Current Protein & Peptide Science[JOURNAL]
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168 papers
Peptides derived from HIV-1 Rev inhibit HIV-1 integrase in a shiftide mechanism.
Hayouka Z, Rosenbluh J, Levin A … +3 more , Maes M, Loyter A, Friedler A
Biopolymers · 2008 · PMID 18219678 · Publisher ↗
The HIV-1 Integrase protein (IN) mediates the integration of the viral cDNA into the host genome. IN is an emerging target for anti-HIV drug design, and the first IN-inhibitor was recently approved by the FDA. We have de...
The HIV-1 Integrase protein (IN) mediates the integration of the viral cDNA into the host genome. IN is an emerging target for anti-HIV drug design, and the first IN-inhibitor was recently approved by the FDA. We have developed a new approach for inhibiting IN by "shiftides": peptides derived from its cellular binding protein LEDGF/p75 that inhibit IN by shifting its oligomerization equilibrium from the active dimer to an inactive tetramer. In addition, we described two peptides derived from the HIV-1 Rev protein that interact with IN and inhibit its activity in vitro and in cells. In the current study, we show that the Rev-derived peptides also act as shiftides. Analytical gel filtration and cross-linking experiments showed that IN was dimeric when bound to the viral DNA, but tetrameric in the presence of the Rev-derived peptides. Fluorescence anisotropy studies revealed that the Rev-derived peptides inhibited the DNA binding of IN. The Rev-derived peptides inhibited IN catalytic activity in vitro in a concentration-dependent manner. Inhibition was much more significant when the peptides were added to free IN before it bound the viral DNA than when the peptides were added to a preformed IN-DNA complex. This confirms that the inhibition is due to the ability of the peptides to shift the oligomerization equilibrium of the free IN toward a tetramer that binds much weaker to the viral DNA. We conclude that protein-protein interactions of IN may serve as a general valuable source for shiftide design.
Back to the future: ribonuclease A.
Marshall GR, Feng JA, Kuster DJ
Biopolymers · 2008 · PMID 17868092 · Publisher ↗
Pancreatic ribonuclease A (EC 3.1.27.5, RNase) is, perhaps, the best-studied enzyme of the 20th century. It was isolated by René Dubos, crystallized by Moses Kunitz, sequenced by Stanford Moore and William Stein, and syn...
Pancreatic ribonuclease A (EC 3.1.27.5, RNase) is, perhaps, the best-studied enzyme of the 20th century. It was isolated by René Dubos, crystallized by Moses Kunitz, sequenced by Stanford Moore and William Stein, and synthesized in the laboratory of Bruce Merrifield, all at the Rockefeller Institute/University. It has proven to be an excellent model system for many different types of experiments, both as an enzyme and as a well-characterized protein for biophysical studies. Of major significance was the demonstration by Chris Anfinsen at NIH that the primary sequence of RNase encoded the three-dimensional structure of the enzyme. Many other prominent protein chemists/enzymologists have utilized RNase as a dominant theme in their research. In this review, the history of RNase and its offspring, RNase S (S-protein/S-peptide), will be considered, especially the work in the Merrifield group, as a preface to preliminary data and proposed experiments addressing topics of current interest. These include entropy-enthalpy compensation, entropy of ligand binding, the impact of protein modification on thermal stability, and the role of protein dynamics in enzyme action. In continuing to use RNase as a prototypical enzyme, we stand on the shoulders of the giants of protein chemistry to survey the future.
Focus on function: single molecule RNA enzymology.
Ditzler MA, Alemán EA, Rueda D … +1 more , Walter NG
Biopolymers · 2007 Dec 5-15 · PMID 17685395 · Publisher ↗
The ability of RNA to catalyze chemical reactions was first demonstrated 25 years ago with the discovery that group I introns and RNase P function as RNA enzymes (ribozymes). Several additional ribozymes were subsequentl...
