Polypeptide Conformation Research Articles

Extraction of native collagen from limed bovine split wastes through improved pretreatment methods

AbstractBACKGROUND: The large amount of limed bovine split wastes discharged by the leather industry has raised concerns regarding their environmental effect. The objective of this work was to perform pilot plant trials to extract high‐value native collagen from these wastes through improved pretreatment methods.RESULTS: EDTA‐ and HCl‐pretreatment gave similar removal percentages of inorganic substances. Owing to the open structure of fibers, the collagen yield of HCl‐pretreated splits (HPS) (41.31%) was higher than that of EDTA‐pretreated splits (EPS) (10.42%). Furthermore, HCl‐pretreated split collagen (HPC) had a more acidic isoelectric point, lower content of primary amino groups, larger Z‐average particle size and higher relative viscosity than EDTA‐pretreated split collagen (EPC). Electrophoretic analysis and circular dichroism spectra revealed the maintenance of polypeptide and triple helix conformation, respectively. In addition, the transition temperatures of EPC (34.7 °C) and HPC (34.6 °C) detected by differential scanning calorimetry (DSC) were close to that of commercial collagen from calfskin (CCC) (35.7 °C).CONCLUSION: A process of native collagen extraction from limed bovine split wastes was proposed. While both EPC and HPC represented similar physicochemical properties to those of CCC, the collagen yield of HPS was much higher than that of EPS. Copyright © 2008 Society of Chemical Industry

Conformational distributions of unfolded polypeptides from novel NMR techniques

How the information content of an unfolded polypeptide sequence directs a protein towards a well-formed three-dimensional structure during protein folding remains one of the fundamental questions in structural biology. Unfolded proteins have recently attracted further interest due to their surprising prevalence in the cellular milieu, where they fulfill not only central regulatory functions, but also are implicated in diseases involving protein aggregation. The understanding of both the protein folding transition and these often natively unfolded proteins hinges on a more detailed experimental characterization of the conformations and conformational transitions in the unfolded state. This description is intrinsically very difficult due to the very large size of the conformational space. In principle, solution NMR can monitor unfolded polypeptide conformations and their transitions at atomic resolution. However, traditional NMR parameters such as chemical shifts, J couplings, and nuclear Overhauser enhancements yield only rather limited and often qualitative descriptions. This situation has changed in recent years by the introduction of residual dipolar couplings and paramagnetic relaxation enhancements, which yield a high number of well-defined, quantitative parameters reporting on the averages of local conformations and long-range interactions even under strongly denaturing conditions. This information has been used to obtain plausible all-atom models of the unfolded state at increasing accuracy. Currently, the best working model is the coil model, which derives amino acid specific local conformations from the distribution of amino acid torsion angles in the nonsecondary structure conformations of the protein data bank. Deviations from the predictions of such models can often be interpreted as increased order resulting from long-range contacts within the unfolded ensemble.

The Blout Laboratory at Harvard Medical School from 1957 to 1972

Elkan R. Blout's laboratory at the Children's Cancer Research Foundation and Harvard Medical School pioneered many approaches to the synthesis, conformation and structural studies of polypeptides, biopolymers and selected proteins. Here the early days (1957-1972) of his research group are remembered.

N-Linked Glycosylation Does Not Impair Proteasomal Degradation but Affects Class I Major Histocompatibility Complex Presentation

