Long-range Hydrogen Bonds Research Articles

Nucleobases Undergo Dynamic Rearrangements during RNA Tertiary Folding

The tertiary structure of the GTPase center (GAC) of 23S ribosomal RNA (rRNA) as seen in cocrystals is extremely compact. It is stabilized by long-range hydrogen bonds and nucleobase stacking and by a triloop that forms within its three-way junction. Its folding pathway from secondary structure to tertiary structure has not been previously observed, but it was shown to require Mg2+ ions in equilibrium experiments. The fluorescent nucleotide 2-aminopurine was substituted at selected sites within the 60-nt GAC. Fluorescence intensity changes upon addition of MgCl2 were monitored over a time-course from 1ms to 100s as the RNA folds. The folding pathway is revealed here to be hierarchical through several intermediates. Observation of the nucleobases during folding provides a new perspective on the process and the pathway, revealing the dynamics of nucleobase conformational exchange during the folding transitions.

The functional exchangeability of pk- and k-turns in RNA structure

Ribonuclease P RNA requires a sharply kinked RNA helix to make a loop-receptor interaction that creates the binding site for the substrate. In some forms of the ribozyme, this is accomplished by a k-turn, while others have a different element called the pk-turn. The structure of the pk-turn in RNase P of Thermotoga maritima is globally very similar to a k-turn, but lacks all the standard features of that structure, including long-range hydrogen bonds between the two helical arms. We show here that in an isolated RNA duplex, the pk-turn fails to adopt a tightly kinked structure, but rather is a flexible element. This suggests that the tertiary contacts of RNase P assist its folding into the required kinked structure. We find that we can replace the k-turn of the SAM-I riboswitch with the pk-turn, such that the resulting RNA retains its ability to bind SAM, although with lower affinity. We also find that we can replace the pk-turn of T. maritima RNase P with a standard k-turn (in either orientation) with retention of ribozyme activity. Thus, although the pk-turn cannot intrinsically fold into the kinked structure, it can be induced to fold correctly in context. And the pk-turn and k-turns can substitute functionally for one another.

RNA structural motifs that entail hydrogen bonds involving sugar–phosphate backbone atoms of RNA

The growing number of high-resolution crystal structures of large RNA molecules provides much information for understanding the principles of structural organization of these complex molecules. Several in-depth analyses of nucleobase-centered RNA structural motifs and backbone conformations have been published based on this information, including a systematic classification of base pairs by Leontis and Westhof. However, hydrogen bonds involving sugar-phosphate backbone atoms of RNA have not been analyzed systematically until recently, although such hydrogen bonds appear to be common both in local and tertiary interactions. Here we review some backbone structural motifs discussed in the literature and analyze a set of eight high-resolution multi-domain RNA structures. The analyzed RNAs are highly structured: among 5372 nucleotides in this set, 89% are involved in at least one "long-range" RNA-RNA hydrogen bond, i.e., hydrogen bonds between atoms in the same residue or sequential residues are ignored. These long-range hydrogen bonds frequently use backbone atoms as hydrogen bond acceptors, i.e., OP1, OP2, O2', O3', O4', or O5', or as a donor (2'OH). A surprisingly large number of such hydrogen bonds are found, considering that neither single-stranded nor double-stranded regions will contain such hydrogen bonds unless additional interactions with other residues exist. Among 8327 long-range hydrogen bonds found in this set of structures, 2811, or about one-third, are hydrogen bonds entailing RNA backbone atoms; they involve 39% of all nucleotides in the structures. The majority of them (2111) are hydrogen bonds entailing ribose hydroxyl groups, which can be used either as a donor or an acceptor; they constitute 25% of all hydrogen bonds and involve 31% of all nucleotides. The phosphate oxygens OP1 or OP2 are used as hydrogen bond acceptors in 12% of all nucleotides, and the ribose ring oxygen O4' and phosphodiester oxygens O3' and O5' are used in 4%, 4%, and 1% of all nucleotides, respectively. Distributions of geometric parameters and some examples of such hydrogen bonds are presented in this report. A novel motif involving backbone hydrogen bonds, the ribose-phosphate zipper, is also identified.