The ability of RNA to catalyze chemical reactions was first demonstrated 25 years ago with the discovery that group I introns and RNase P function as RNA enzymes (ribozymes). Several additional ribozymes were subsequently identified, most notably the ribosome, followed by intense mechanistic studies. More recently, the introduction of single molecule tools has dissected the kinetic steps of several ribozymes in unprecedented detail and has revealed surprising heterogeneity not evident from ensemble approaches. Still, many fundamental questions of how RNA enzymes work at the molecular level remain unanswered. This review surveys the current status of our understanding of RNA catalysis at the single molecule level and discusses the existing challenges and opportunities in developing suitable assays.
Time-resolved NMR studies of RNA folding.
Fürtig B, Buck J, Manoharan V … +5 more , Bermel W, Jäschke A, Wenter P, Pitsch S, Schwalbe H
Biopolymers · 2007 Aug 5-15 · PMID 17595685 · Publisher ↗
The application of real-time NMR experiments to the study of RNA folding, as reviewed in this article, is relatively new. For many RNA folding events, current investigations suggest that the time scales are in the second...
The application of real-time NMR experiments to the study of RNA folding, as reviewed in this article, is relatively new. For many RNA folding events, current investigations suggest that the time scales are in the second to minute regime. In addition, the initial investigations suggest that different folding rates are observed for one structural transition may be due to the hierarchical folding units of RNA. Many of the experiments developed in the field of NMR of protein folding cannot directly be transferred to RNA: hydrogen exchange experiments outside the spectrometer cannot be applied since the intrinsic exchange rates are too fast in RNA, relaxation dispersion experiments on the other require faster structural transitions than those observed in RNA. On the other hand, information derived from time-resolved NMR experiments, namely the acquisition of native chemical shifts, can be readily interpreted in light of formation of a single long-range hydrogen bonding interaction. Together with mutational data that can readily be obtained for RNA and new ligation technologies that enhance site resolution even further, time-resolved NMR may become a powerful tool to decipher RNA folding. Such understanding will be of importance to understand the functions of coding and non-coding RNAs in cells.
Amphetamine effects on brain protein structure and oxidative stress as revealed by FTIR microspectroscopy.
Rodríguez-Casado A, Alvarez I, Toledano A … +2 more , de Miguel E, Carmona P
Biopolymers · 2007 Aug 5-15 · PMID 17480001 · Publisher ↗
Amphetamines are psychostimulants abused by man, that eventually leads to drug dependence. Amphetamine administration to rodents has been shown to provoke significant neurotoxicity involving dopaminergic nerve terminal d...
Amphetamines are psychostimulants abused by man, that eventually leads to drug dependence. Amphetamine administration to rodents has been shown to provoke significant neurotoxicity involving dopaminergic nerve terminal degeneration. However, little information related to the effect of amphetamines on reactive oxygen species (ROS) production and neurotoxicity in brain is currently available. Herein we report the biochemical alterations of lipids and proteins in brain sections from amphetamine-treated rodents using infrared microspectroscopy, immunohistochemistry, and immunoblotting. The spectroscopic changes reveal for the first time the formation of beta-sheet-rich proteins in the cortex, but no significant protein alterations are visible in hippocampus region where hydroperoxide concentration is found to be lower relative to cortex. These result suggest that ROS generated by amphetamine-mediated oxidative stress induce formation beta-sheet-rich proteins which can be of amyloid beta-like character.
Reconstitution of bluetongue virus polymerase activity from isolated domains based on a three-dimensional structural model.
Wehrfritz JM, Boyce M, Mirza S … +1 more , Roy P
Biopolymers · 2007 May · PMID 17323325 · Full text
Bluetongue virus (BTV) is a double-stranded RNA virus of the Reoviridae family. The VP1 protein of BTV is the viral RNA-dependent RNA polymerase (RdRp), which is responsible for the replication of the viral genome. Curre...