The addition of N-linked glycans to nascent polypeptides occurs cotranslationally in the endoplasmic reticulum (ER). For many proteins the state of the glycans serves as an indicator, which allows the ER quality control system to monitor the conformation of polypeptides upon folding. Proteins that fail to fold in the ER are often dislocated to the cytoplasm, where they are subjected to proteasomal degradation. Although the addition of N-linked glycans occurs within the ER, non-lysosomal removal of the glycans occurs in the cytosol by the action of peptide N-glycanase (PNGase). In this study, we investigated the interplay between PNGase action and proteasomal degradation of ER misfolded proteins (i.e. whether PNGase acts prior to or following proteasomal degradation). Interestingly, we found that glycan removal from N-terminally extended peptides modulates the presentation of class I major histocompatibility complex-restricted epitopes. Our findings provide direct evidence that the proteasome is capable of degrading glycoproteins without prior removal of their glycans. This degradation is independent of either the identity of the glycosylated protein or the type and number of N-linked glycans it harbors. We also captured and characterized glycopeptides generated following proteasomal degradation of RNaseB. Although the carbohydrate moiety reduced the variability of the degradation products that include the glycosylated residue (local effect), the overall global digestion pattern of RNaseB was unaffected. Together with earlier findings by others, our data support a model in which PNGase may act both upstream and downstream to proteasomal degradation and demonstrates its important role in class I major histocompatibility complex antigen presentation.

Surveying polypeptide and protein domain conformation and association with FlAsH and ReAsH

Recombinant polypeptides and protein domains containing two cysteine pairs located distal in primary sequence but proximal in the native folded or assembled state are labeled selectively in vitro and in mammalian cells using the profluorescent biarsenical reagents FlAsH-EDT2 and ReAsH-EDT2. This strategy, termed bipartite tetracysteine display, enables the detection of protein-protein interactions and alternative protein conformations in live cells. As proof of principle, we show that the equilibrium stability and fluorescence intensity of polypeptide-biarsenical complexes correlates with the thermodynamic stability of the protein fold or assembly. Destabilized protein variants form less stable and less bright biarsenical complexes, which allows discrimination of live cells expressing folded polypeptide and protein domains from those containing disruptive point mutations. Bipartite tetracysteine display may provide a means to detect early protein misfolding events associated with Alzheimer's disease, Parkinson's disease and cystic fibrosis; it may also enable high-throughput screening of compounds that stabilize discrete protein folds.

Expected and Unexpected Effects of Amino Acid Substitutions on Polypeptide-Directed Crystal Growth

Nature's use of biomineralization polypeptides to control and modulate the growth of biogenic minerals is an important process that, if properly understood, could have significant implications for designing and creating new inorganic-based materials. Although the sequences for a number of biomineralization proteins exist, very little is known about the participation of specific amino acids in the mineral modulation process. In this letter, we investigate the impact of global Asp --> Asn and Glu --> Gln substitutions on two mollusk shell nacre polypeptides, AP7N and n16N. We find that these global substitutions, which remove all anionic Ca(II) binding sites, abolish the expected in vitro mineralization activities associated with each native polypeptide. In addition, the ability of substituted peptides to form complexes with both Ca(II) and Ca(II) metal ion analogs is also abolished. However, some unexpected effects were noted. First, the Asp --> Asn, Glu --> Gln substituted n16N polypeptide is observed to self-assemble and form biofilms or coatings that appear to mineralize in vitro. Second, both polypeptides are structurally affected by these substitutions, with Asp --> Asn substituted AP7N transforming to an alpha helix and Asp --> Asn, Glu --> Gln substituted n16N transforming to a more unfolded random-coil-like structure. We find that the participation of Asp and Glu residues is crucial to the inherent mineralization activities and conformations of AP7N and n16N polypeptides. Surprisingly, we find that the replacement of anionic residues within biomineralization polypeptides such as n16N still permits mineral modulation, but in a different form that now involves peptide self-association and biofilm formation.

Dendrimer Effects on Peptide and Protein Fibrillation

Dendrimers are synthetic, symmetrically branched polymers that can be manufactured to a high degree of definition and therefore present themselves as monodisperse entities. Flexible and globular in shape and compartementalized into a partly inaccessible interior and a highly exposed surface, they offer numerous possibilities for interactions with and responses to biological macromolecules and biostructures including cell membranes and proteins. By way of their multiple functional surface groups, they allow the design of surfaces carrying a multitude of biological motifs and/or charges giving rise to quite significant biological and physico-chemical effects. Here we describe the surprising ability of dendrimers to interact with and perturb polypeptide conformations, particularly efficiently towards amyloid structures; that is, the structures of highly insoluble polypeptide aggregates involved in a range of serious and irreversibly progressive pathological conditions (protein-misfolding diseases). Interesting as this may be, the interaction of dendrimers with such generic peptidic aggregates also offers a new perspective on the molecular mechanisms governing assembly and disassembly of amyloid structures and thereby on determinants of protein and peptide folding. Despite the potent disaggregative nature of various dendrimers, they have variable effects on the stability of different proteins, suggesting that they do not act as generic denaturants, but rather exert their effects via specific interactions with individual parts of each protein.

