred β-sheet arrangements. Finally, the generated topologies are re-ranked using a fully atomistic approach involving torsion angle dynamics and clustering. For a large, non-redundant data set of 2102 β and mixed α/β proteins with at least 3 strands taken from the PDB, the proposed approach provides the top 5 solutions with average precision and recall greater than 78%. Consistent results are obtained in the β-sheet topology prediction for blind targets provided during the CASP8 and CASP9 experiments, as well as for actual and predicted secondary structures. The β-sheet topology prediction algorithm, BeST, is available to the scientific community at http://selene.princeton.edu/BeST/.     

Effect of monolayer lipid charges on the structure and orientation of protein VAMP1 at the air-water interface

SNARE proteins are implicated in membrane fusion during neurotransmission and peptide hormone secretion. Relatively little is known about the molecular interactions of their trans- and juxtamembrane domains with lipid membranes. Here, we report the structure and the assembling behavior of one of the SNARE proteins, VAMP1/synaptobrevin1 incorporated in a lipid monolayer at an air-water interface which mimics the membrane environment. Our results show that the protein is extremely sensitive to surface pressure as well as the lipid composition. Monolayers of proteins alone or in the presence of the neutral phospholipid DMPC underwent structural transition from α-helix to β-sheet upon surface compression. In contrast, the anionic phospholipid DMPG inhibited this transition in a concentration-dependent manner. Moreover, the orientation of the proteins was highly sensitive to the charge density of the lipid layers. Thus, the structure of VAMP1 is clearly controlled by protein-lipid interactions.     

Increased sequence hydrophobicity reduces conformational specificity: A mutational case study of the Arc repressor protein

The amino-acid sequences of soluble, globular proteins must have hydrophobic residues to form a stable core, but excess sequence hydrophobicity can lead to loss of native state conformational specificity and aggregation. Previous studies of polar-to-hydrophobic mutations in the β-sheet of the Arc repressor dimer showed that a single substitution at position 11 (N11L) leads to population of an alternate dimeric fold in which the β-sheet is replaced by helix. Two additional hydrophobic mutations at positions 9 and 13 (Q9V and R13V) lead to population of a differently folded octamer along with both dimeric folds. Here we conduct a comprehensive study of the sequence determinants of this progressive loss of fold specificity. We find that the alternate dimer-fold specifically results from the N11L substitution and is not promoted by other hydrophobic substitutions in the β-sheet. We also find that three highly hydrophobic substitutions at positions 9, 11, and 13 are necessary and sufficient for oligomer formation, but the oligomer size depends on the identity of the hydrophobic residue in question. The hydrophobic substitutions increase thermal stability, illustrating how increased hydrophobicity can increase folding stability even as it degrades conformational specificity. The oligomeric variants are predicted to be aggregation-prone but may be hindered from doing so by proline residues that flank the β-sheet region. Loss of conformational specificity due to increased hydrophobicity can manifest itself at any level of structure, depending upon the specific mutations and the context in which they occur.     

The group II chaperonin Mm-Cpn binds and refolds human γD crystallin

Chaperonins assist in the folding of nascent and misfolded proteins, though the mechanism of folding within the lumen of the chaperonin remains poorly understood. The archeal chaperonin from Methanococcus marapaludis, Mm-Cpn, shares the eightfold double barrel structure with other group II chaperonins, including the eukaryotic TRiC/CCT, required for actin and tubulin folding. However, Mm-Cpn is composed of a single species subunit, similar to group I chaperonin GroEL, rather than the eight subunit species needed for TRiC/CCT. Features of the β-sheet fold have been identified as sites of recognition by group II chaperonins. The crystallins, the major components of the vertebrate eye lens, are β-sheet proteins with two homologous Greek key domains. During refolding in vitro a partially folded intermediate is populated, and partitions between productive folding and off-pathway aggregation. We report here that in the presence of physiological concentrations of ATP, Mm-Cpn suppressed the aggregation of HγD-Crys by binding the partially folded intermediate. The complex was sufficiently stable to permit recovery by size exclusion chromatography. In the presence of ATP, Mm-Cpn promoted the refolding of the HγD-Crys intermediates to the native state. The ability of Mm-Cpn to bind and refold a human β-sheet protein suggests that Mm-Cpn may be useful as a simplified model for the substrate recognition mechanism of TRiC/CCT.     