Bluetongue virus (BTV) is a double-stranded RNA virus of the Reoviridae family. The VP1 protein of BTV is the viral RNA-dependent RNA polymerase (RdRp), which is responsible for the replication of the viral genome. Currently there is no structural information available for VP1. By manual alignment of BTV, Reovirus and other viral RdRps we have generated a model for the structure of VP1, the RdRp of BTV. The structure can be divided into three domains: an N-terminal domain, a C-terminal domain, and a central polymerase domain. Mutation of the putative catalytic site in the central polymerase domain by site-directed mutagenesis abrogated in vitro replicase activity. Each of the domains was expressed individually and subsequently partially purified to obtain direct evidence for the location of polymerase activity and the nucleoside triphosphate binding site. The nucleoside triphosphate binding site was located by showing that CTP only bound to the full-length protein or to the polymerase domain and not to either of the other two domains. None of the domains had catalytic activity when tested individually or in tandem but when all three domains were mixed together the RdRp activity was reconstituted. This is the first report of the reconstitution of a functional viral RdRp in vitro from individual domains.
Structural flexibility of the nucleosome core particle at atomic resolution studied by molecular dynamics simulation.
Roccatano D, Barthel A, Zacharias M
Biopolymers · 2007 Apr 5-15 · PMID 17252562 · Publisher ↗
Comparative explicit solvent molecular dynamics (MD) simulations have been performed on a complete nucleosome core particle with and without N-terminal histone tails for more than 20 ns. Main purpose of the simulations w...
Comparative explicit solvent molecular dynamics (MD) simulations have been performed on a complete nucleosome core particle with and without N-terminal histone tails for more than 20 ns. Main purpose of the simulations was to study the dynamics of mobile elements such as histone N-terminal tails and how packing and DNA-bending influences the fine structure and dynamics of DNA. Except for the tails, histone and DNA molecules stayed on average close to the crystallographic start structure supporting the quality of the current force field approach. Despite the packing strain, no increase of transitions to noncanonical nucleic acid backbone conformations compared to regular B-DNA was observed. The pattern of kinks and bends along the DNA remained close to the experiment overall. In addition to the local dynamics, the simulations allowed the analysis of the superhelical mobility indicating a limited relative mobility of DNA segments separated by one superhelical turn (mean relative displacement of approximately +/-0.2 nm, mainly along the superhelical axis). An even higher rigidity was found for relative motions (distance fluctuations) of segments separated by half a superhelical turn (approximately +/-0.1 nm). The N-terminal tails underwent dramatic conformational rearrangements on the nanosecond time scale toward partially and transiently wrapped states around the DNA. Many of the histone tail changes corresponded to coupled association and folding events from fully solvent-exposed states toward complexes with the major and minor grooves of DNA. The simulations indicate that the rapid conformational changes of the tails can modulate the DNA accessibility within a few nanoseconds.
NMR analysis of G7-18NATE, a nonphosphorylated cyclic peptide inhibitor of the Grb7 adapter protein.
Porter CJ, Wilce JA
Biopolymers · 2007 · PMID 17206629 · Publisher ↗
G7-18NATE is a nonphosphorylated, cyclic peptide that specifically inhibits the Grb7 adapter protein implicated in several pathways critical to cell proliferation and migration. It has been shown that G7-18NATE is able t...
G7-18NATE is a nonphosphorylated, cyclic peptide that specifically inhibits the Grb7 adapter protein implicated in several pathways critical to cell proliferation and migration. It has been shown that G7-18NATE is able to compete with natural ligands for the Grb7 SH2 phosphotyrosine binding site, and to attenuate cell migration in a pancreatic cancer cell line. It is thus an important lead in the development of a selective inhibitor of Grb7 and potential novel anticancer therapeutics. The current study reports the solution properties of G7- 18NATE determined using NMR spectroscopy, in both water (pH 2-3) and phosphate buffer (pH 6.0), with 100 mM NaCl. The spectra reveal that G7-18NATE exists in two distinguishable conformational states on the NMR timescale, most likely due to cis-trans proline isomerization. In addition, the chemical shift data are consistent with a tendency of G7-18NATE to form a turn about the YDN motif, known to be important for binding, and suggest that this turn is stabilized in low salt and low pH conditions. Low NH temperature coefficients of Tyr-5 and Asn-7 amide protons may reflect their involvement in the formation of hydrogen bonds that stabilize such a turn. Overall, however, the peptide does not form a rigid structure, but exists in a highly flexible state in solution. Averaged 3JNH-H coupling constants and a lack of interresidue NOEs are characteristic of such peptide solution behavior. This suggests that there is scope for increasing the rigidity of the peptide that may enhance its binding affinity and specificity for Grb7.