Disruption Mechanism in the Helix of SPF Peptide by Interchanging E5 and K10 Residues: Inference from Molecular Dynamics Study

Seminalplasmin (SPLN) is a 47-residue peptide (SDEKASPDKHHRFSLSRYAKLANR LANPKLLETFLSKWIGDRGNRSV) from bovine seminal plasma. It has broad spectrum antimicrobial activity, without any hemolytic activity. The 28–40 segment of SPLN with the sequence PKLLETFLSKWIG, designated as SPF, is the most hydrophobic stretch of SPLN and primarily responsible for the membrane-perturbing activity of SPLN. It was reported that SPF has a helical structure and the interchange of E5 and K10 residues disrupted the helical structure. The present paper reports a possible mechanism of disruption of the helical structure of SPF peptide during the interchange of E5 and K10 residues. The result is based on simulated annealing and molecular dynamics simulation studies on SPF and its four analogues with K10E, K10D, E5K, and E5K & K10E substitutions. It showed that K10 residue has a critical role in maintaining the highest helical content and the positions of charged residues are also very important for maintaining the helical structure of the SPF peptide. Formation of some new long-range hydrogen bonds and the rupture of some shortrange hydrogen bonds involving the tenth residue led to the disruption of helical structure of SPF peptide when E5 and K10 residues are interchanged.

Long‐range side‐chain–main‐chain interactions play crucial roles in stabilizing the (βα)8 barrel motif of the alpha subunit of tryptophan synthase

The role of hither-to-fore unrecognized long-range hydrogen bonds between main-chain amide hydrogens and polar side chains on the stability of a well-studied (betaalpha)8, TIM barrel protein, the alpha subunit of tryptophan synthase (alphaTS), was probed by mutational analysis. The F19-D46 and I97-D124 hydrogen bonds link the N terminus of a beta-strand with the C terminus of the succeeding antiparallel alpha-helix, and the A103-D130 hydrogen bond links the N terminus of an alpha-helix with the C terminus of the succeeding antiparallel beta-strand, forming clamps for the respective betaalpha or alphabeta hairpins. The individual replacement of these aspartic acid side chains with alanine leads to what appear to be closely related partially folded structures with significantly reduced far-UV CD ellipticity and thermodynamic stability. Comparisons with the effects of eliminating another main-chain-side-chain hydrogen bond, G26-S33, and two electrostatic side-chain-side-chain hydrogen bonds, D38-H92 and D112-H146, all in the same N-terminal folding unit of alphaTS, demonstrated a unique role for the clamp interactions in stabilizing the native barrel conformation. Because neither the asparagine nor glutamic acid variant at position 46 can completely reproduce the spectroscopic, thermodynamic, or kinetic folding properties of aspartic acid, both size and charge are crucial to its unique role in the clamp hydrogen bond. Kinetic studies suggest that the three clamp hydrogen bonds act in concert to stabilize the transition state leading to the fully folded TIM barrel motif.

Promoting helicity in carbohydrate-containing foldamers through long-range hydrogen bonds

A sensor system to probe the propensity of carbohydrates to induce helical structures through long-range hydrogen bonds, based on a C(2)-symmetric xylylene bis(thiourea) arrangement, is reported; the formation of intermolecular complexes with benzoate anion promotes helix uncoiling, the free energy of the process being related to helix stability.

Understanding protein hydrogen bond formation with kinetic H/D amide isotope effects.

Through the development of a procedure to measure when hydrogen bonds form under two-state folding conditions, alpha-helices have been determined to form proportionally to denaturant-sensitive surface area buried in the transition state. Previous experiments assessing H/D isotope effects are applied to various model proteins, including lambda and Arc repressor variants, a coiled coil domain, cytochrome c, colicin immunity protein 7, proteins L and G, acylphosphatase, chymotrypsin inhibitor II and a Src SH3 domain. The change in free energy accompanied by backbone deuteration is highly correlated to secondary structure composition when hydrogen bonds are divided into two classes. The number of helical hydrogen bonds correlates with an average equilibrium isotope effect of 8.6 +/- 0.9 cal x mol(-1) x site(-1). However, beta-sheet and long-range hydrogen bonds have little isotope effect. The kinetic isotope effects support our hypothesis that, for helical proteins, hydrophobic association cannot be separated from helix formation in the transition state. Therefore, folding models that describe an incremental build-up of structure in which hydrophobic burial and hydrogen bond formation occur commensurately are more consistent with the data than are models that posit the extensive formation of one quantity before the other.