Structural properties of an engineered outer membrane protein G mutant,OmpG-16SL,investigated with infrared spectroscopy

Abstract

The structural and functional differences between wild type (WT) outer membrane protein G and its two mutants are investigated with Fourier transform infrared spectroscopy. Both mutants have a long extension to the primary sequence to increase the number of β-strands from 14 (wild type) to 16 in an attempt to enlarge the pore diameter. The comparison among proteins is made in terms of pH-dependent conformational changes and thermal stability. Results show that all proteins respond to pH change but at different degrees. At acidic environment, all proteins contain the same number of residues participated in β-sheet structure. However, at neutral pH, the mutants have less ordered structure compared to WT porin. Thermal stability tests show that mutants differ significantly from WT porin at neutral pH. Although the transition temperature is directly proportional with the amount of β-sheet content, the changes in the pre-transition phase that pave the way to structural breakdown are shown to involve interactions among charged residues by two-dimensional correlation spectroscopy analysis. Results of the analysis show that side chain interactions play an active role under increasing temperature. Both mutants have more unordered secondary structure but they respond to pH change in tertiary structure level. Findings of this study provided deeper insight on the active players in structural stability of the WT porin.

Communicated by Ramaswamy H. Sarma

     

昆虫对低温的适应——抗冻蛋白研究进展

昆虫抗冻蛋白的研究主要在几种昆虫中展开,到目前为止已有二十多种昆虫抗冻蛋白被分离纯化。本文综述了关于昆虫抗冻蛋白的结构、组成、生物学活性及功能等方面的研究进展。昆虫抗冻蛋白的二级结构为β折叠和β转角,在其特殊的氨基酸序列结构中,半胱氨酸形成的二硫键对稳定其结构和活性起着很重要的作用。影响昆虫抗冻蛋白的因子,如活化蛋白及低分子量溶质的发现开辟了昆虫抗冻蛋白研究的新领域。     

Amyloid-like fibrils from a domain-swapping protein feature a parallel, in-register conformation without native-like interactions

The formation of amyloid-like fibrils is characteristic of various diseases, but the underlying mechanism and the factors that determine whether, when, and how proteins form amyloid, remain uncertain. Certain mechanisms have been proposed based on the three-dimensional or runaway domain swapping, inspired by the fact that some proteins show an apparent correlation between the ability to form domain-swapped dimers and a tendency to form fibrillar aggregates. Intramolecular β-sheet contacts present in the monomeric state could constitute intermolecular β-sheets in the dimeric and fibrillar states. One example is an amyloid-forming mutant of the immunoglobulin binding domain B1 of streptococcal protein G, which in its native conformation consists of a four-stranded β-sheet and one α-helix. Under native conditions this mutant adopts a domain-swapped dimer, and it also forms amyloid-like fibrils, seemingly in correlation to its domain-swapping ability. We employ magic angle spinning solid-state NMR and other methods to examine key structural features of these fibrils. Our results reveal a highly rigid fibril structure that lacks mobile domains and indicate a parallel in-register β-sheet structure and a general loss of native conformation within the mature fibrils. This observation contrasts with predictions that native structure, and in particular intermolecular β-strand interactions seen in the dimeric state, may be preserved in "domain-swapping" fibrils. We discuss these observations in light of recent work on related amyloid-forming proteins that have been argued to follow similar mechanisms and how this may have implications for the role of domain-swapping propensities for amyloid formation.     

Temporary secondary structures in tau,an intrinsically disordered protein

The tau protein belongs to the category of intrinsically disordered proteins, which in their native state do not have an average stable structure and fluctuate between many conformations. In its physiological state, tau helps nucleating and stabilising the microtubules in the axons of the neurons. On the other hand, the same tau is involved in the development of Alzheimer disease, when it aggregates in paired helical filaments forming fibrils, which form insoluble tangles. The beginning of the pathological aggregation of tau has been attributed to a local transition of protein portions from random coil to a β-sheet. These structures would very likely be transient; therefore, we performed a molecular dynamics simulation of tau to gather information on the existence of segments of tau endowed with a secondary structure. We combined the results of our simulation with small-angle X-ray scattering experimental data to extract from the dynamics a set of most probable conformations of tau. The analysis of these conformations highlights the presence of transient secondary structures such as turns, β-bridges, β-sheets and α-helices. It also shows that a large segment of the N-terminal region is found near the repeats domain in a globular-like shape.     