Molecular dynamics simulations of RNA: an in silico single molecule approach.
McDowell SE, Spacková N, Sponer J … +1 more , Walter NG
Biopolymers · 2007 Feb · PMID 17080418 · Full text
RNA molecules are now known to be involved in the processing of genetic information at all levels, taking on a wide variety of central roles in the cell. Understanding how RNA molecules carry out their biological functio...
RNA molecules are now known to be involved in the processing of genetic information at all levels, taking on a wide variety of central roles in the cell. Understanding how RNA molecules carry out their biological functions will require an understanding of structure and dynamics at the atomistic level, which can be significantly improved by combining computational simulation with experiment. This review provides a critical survey of the state of molecular dynamics (MD) simulations of RNA, including a discussion of important current limitations of the technique and examples of its successful application. Several types of simulations are discussed in detail, including those of structured RNA molecules and their interactions with the surrounding solvent and ions, catalytic RNAs, and RNA-small molecule and RNA-protein complexes. Increased cooperation between theorists and experimentalists will allow expanded judicious use of MD simulations to complement conceptually related single molecule experiments. Such cooperation will open the door to a fundamental understanding of the structure-function relationships in diverse and complex RNA molecules. .
Effect of pH, urea, peptide length, and neighboring amino acids on alanine alpha-proton random coil chemical shifts.
Carlisle EA, Holder JL, Maranda AM … +3 more , de Alwis AR, Selkie EL, McKay SL
Biopolymers · 2007 Jan · PMID 17054116 · Publisher ↗
Accurate random coil alpha-proton chemical shift values are essential for precise protein structure analysis using chemical shift index (CSI) calculations. The current study determines the chemical shift effects of pH, u...
Accurate random coil alpha-proton chemical shift values are essential for precise protein structure analysis using chemical shift index (CSI) calculations. The current study determines the chemical shift effects of pH, urea, peptide length and neighboring amino acids on the alpha-proton of Ala using model peptides of the general sequence GnXaaAYaaGn, where Xaa and Yaa are Leu, Val, Phe, Tyr, His, Trp or Pro, and n = 1-3. Changes in pH (2-6), urea (0-1M), and peptide length (n = 1-3) had no effect on Ala alpha-proton chemical shifts. Denaturing concentrations of urea (8M) caused significant downfield shifts (0.10 +/- 0.01 ppm) relative to an external DSS reference. Neighboring aliphatic residues (Leu, Val) had no effect, whereas aromatic amino acids (Phe, Tyr, His and Trp) and Pro caused significant shifts in the alanine alpha-proton, with the extent of the shifts dependent on the nature and position of the amino acid. Smaller aromatic residues (Phe, Tyr, His) caused larger shift effects when present in the C-terminal position (approximately 0.10 vs. 0.05 ppm N-terminal), and the larger aromatic tryptophan caused greater effects in the N-terminal position (0.15 ppm vs. 0.10 C-terminal). Proline affected both significant upfield (0.06 ppm, N-terminal) and downfield (0.25 ppm, C-terminal) chemical shifts. These new Ala correction factors detail the magnitude and range of variation in environmental chemical shift effects, in addition to providing insight into the molecular level interactions that govern protein folding.
Toward understanding the inactivation mechanism of monooxygenase P450 BM-3 by organic cosolvents: a molecular dynamics simulation study.