Evaluation of conformation and association behavior of multivalent alanine-rich polypeptides.

Helical alanine-rich polypeptides with functional groups displayed along the backbone can display desired molecules such as saccharides or therapeutic molecules at a prescribed spacing. Because these polypeptides have promise for application as biomaterials, the conformation and association of these molecules have been investigated under biologically relevant conditions. Three polypeptide sequences, 17-H-3, 17-H-6, and 35-H-6, have been produced through recombinant techniques. Circular dichroic (CD) spectroscopy was used to monitor the secondary structure of the polypeptides in PBS (phosphate buffered saline, pH 7.4). The aggregation behavior in PBS was monitored via analytical ultracentrifugation and non-denaturing polyacrylamide gel electrophoresis. The three polypeptides adopt a highly helical structure at low and ambient temperatures, and when heated, undergo a helix-to-coil transition, typical of other alanine-rich peptide sequences. The melting temperatures and van't Hoff enthalpies, extracted from the CD data, suggest similar stability of the sequences. Although alanine-rich sequences can be prone to aggregation, there is no indication of aggregation for the three polypeptides at a range of concentrations relevant for possible biological applications. The helical polypeptides are monomeric under biologically relevant conditions enabling application of these polypeptides as useful scaffolds for ligand or drug display.

Folding of the 25 Residue Aβ(12−36) Peptide in TFE/Water: Temperature-Dependent Transition from a Funneled Free-Energy Landscape to a Rugged One

The free-energy landscape of the Alzheimer beta-amyloid peptide Abeta(12-36) in a 40% (v/v) 2,2,2-trifluoroethanol (TFE)/water solution was determined by using multicanonical molecular dynamics simulations. Simulations using this enhanced conformational sampling technique were initiated from a random unfolded polypeptide conformation. Our simulations reliably folded the peptide to the experimental NMR structure, which consists of two linked helices. The shape of the free energy landscape for folding was found to be strongly dependent on temperature: Above 325 K, the overall shape was funnel-like, with the bottom of the funnel coinciding exactly with the NMR structure. Below 325 K, on the other hand, the landscape became increasingly rugged, with the emergence of new conformational clusters connected by low free-energy pathways. Finally, our simulations reveal that water and TFE solvate the polypeptide in different ways: The hydrogen bond formation between TFE and Abeta was enhanced with decreasing temperature, while that between water and Abeta was depressed.

Underlying source of the relation between polypeptide conformation and strength of NH···O hydrogen bonds

Previous calculations have revealed that the NH···O bond between a dipeptide NH and a proton acceptor is considerably weaker when the dipeptide is in its extended C5 conformation than in the C7 structure. The present work uses quantum chemical methods to determine the underlying reason for this different behavior. With regard to the pertinent NH group, the two dipeptide conformers show very little difference in total electron density or in the character of the low-lying vacant molecular orbitals which might act as sinks for electron density from the proton acceptor. There is also little evidence that a weak intramolecular H-bond within the C5 structure is able to influence the ability of the NH group to participate in an intermolecular bond. Energy decomposition analysis points toward electrostatic interaction as the chief factor in the different behavior of the C5 and C7 structures. Further analysis reveals that the proximity of the carbonyl O atom of the dipeptide, with its partial negative charge, toward the NH group changes the character of the electrostatic potential in the C5 geometry, making NH a less attractive target for an incoming proton acceptor.