The Structure of Physarum polycephalum hemagglutinin I suggests a minimal carbohydrate recognition domain of legume lectin fold

Physarum polycephalum hemagglutinin I (HA1) is a 104-residue protein that is secreted to extracellular space. The crystal structure of HA1 has a β-sandwich fold found among lectin structures, such as legume lectins and galectins. Interestingly, the β-sandwich of HA1 lacks a jelly roll motif and is essentially composed of two simple up-and-down β-sheets. This up-and-down β-sheet motif is well conserved in other legume lectin-like proteins derived from animals, plants, bacteria, and viruses. It is more noteworthy that the up-and-down β-sheet motif includes many residues that make contact with the target carbohydrates. Our NMR data demonstrate that HA1 lacking a jelly roll motif also binds to its target glycopeptide. Taken together, these data show that the up-and-down β-sheet motif provides a fundamental scaffold for the binding of legume lectin-like proteins to the target carbohydrates, and the structure of HA1 suggests a minimal carbohydrate recognition domain.     

Unusually stable β-sheet formation in an ionic self-complementary oligopeptide

A 16-residue amphiphilic oligopeptide (EAK16) with every other residue alanine and also containing glutamic acid and lysine (Ac-NH-AEAEAKAKAEAEAKAK-CONH₂) is able to form an unusually stable β-sheet structure. The β-sheet structure is stable at very low concentrations in water and at high temperatures. Various pH changes at 1.5, 3, 7, and 11 had little effect on the stability of the β-sheet structure. The β-sheet structure was not altered significantly even in the presence of 0.1% SDS, 7 molar guanidine hydrochloride, or 8 molar urea. One of the structural characteristics of the EAK16 is its ionic self-complementarity in that ionic bonds and hydrogen bonds between Glu and Lys can form readily between two oligopeptide β-sheet structures. This structural feature is probably one of the factors that promotes its extreme stability. This is the first example of such an extended ionic self-complementarity in a protein structure. EAK16 and its related peptides may have applications as useful biomaterials. It also offers a good model for studying the mechanism of β-sheet formation. Because the oligopeptide can self-assemble to form a membranous structure, it may have relevance to origin of life research. © 1994 John Wiley & Sons, Inc.     

The signal distinguishing between targeting of outer membrane β-barrel protein to plastids and mitochondria in plants

The proteome of the outer membrane of mitochondria and chloroplasts consists of membrane proteins anchored by α-helical or β-sheet elements. While proteins with α-helical transmembrane domains are present in all cellular membranes, proteins with β-barrel structure are specific for these two membranes. The organellar β-barrel proteins are encoded in the nuclear genome and thus, have to be targeted to the outer organellar membrane where they are recognized by surface exposed translocation complexes. In the last years, the signals that ensure proper targeting of these proteins have been investigated as essential base for an understanding of the regulation of cellular protein distribution. However, the organellar β-barrel proteins are unique as most of them do not contain a typical targeting information in form of an N-terminal cleavable targeting signal. Recently, it was discovered that targeting and surface recognition of mitochondrial β-barrel proteins in yeast, humans and plants depends on the hydrophobicity of the last β-hairpin of the β-barrel. However, we demonstrate that the hydrophobicity is not sufficient for the discrimination of targeting to chloroplasts or mitochondria. By domain swapping between mitochondrial and chloroplast targeted β-barrel proteins atVDAC1 and psOEP24 we demonstrate that the presence of a hydrophilic amino acid at the C-terminus of the penultimate β-strand is required for mitochondrial targeting. A mutation of the chloroplast β-barrel protein psOEP24 which mimics such profile is efficiently targeted to mitochondria. Thus, we present the properties of the signal for mitochondrial targeting of β-barrel proteins in plants.     

Secondary structural changes in the intact and the disulfide Bridges cleaved β-lactoglobulin A and B in solutions of urea,guanidine hydrochloride,and sodium dodecyl sulfate

The relative proportions of α-helix, β-sheet, and unordered form in β-lactoglobulin A and B were examined in solutions of urea, guanidine, and sodium dodecyl sulfate (SDS). In the curve-fitting method of circular dichroism (CD) spectra, the reference spectra of the corresponding structures determined by Chen et al. (1974) were modified essentially according to the secondary structure of β-lactoglobulin B predicted by Creamer et al. (1983), i.e., that the protein has 17% α-helix and 41% β-sheet. The two variants showed no appreciable difference in structural changes. The reduction of disulfide bridges in the proteins increased β-sheet up to 48% but did not affect the α-helical proportion. The α-helical proportions of nonreduced β-lactoglobulin A and B were not affected below 2 M guanidine or below 3 M urea, but those of the reduced proteins began to decrease in much lower concentrations of these denaturants. By contrast, the α-helical proportions of the nonreduced and reduced proteins increased to 40–44% in SDS. The β-sheet proportions of both nonreduced and reduced proteins, which remained unaffected even in 6 M guanidine and 9 M urea, decreased to 24–25% in SDS.