Roccatano D, Wong TS, Schwaneberg U … +1 more , Zacharias M
Biopolymers · 2006 Dec · PMID 16862534 · Publisher ↗
Cytochrome P450 BM-3 from Bacillus megaterium is an extensively studied enzyme for industrial applications. A major focus of current protein engineering research is directed to improving the catalytic performance of P450...
Cytochrome P450 BM-3 from Bacillus megaterium is an extensively studied enzyme for industrial applications. A major focus of current protein engineering research is directed to improving the catalytic performance of P450 BM-3 toward nonnatural substrates of industrial importance in the presence of organic solvents or cosolvents. For the latter reason, it is important to study the effect of organic cosolvent molecules on the structure and dynamics of the enzyme, in particular, the effect of cosolvent molecules on the active site's structure and dynamics. In this paper, we have studied, using molecular dynamics (MD) simulations, the F87A mutant of P450 BM-3 in the presence of DMSO as cosolvent, to understand the role of the F87A substitution for its catalytic activity. This mutant exhibits an altered regioselectivity and substrate specificity compared with wild-type; however, it has lower tolerance toward DMSO. The simulation results offer an explanation for the DMSO sensitivity of the F87A mutant. Our simulation results show that the F87 side chain prevents the disturbance of the water molecule bound to the heme iron by DMSO molecules. The absence of the phenyl ring in F87A mutant promotes interactions of the DMSO molecule with the heme iron resulting in water displacement by DMSO at the catalytic heme center.
How strongly do sequence conservation patterns and empirical scales correlate with exposure patterns of transmembrane helices of membrane proteins?
Park Y, Helms V
Biopolymers · 2006 Nov · PMID 16838301 · Publisher ↗
Given the difficulty in determining high-resolution structures of helical membrane proteins, sequence-based prediction methods can be useful in elucidating diverse physiological processes mediated by this important class...
Given the difficulty in determining high-resolution structures of helical membrane proteins, sequence-based prediction methods can be useful in elucidating diverse physiological processes mediated by this important class of proteins. Predicting the angular orientations of transmembrane (TM) helices about the helix axes, based on the helix parameters from electron microscopy data, is a classical problem in this regard. This problem has triggered the development of a number of different empirical scales. Recently, sequence conservation patterns were also made use of for improved predictions. Empirical scales and sequence conservation patterns (collectively termed as "prediction scales") have also found frequent applications in other research areas of membrane proteins: for example, in structure modeling and in prediction of buried TM helices. This trend is expected to grow in the near future unless there are revolutionary developments in the experimental characterization of membrane proteins. Thus, it is timely and imperative to carry out a comprehensive benchmark test over the prediction scales proposed so far to determine their pros and cons. In the current analysis, we use exposure patterns of TM helices as a golden standard, because if one develops a prediction scale that correlates perfectly with exposure patterns of TM helices, it will enable one to predict buried residues (or buried faces) of TM helices with an accuracy of 100%. Our analysis reveals several important points. (1) It demonstrates that sequence conservation patterns are much more strongly correlated with exposure patterns of TM helices than empirical scales. (2) Scales that were specifically parameterized using structure data (structure-based scales) display stronger correlation than hydrophobicity-based scales, as expected. (3) A nonnegligible difference is observed among the structure-based scales in their correlational property, suggesting that not every learning algorithm is equally effective. (4) A straightforward framework of optimally combining sequence conservation patterns and empirical scales is proposed, which reveals that improvements gained from combining the two sources of information are not dramatic in almost all cases. In turn, this calls for the development of fundamentally different scales that capture the essentials of membrane protein folding for substantial improvements.
The photochemistry of the light-, oxygen-, and voltage-sensitive domains in the algal blue light receptor phot.
Kottke T, Hegemann P, Dick B … +1 more , Heberle J
Biopolymers · 2006 Jul · PMID 16552739 · Publisher ↗
Phot proteins are blue light photoreceptors in plants and algae that mainly regulate photomovement responses. They contain two light-, oxygen-, and voltage-sensitive (LOV) domains and a serine/threonine kinase domain. Bo...
