Backbone dynamics of free barnase and its complex with barstar determined by 15N NMR relaxation study

Backbone dynamics of uniformly ¹⁵N-labeled free barnase and its complex with unlabelled barstar have been studied at 40°C, pH 6.6, using ¹⁵N relaxation data obtained from proton-detected 2D {¹H}-¹⁵N NMR spectroscopy. ¹⁵N spin-lattice relaxation rate constants (R₁), spin-spin relaxation rate constants (R₂), and steady-state heteronuclear {¹H}-¹⁵N NOEs have been measured at a magnetic field strength of 14.1 Tesla for 91 residues of free barnase and for 90 residues out of a total of 106 in the complex (excluding three prolines and the N-terminal residue) backbone amide ¹⁵N sites of barnase. The primary relaxation data for both the cases have been analyzed in the framework of the model-free formalism using both isotropic and axially symmetric models of the rotational diffusion tensor. As per the latter, the overall rotational correlation times (_m) are 5.0 and 9.5 ns for the free and complexed barnase, respectively. The average order parameter is found to be 0.80 for free barnase and 0.86 for the complex. However, the changes are not uniform along the backbone and for about 5 residues near the binding interface there is actually a significant decrease in the order parameters on complex formation. These residues are not involved in the actual binding. For the residues where the order parameter increases, the magnitudes vary significantly. It is observed that the complex has much less internal mobility, compared to free barnase. From the changes in the order parameters, the entropic contribution of NH bond vector motion to the free energy of complex formation has been calculated. It is apparent that these motions cause significant unfavorable contributions and therefore must be compensated by many other favorable contributions to effect tight complex formation. The observed variations in the motion and their different locations with regard to the binding interface may have important implications for remote effects and regulation of the enzyme action.     

The amide III vibrational circular dichroism band as a probe to detect conformational preferences of alanine dipeptide in water

The conformational preferences of blocked alanine dipeptide (ADP), Ac‐Ala‐NHMe, in aqueous solution were studied using vibrational circular dichroism (VCD) together with density functional theory (DFT) calculations. DFT calculations of three most representative conformations of ADP surrounded by six explicit water molecules immersed in a dielectric continuum have proven high sensitivity of amide III VCD band shape that is characteristic for each conformation of the peptide backbone. The polyproline II (P_II) and α_R conformation of ADP are associated with a positive VCD band while β conformation has a negative VCD band in amide III region. Knowing this spectral characteristic of each conformation allows us to assign the experimental amide III VCD spectrum of ADP. Moreover, the amide III region of the VCD spectrum was used to determine the relative populations of conformations of ADP in water. Based on the interpretation of the amide III region of VCD spectrum we have shown that dominant conformation of ADP in water is P_II which is stabilized by hydrogen bonded water molecules between CO and NH groups on the peptide backbone. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 814–818, 2014.     

On the use of N‐dicyclopropylmethyl aspartyl‐glycine synthone for backbone amide protection

To prevent aspartimide formation and related side products in Asp‐Xaa, particularly Asp‐Gly‐containing peptides, usually the 2‐hydroxy‐4‐methoxybenzyl (Hmb) backbone amide protection is applied for peptide synthesis according to the Fmoc‐protocols. In the present study, the usefulness of the recently proposed acid‐labile dicyclopropylmethyl (Dcpm) protectant was analyzed. Despite the significant steric hindrance of this bulky group, N‐terminal H‐(Dcpm)Gly‐peptides are quantitatively acylated by potent acylating agents, and alternatively the dipeptide Fmoc‐Asp(OtBu)‐(Dcpm)Gly‐OH derivative can be used as a building block. In contrast to the Hmb group, Dcpm is inert toward acylations, but is readily removed in the acid deprotection and resin‐cleavage step. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Novel method for the synthesis of urea backbone cyclic peptides using new Alloc‐protected glycine building units

Cyclization of bioactive peptides, utilizing functional groups serving as natural pharmacophors, is often accompanied with loss of activity. The backbone cyclization approach was developed to overcome this limitation and enhance pharmacological properties. Backbone cyclic peptides are prepared by the incorporation of special building units, capable of forming amide, disulfide and coordinative bonds. Urea bridge is often used for the preparation of cyclic peptides by connecting two amine functionalized side chains. Here we present urea backbone cyclization as an additional method for the preparation of backbone cyclic peptide libraries. A straightforward method for the synthesis of crystalline Fmoc‐N^α [ω‐amino(Alloc)‐alkyl] glycine building units is presented. A set of urea backbone cyclic Glycogen Synthase Kinase 3 analogs was prepared and assessed for protein kinase B inhibition as anticancer leads. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Formation of cyclotides and variations in cyclotide expression in <Emphasis Type="Italic">Oldenlandia affinis</Emphasis> suspension cultures