Structural Features That Distinguish Kinetically Distinct Biomineralization Polypeptides

AP7 and AP24 are mollusk shell proteins which are responsible for aragonite polymorph formation and stabilization within the nacre layer of the Pacific red abalone, Haliotis rufescens. It is known that the 30-AA N-terminal mineral modification domains of both proteins (AP7N, AP24N) possess identical multifunctional mineralization capabilities within in vitro assays but differ in terms of rate kinetics, with AP24N > AP7N. In this report, we identify previously unreported molecular features of AP24N and contrast the lowest energy polypeptide backbone structures of AP24N (planar configuration) with that of AP7N ("bent paper clip" configuration) using NMR data and simulated annealing molecular dynamics structure refinement. Like AP7N, we find that AP24N possesses an unfolded conformation, can sequester Ca(II) and other multivalent metal ions, can adsorb onto or within calcite crystals, and possesses anionic and cationic electrostatic "pocket" regions on its molecular surfaces. However, AP24N has some unique features: greater conformational responsiveness to Ca(II), the tendency to form a more planar backbone configuration, and longer anionic and hydrogen-bonding donor/acceptor sequence blocks. We conclude that the presence of unfolded polypeptide conformation, electrostatic surface pockets, and interactive sequence clustering endow both AP7N and AP24N with similar features that lead to comparable effects on crystal morphology and nucleation. However, AP24N possesses longer anionic and hydrogen-bonding sequence clusters and exhibits a tendency to adopt a more planar backbone configuration than AP7N does. We believe that these features facilitate peptide-mineral, peptide-ion, or water cluster interactions, thereby enhancing the mineralization kinetics of AP24N over AP7N.

Comparison of cytochromes b5 from insects and vertebrates

We report a 1.55 A X-ray crystal structure of the heme-binding domain of cytochrome b(5) from Musca domestica (house fly; HF b(5)), and compare it with previously published structures of the heme-binding domains of bovine microsomal cytochrome b(5) (bMc b(5)) and rat outer mitochondrial membrane cytochrome b(5) (rOM b(5)). The structural comparison was done in the context of amino acid sequences of all known homologues of the proteins under study. We show that insect b(5)s contain an extended hydrophobic patch at the base of the heme binding pocket, similar to the one previously shown to stabilize mammalian OM b(5)s relative to their Mc counterparts. The hydrophobic patch in insects includes a residue with a bulky hydrophobic side chain at position 71 (Met). Replacing Met71 in HF b(5) with Ser, the corresponding residue in all known mammalian Mc b(5)s, is found to substantially destabilize the holoprotein. The destabilization is a consequence of two related factors: (1) a large decrease in apoprotein stability and (2) extension of conformational disruption in the apoprotein beyond the empty heme binding pocket (core 1) and into the heme-independent folding core (core 2). Analogous changes have previously been shown to accompany replacement of Leu71 in rOM b(5) with Ser. That the stabilizing role of Met71 in HF b(5) is manifested primarily in the apo state is highlighted by the fact that its crystallographic Calpha B factor is modestly larger than that of Ser71 in bMc b(5), indicating that it slightly destabilizes local polypeptide conformation when heme is in its binding pocket. Finally, we show that the final unit of secondary structure in the cytochrome b(5) heme-binding domain, a 3(10) helix known as alpha6, differs substantially in length and packing interactions not only for different protein isoforms but also for given isoforms from different species.

Expanding the polypeptide backbone: hydrogen-bonded conformations in hybrid polypeptides containing the higher homologues of alpha-amino acids.