Phot proteins are blue light photoreceptors in plants and algae that mainly regulate photomovement responses. They contain two light-, oxygen-, and voltage-sensitive (LOV) domains and a serine/threonine kinase domain. Both LOV domains noncovalently bind a flavin mononucleotide (FMN) as chromophore. Upon blue light illumination, the LOV domains undergo a photocycle, transiently forming a covalent adduct of the FMN moiety with a nearby cysteine residue. The presence of two light-sensitive domains in the photoreceptor raises the question about the differences in properties and function between LOV1 and LOV2. As a model system, the photocycles of the LOV1 and LOV2 domains from phot of the green alga Chlamydomonas reinhardtii have been studied in detail, both separately and in a tandem construct. Here we give an overview about the results on the individual behavior of the domains and their interaction. Furthermore, the current status in the understanding of the role of LOV1 in phot in general is presented.
Heat capacity changes associated with nucleic acid folding.
Mikulecky PJ, Feig AL
Biopolymers · 2006 May · PMID 16429398 · Full text
Whereas heat capacity changes (DeltaCPs) associated with folding transitions are commonplace in the literature of protein folding, they have long been considered a minor energetic contributor in nucleic acid folding. Rec...
Whereas heat capacity changes (DeltaCPs) associated with folding transitions are commonplace in the literature of protein folding, they have long been considered a minor energetic contributor in nucleic acid folding. Recent advances in the understanding of nucleic acid folding and improved technology for measuring the energetics of folding transitions have allowed a greater experimental window for measuring these effects. We present in this review a survey of current literature that confronts the issue of DeltaCPs associated with nucleic acid folding transitions. This work helps to gather the molecular insights that can be gleaned from analysis of DeltaCPs and points toward the challenges that will need to be overcome if the energetic contribution of DeltaCP terms are to be put to use in improving free energy calculations for nucleic acid structure prediction.
Oligoarginine vectors for intracellular delivery: design and cellular-uptake mechanisms.
Futaki S
Biopolymers · 2006 · PMID 16333858 · Publisher ↗
Intracellular delivery using membrane-permeable peptide vectors is a recently developed methodology that has been employed successfully to transport various bioactive molecules into cells to modify cell functions. The ef...
Intracellular delivery using membrane-permeable peptide vectors is a recently developed methodology that has been employed successfully to transport various bioactive molecules into cells to modify cell functions. The efficient delivery of proteins, peptides, nucleic acids, liposomes, and so on has been accomplished using this methodology by conjugation of a peptide vector with the cargo molecules. The potentials of this approach for medical and pharmaceutical applications has also attracted our attention. Arginine-rich peptides, including a basic peptide segment derived from the human immunodeficiency virus type 1 (HIV-1) Tat protein, are categorized into one of the most frequently used peptide vectors, and the efforts of designing novel vectors have been ongoing. Internalization of these peptides has previously been regarded as not employing endocytosis. However, recent reevaluations have demonstrated the significant involvement of endocytosis in the cellular uptake of these peptides. These arginine-rich peptide vectors share many common features in internalization. However, there seem to be certain simultaneous dissimilarities observed in the modes of internalization among these peptides. In this review, the structural features of these arginine-rich peptide vectors have been focused on and the current understandings of their internalization mechanisms are summarized.
Secondary conformation of short lysine- and leucine-rich peptides assessed by optical spectroscopies: effect of chain length, concentration, solvent, and time.
Hernández B, Boukhalfa-Heniche FZ, Seksek O … +2 more , Coïc YM, Ghomi M
Biopolymers · 2006 Jan · PMID 16134172 · Publisher ↗
Solution secondary structures of three synthetic cationic peptides, currently used in antisense oligonucleotide delivery into living cells, have been analyzed by means of circular dichroism (CD) and Raman scattering in d...