Cyclotides, a family of disulfide-rich mini-proteins, show a wide range of biological activities, making them interesting targets for pharmaceutical and agrochemical applications, but little is known about their natural function and the events that trigger their expression. An investigation of nutritional variations and irradiation during a batch process involving plant cell cultures has been performed, using the native African medical herb, Oldenlandia affinis, as a model plant. The results demonstrated the biosynthesis of kalata B1, the main cyclotide in O. affinis, in a combined growth/nongrowth-associated pattern. The highest concentration, 0.37 mg g⁻¹ dry weight, was accumulated in irradiated cells at 35 μmol m⁻² s⁻¹. Furthermore, 12 novel cyclotides were identified and the expression of various cyclotides compared in irradiated vs non-irradiated cultures. The results indicate that cyclotide expression varies greatly depending on physiological conditions and environmental stress. Kalata B1 is the most abundant cyclotide in plant suspension cultures, which underlies its importance as a natural defense molecule. The identification of novel cyclotides in suspension cultures, compared to whole plants, indicates that there may be more novel cyclotides to be discovered and that the genetic network regulating cyclotide expression is a very sensitive system, ready to adapt to the current environmental growth condition.     

Pulling apart catalytically active Tn5 synaptic complexes using magnetic tweezers

The Tn5 transposase is an example of a class of proteins that move DNA sequences (transposons) via a process called transposition. DNA transposition is a widespread genetic mobility mechanism that has profoundly affected the genomes of nearly all organisms. We have used single-DNA micromanipulation experiments to study the process by which Tn5 DNA transposons are identified and processed by their transposase protein. We have determined that the energy barrier to disassemble catalytically active synaptic complexes is 16 kcal mol(-1). However, we have found that the looping organization of DNA segments by transposase is less sequence-driven than previously thought. Loops anchored at some non-transposon end sequences display a disassembly energy barrier of 14 kcal mol(-1), nearly as stable as the synapses formed at known transposon end sequences. However, these non-transposon end sequence independent complexes do not mediate DNA cleavage. Therefore, the sequence-sensitivity for DNA binding and looping by Tn5 transposase is significantly less than that required for DNA cleavage. These results have implications for the in vivo down regulation of transposition and the cis-transposition bias of transposase.     

On interpretation of protein X‐ray structures: Planarity of the peptide unit

Pauling's mastery of peptide stereochemistry—based on small molecule crystal structures and the theory of chemical bonding—led to his realization that the peptide unit is planar and then to the Pauling–Corey–Branson model of the α‐helix. Similarly, contemporary protein structure refinement is based on experimentally determined diffraction data together with stereochemical restraints. However, even an X‐ray structure at ultra‐high resolution is still an under‐determined model in which the linkage among refinement parameters is complex. Consequently, restrictions imposed on any given parameter can affect the entire structure. Here, we examine recent studies of high resolution protein X‐ray structures, where substantial distortions of the peptide plane are found to be commonplace. Planarity is assessed by the ω‐angle, a dihedral angle determined by the peptide bond (C? N) and its flanking covalent neighbors; for an ideally planar trans peptide, ω = 180°. By using a freely available refinement package, Phenix [Afonine et al. (2012) Acta Cryst. D, 68:352–367], we demonstrate that tightening default restrictions on the ω‐angle can significantly reduce apparent deviations from peptide unit planarity without consequent reduction in reported evaluation metrics (e.g., R‐factors). To be clear, our result does not show that substantial non‐planarity is absent, only that an equivalent alternative model is possible. Resolving this disparity will ultimately require improved understanding of the deformation energy. Meanwhile, we urge inclusion of ω‐angle statistics in new structure reports in order to focus critical attention on the usual practice of assigning default values to ω‐angle constraints during structure refinement. Proteins 2015; 83:1687–1692. © 2015 Wiley Periodicals, Inc.     

Optimization of rotamers prior to template minimization improves stability predictions made by computational protein design

Computational protein design (CPD) predictions are highly dependent on the structure of the input template used. However, it is unclear how small differences in template geometry translate to large differences in stability prediction accuracy. Herein, we explored how structural changes to the input template affect the outcome of stability predictions by CPD. To do this, we prepared alternate templates by Rotamer Optimization followed by energy Minimization (ROM) and used them to recapitulate the stability of 84 protein G domain β1 mutant sequences. In the ROM process, side-chain rotamers for wild-type (WT) or mutant sequences are optimized on crystal or nuclear magnetic resonance (NMR) structures prior to template minimization, resulting in alternate structures termed ROM templates. We show that use of ROM templates prepared from sequences known to be stable results predominantly in improved prediction accuracy compared to using the minimized crystal or NMR structures. Conversely, ROM templates prepared from sequences that are less stable than the WT reduce prediction accuracy by increasing the number of false positives. These observed changes in prediction outcomes are attributed to differences in side-chain contacts made by rotamers in ROM templates. Finally, we show that ROM templates prepared from sequences that are unfolded or that adopt a nonnative fold result in the selective enrichment of sequences that are also unfolded or that adopt a nonnative fold, respectively. Our results demonstrate the existence of a rotamer bias caused by the input template that can be harnessed to skew predictions toward sequences displaying desired characteristics.