Half a century has passed since the hydrogen-bonded secondary structures of polypeptides and proteins were first recognized. An extraordinary wealth of conformational information is now available on peptides and proteins, which are formed of alpha-amino acid residues. More recently, the discovery of well-folded structures in oligopeptides containing beta-amino acids has focused a great deal of current interest on the conformational properties of peptides constructed from higher homologues (omega) of alpha-amino acids. This review examines the nature of intramolecularly hydrogen-bonded conformations of hybrid peptides formed by amino acid residues, with a varying number of backbone atoms. The beta-turn, a ubiquitous structural feature formed by two residue (alphaalpha) segments in proteins and peptides, is stabilized by a 10-atom (C10) intramolecular 4-->1 hydrogen bond. Hybrid turns may be classified by comparison with their alphaalpha counterparts. The available crystallographic information on hydrogen-bonded hybrid turns is surveyed in this review. Several recent examples demonstrate that individual omega-amino acid residues and hybrid dipeptide segments may be incorporated into the regular structures of alpha-peptides. Examples of both peptide helices and hairpins are presented. The present review explores the relationships between folded conformations in hybrid sequences and their counterparts in all alpha-residue sequences. The use of stereochemically constrained omega-residues promises to expand the range of peptide design strategies to include omega-amino acids. This approach is exemplified by well-folded structures like the C12 (alphagamma) and C14 (gammagamma) helices formed in short peptides containing multiply substituted gamma-residues. The achiral gamma-residue gabapentin is a readily accessible building block in the design of peptides containing gamma-amino acids. The construction of globular polypeptide structures using diverse hybrid sequences appears to be a realistic possibility.

Secondary Structure Mapping of DnaK-Bound Protein Fragments: Chain Helicity and Local Helix Unwinding at the Binding Site

Little is known about polypeptide conformation and folding in the presence of molecular chaperones participating in protein biosynthesis. In vitro studies on chaperone-substrate complexes have been mostly carried out with small peptide ligands. However, the technical challenges associated with either competing aggregation or spectroscopically unfavorable size and exchange rates have typically prevented analysis of larger substrates. Here, we report the high-resolution secondary structure of relatively large N-terminal protein fragments bound to the substrate-binding domain of the cotranslationally active chaperone DnaK. The all-alpha-helical protein apomyoglobin (apoMb), bearing the ubiquitous globin fold, has been chosen as a model substrate. On the basis of NMR secondary chemical shift analysis, we identify, for the first time, weak helical content (similar to that found in the chemically unfolded full-length protein) for the assigned residues of the chaperone-bound chain away from the chaperone binding sites. In contrast, we found that the residues corresponding to the strongest specific binding site for DnaK, examined via a short 13-mer apoMb peptide fragment matching the binding site sequence, display highly reduced helical content in their chaperone-bound form. Given that the free state of the peptide is weakly helical in isolation, we conclude that the substrate residues corresponding to the chaperone binding site undergo helix unwinding upon chaperone binding.

Proline Isomerization of Histone H3 Regulates Lysine Methylation and Gene Expression

The cis-trans isomerization of proline serves as a regulatory switch in signaling pathways. We identify the proline isomerase Fpr4, a member of the FK506 binding protein family in Saccharomyces cerevisiae, as an enzyme which binds the amino-terminal tail of histones H3 and H4 and catalyses the isomerization of H3 proline P30 and P38 in vitro. We show that P38 is necessary for methylation of K36 and that isomerization by Fpr4 inhibits the ability of Set2 to methylate H3 K36 in vitro. These results suggest that the conformational state of P38, controlled by Fpr4, is important for methylation of H3K36 by Set2. Consistent with such an antagonistic role, abrogation of Fpr4 catalytic activity in vivo results in increased levels of H3K36 methylation and delayed transcriptional induction kinetics of specific genes in yeast. These results identify proline isomerization as a novel noncovalent histone modification that regulates transcription and provides evidence for crosstalk between histone lysine methylation and proline isomerization.

The interplay of sequence and stereochemistry in defining conformation in proteins and polypeptides