Solution secondary structures of three synthetic cationic peptides, currently used in antisense oligonucleotide delivery into living cells, have been analyzed by means of circular dichroism (CD) and Raman scattering in different buffers as a function of concentration and time. All three peptides are of minimalist conception, i.e., formed by only two types of amino acids (leucine: L and lysine: K). Two of these peptides contain 15 aminoacids: N(ter)- KLLKLLLKLLLKLLK (L(10)K(5)), N(ter)-KLKLKLKLKLKLKLK (L(7)K(8)), and the third one has only 9 residues: N(ter)-KLKLKLKLK (L(4)K(5)). The conformational behavior of the 15-mers in pure water differs considerably one from another. Although both of them are initially disordered in the 50-350 microM range, L(10)K(5) gradually undergoes a disordered to alpha-helix transition for molecular concentrations above 100 microM. In all other solvents used, L(10)K(5) adopts a stable alpha-helical conformation. In methanol and methanol/Tris mixture, nonnative alpha-helices can be induced in both KL-alternating peptides, i.e., L(7)K(8) and L(4)K(5). However, in major cases and with a time delay depending on peptide concentration, beta-like structures can be gradually formed in both solutions. In PBS and methanol/PBS mixture, the tendency for L(7)K(8) and L(4)K(5) is to form structures belonging to beta-family. A discussion has been undertaken on the effect of counterions as well as their nature in the stabilization of ordered structures in both KL-alternating peptides.
Beta-hairpin formation in aqueous solution and in the presence of trifluoroethanol: a (1)H and (13)C nuclear magnetic resonance conformational study of designed peptides.
Santiveri CM, Pantoja-Uceda D, Rico M … +1 more , Jiménez MA
Biopolymers · 2005 Oct · PMID 16078190 · Publisher ↗
In order to check our current knowledge on the principles involved in beta-hairpin formation, we have modified the sequence of a 3:5 beta-hairpin forming peptide with two different purposes, first to increase the stabili...
In order to check our current knowledge on the principles involved in beta-hairpin formation, we have modified the sequence of a 3:5 beta-hairpin forming peptide with two different purposes, first to increase the stability of the formed 3:5 beta-hairpin, and second to convert the 3:5 beta-hairpin into a 2:2 beta-hairpin. The conformational behavior of the designed peptides was investigated in aqueous solution and in 30% trifluoroethanol (TFE) by analysis of the following nuclear magnetic resonance (NMR) parameters: nuclear Overhauser effect (NOE) data, and C(alpha)H, (13)C(alpha), and (13)C(beta) conformational shifts. From the differences in the ability to adopt beta-hairpin structures in these peptides, we have arrived to the following conclusions: (i) beta-Hairpin population increases with the statistical propensity of residues to occupy each turn position. (ii) The loop length, and in turn, the beta-hairpin type, can be modified as a function of the type of turn favored by the loop sequence. These two conclusions reinforce previous results about the importance of beta-turn sequence in beta-hairpin folding. (iii) Side-chain packing on each face of the beta-sheet may play a major role in beta-hairpin stability; hence simplified analysis in terms of isolated pair interactions and intrinsic beta-sheet propensities is insufficient. (iv) Contributions to beta-hairpin stability of turn and strand sequences are not completely independent. (v) The burial of hydrophobic surface upon beta-hairpin formation that, in turn, depends on side-chain packing also contributes to beta-hairpin stability. (vi) As previously observed, TFE stabilizes beta-hairpin structures, but the extent of the contribution of different factors to beta-hairpin formation is sometimes different in aqueous solution and in 30% TFE.
Solution structure of chi-conopeptide MrIA, a modulator of the human norepinephrine transporter.
Nilsson KP, Lovelace ES, Caesar CE … +7 more , Tynngård N, Alewood PF, Johansson HM, Sharpe IA, Lewis RJ, Daly NL, Craik DJ
Biopolymers · 2005 · PMID 15931669 · Publisher ↗
The chi-conopeptides MrIA and MrIB are 13-residue peptides with two disulfide bonds that inhibit human and rat norepinephrine transporter systems and are of significant interest for the design of novel drugs involved in...