Sequential specification of conformation in proteins and polypeptides is a triangular interplay involving the system of linked peptides, the sequences in side chains, and water as solvent. Stereochemistry in side chain linkages is obviously important in the interaction between all of the players, but no specification of its explicit role, if any, in linking sequence with conformation has been made. Flory and coworkers made a puzzling observation in 1967 that, when mutated from poly-L to alternating-L,D stereochemical structure, polypeptides will suffer a reduction in overall dimension or characteristic ratio by an astonishing factor of 10 and to a value even lower than that predicted for free rotation (Miller, W. G.; Brant, D. A.; Flory, P. J. J Mol Biol 1967, 23, 67-80). Enquiring into this longstanding puzzle, Durani and coworkers found that the stereochemical modification will also abolish conformational sensitivity in polypeptide structure to solvent, because electrostatic interactions in the system of linked peptides are transformed from a condition of mutual conflict to one of harmony (Ramakrishnan, V.; Ranbhor, R.; Kumar, A.; Durani, S. J Phys Chem B 2006, 110, 9314-9323). Thus, poly-L stereochemistry could be the fulcrum linking sequences with phi,psis in protein and polypeptide structures, via dielectric arbitrations in a conflicting type of interpeptide electrostatics, in agreement with the electrostatic screening model of Avbelj and Moult (Avbelj, F.; Moult, J. Biochemistry 1995, 34, 755-764).

PH Sensitive Adsorption of Polypeptide/Sodium Dodecyl Sulfate Mixtures

Neutron reflectivity and surface tension have been used to investigate the pH sensitivity of the adsorption of poly-L-lysine hydrobromide and sodium dodecyl sulfate mixtures at the air-solution interface. The surface tension variation with surfactant concentration is complex, and between the critical aggregation concentration and critical micellar concentration there is a marked increase in the surface tension. The neutron reflectivity results show that this is associated with a depletion of the surface of polypeptide/surfactant complexes. The variations in the adsorption and surface tension with pH are attributed to changes in the polypeptide conformation at the interface and in solution.

UV Resonance Raman Investigation of a 310-Helical Peptide Reveals a Rough Energy Landscape

We used UVRRS at 194 and 204 nm excitation to examine the backbone conformation of a 13-residue polypeptide (gp41(659-671)) that has been shown by NMR to predominantly fold into a 3(10)-helix. Examination of the conformation sensitive AmIII(3) region indicates the peptide has significant populations of beta-turn, PPII, 3(10)-helix, and pi-helix-like conformations but little alpha-helix. We estimate that at 1 degree C on average six of the 12 peptide bonds are in folded conformations (predominantly 3(10)- and pi-helix), while the other six are in unfolded (beta-turn/PPII) conformations. The folded and unfolded populations do not change significantly as the temperature is increased from 1 to 60 degrees C, suggesting a unique energy landscape where the folded and unfolded conformations are essentially degenerate in energy and exhibit identical temperature dependences.

Crystal structure of TM1367 from Thermotoga maritima at 1.90 Å resolution reveals an atypical member of the cyclophilin (peptidylprolyl isomerase) fold

Crystal structure of TM1367 from <i>Thermotoga maritima</i> at 1.90 Å resolution reveals an atypical member of the cyclophilin (peptidylprolyl isomerase) fold

Modelling NifH2 protein of Clostridium pasteurianum reveals clues about its physiological function

Clostridium pasteurianum is an anaerobic free-living nitrogen fixer. As a unique feature, the organism contains five extra nifH-like genes in addition to nifH1. Detailed analysis with respect to the structure and function of the nifH-like gene products is missing due to the lack of information about the presence of their translation products in the cell. Recent work indicates that the nifH2 gene is transcribed and translated into a polypeptide of expected size in nitrogen-fixing cells of C. pasteurianum and is regulated both at the transcriptional and translational levels. However, the current data do not reveal the physiological function of the NifH2 protein. In this study, we have used computer tools and the NifH1 of C. pasteurianum as the template to predict a possible tertiary structure and assign a putative function for NifH2 protein. A comparison of the structures of the NifH1 and modelled NifH2 revealed similarities in the polypeptide conformations for both monomers. Analysis of the properties of nucleotide binding, dimer interacting and cluster containing regions did not reveal major differences between NifH1 and modelled NifH2, although minor differences were observed. Rigid docking results revealed the possibility of formation of a NifH1–NifH2 heterodimer as well as formation of a NifH2 homodimer. We, therefore, propose that NifH2 can form a dimer with NifH1, albeit less efficiently and may function as a regulatory Fe-protein.