The chi-conopeptides MrIA and MrIB are 13-residue peptides with two disulfide bonds that inhibit human and rat norepinephrine transporter systems and are of significant interest for the design of novel drugs involved in pain treatment. In the current study we have determined the solution structure of MrIA using NMR spectroscopy. The major element of secondary structure is a beta-hairpin with the two strands connected by an inverse gamma-turn. The residues primarily involved in activity have previously been shown to be located in the turn region (Sharpe, I. A.; Palant, E.; Schroder, C. I.; Kaye, D. M.; Adams, D. J.; Alewood, P. F.; Lewis, R. J. J Biol Chem 2003, 278, 40317-40323), which appears to be more flexible than the beta-strands based on disorder in the ensemble of calculated structures. Analogues of MrIA with N-terminal truncations indicate that the N-terminal residues play a role in defining a stable conformation and the native disulfide connectivity. In particular, noncovalent interactions between Val3 and Hyp12 are likely to be involved in maintaining a stable conformation. The N-terminus also affects activity, as a single N-terminal deletion introduced additional pharmacology at rat vas deferens, while deleting the first two amino acids reduced chi-conopeptide potency.
Control of protein functional dynamics by peptide linkers.
Wriggers W, Chakravarty S, Jennings PA
Biopolymers · 2005 · PMID 15880774 · Publisher ↗
Control of structural flexibility is essential for the proper functioning of a large number of proteins and multiprotein complexes. At the residue level, such flexibility occurs due to local relaxation of peptide bond an...
Control of structural flexibility is essential for the proper functioning of a large number of proteins and multiprotein complexes. At the residue level, such flexibility occurs due to local relaxation of peptide bond angles whose cumulative effect may result in large changes in the secondary, tertiary or quaternary structures of protein molecules. Such flexibility, and its absence, most often depends on the nature of interdomain linkages formed by oligopeptides. Both flexible and relatively rigid peptide linkers are found in many multidomain proteins. Linkers are thought to control favorable and unfavorable interactions between adjacent domains by means of variable softness furnished by their primary sequence. Large-scale structural heterogeneity of multidomain proteins and their complexes, facilitated by soft peptide linkers, is now seen as the norm rather than the exception. Biophysical discoveries as well as computational algorithms and databases have reshaped our understanding of the often spectacular biomolecular dynamics enabled by soft linkers. Absence of such motion, as in so-called molecular rulers, also has desirable functional effects in protein architecture. We review here the historic discovery and current understanding of the nature of domains and their linkers from a structural, computational, and biophysical point of view. A number of emerging applications, based on the current understanding of the structural properties of peptides, are presented in the context of domain fusion of synthetic multifunctional chimeric proteins.
Structural and dynamic properties of cytochrome P450 BM-3 in pure water and in a dimethylsulfoxide/water mixture.
Roccatano D, Wong TS, Schwaneberg U … +1 more , Zacharias M
Biopolymers · 2005 Aug · PMID 15880388 · Publisher ↗
Solvent molecules play an important role for the structural and dynamical properties of proteins. A major focus of current protein engineering is the development of enzymes that are catalytically active in the presence o...
Solvent molecules play an important role for the structural and dynamical properties of proteins. A major focus of current protein engineering is the development of enzymes that are catalytically active in the presence of organic solvents. The monooxygenase P450 BM-3 is one of the best-studied enzymes and promising for industrial applications but with limited activity in the presence of organic solvents or cosolvents. To gain insights into the structural and dynamical properties of the heme domain of this enzyme in solution, molecular dynamics simulations in pure water and in a 14% DMSO/water mixture were performed. The results of the simulations show overall similar structural fluctuations in both solvent systems, with no indication of partial or global unfolding. In 14% DMSO, the regions comprising the helices E, F, and the EF loop (implicated in controlling the entry to the active site channel) undergo a large shift. Significant changes were also observed near the active site access channel at the residue R47. During the simulation, no DMSO molecule penetrated the active site. However, a significant accumulation of DMSO molecules close to the substrate-binding site and to the Flavin Mononucleotide (FMN) reductase domain interface was observed.
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