Hydrogen bonding and morphological structure of segmented polyurethanes based on hydroquinone- bis(?-hydroxyethy)ether as a chain extender

Two series of polyether polyurethanes based on hydroquinone bis(β-hydroxyethy) ether (HQEE) or 1,4-butanediol (BDO) as a chain extender were prepared by a one-step bulk polymerization process. Their hydrogen bonding and morphological structure were studied by means of Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and transmission electron microscopy (TEM). Compared with BDO-based polyurethanes, HQEE-based polyurethanes showed better phase segregation and higher extent of ordering in the hard domain. Besides short-range hydrogen bonds, a considerable amount of long-range hydrogen bonds existed in amorphous phase of the hard domain. The soft segment was easier to crystallize, and the melting temperature of the hard domain was elevated in HQEE-based polyurethanes. Its hard domain displayed a banded structure, rather than spherulitic structure in BDO-based polyurethanes. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 73: 2895–2902, 1999

Molecular Modeling of HIV-1 Coreceptor CCR5 and Exploring of Conformational Space of its Extracellular Domain in Molecular Dynamics Simulation

The chemokine receptor CCR5 functions as a major fusion coreceptor for macrophage-tropic human immunodeficiency virus entry into cell. Here we report a three-dimensional model of CCR5 built using molecular modeling approach. Because the virus binds to extracellular domain of the receptor, special attention was given to conformational flexibility, hydrogen bonding, and environmental polarity properties of this protein part. Such data were obtained in the result of molecular dynamics study of the extracellular domain. It was shown that during the simulation the extracellular segments form a compact globular domain with numerous long-range hydrogen bonds between them. First loop of the receptor stays quite rigid while N- terminal region and loops 2, 3 are rather flexible. A number of amino acid residues disposed in unfavourable environment and, therefore, potentially involved in binding of CCR5 to viral glycoproteins and chemokines, was delineated. Comparison of the results with available experimental data permits a proposal that such residues in loop-1 and N-terminal part of the receptor are important for HIV-1 entry, while those in loops 2 and 3 participate in ligand binding. Perspectives of rational alteration of virus-binding activity of CCR5 are discussed.

The NMR solution structure of the non-classical homeodomain from the rat liver LFB1/HNF1 transcription factor

The nuclear magnetic resonance (NMR) solution structure of the non-classical homeodomain from the rat liver LFB1/HNF1 transcription factor was determined with the program DIANA from an input of 1356 nuclear Overhauser enhancement (NOE) upper distance constraints and 228 dihedral angle constraints collected using experiments with the unlabelled, the uniformly 15N-labelled and the uniformly 13C-labelled protein. Out of a group of 50 independently calculated conformers the 20 conformers with the smallest residual DIANA target function values were refined by energy minimization with the program OPAL and are used to represent the NMR structure. The average of the pairwise root-mean-square deviations (r.m.s.d.) of these 20 individual NMR conformers relative to the mean coordinates is 0.73 Å (1 Å = 0.1 nm) for the backbone atoms N, Cα and C′ of residues 15 to 82. The chain-terminal polypeptide segments 1–14 and 90–99 are disordered in solution. The globular fold contains three well-defined helices comprising the residues 19 to 29, 37 to 53 and 71 to 81, and the third helix is extended by a less well-ordered fourth helix with residues 82 to 89, which coincides with corresponding observations in “classical” homeodomains. Side-chain analysis resulted in 33 “best-defined” side-chains, with global displacements smaller than 1.1 Å, and addition of these side-chains to the global superposition of residues 15 to 82 resulted in a r.m.s.d of 0.81 Å. The protein contains two hydrophobic cores, one of which corresponds to the helical packing seen in classical homeodomains, while the other one stabilizes the conformation of the 21-residue insertion between helices II and III. The individual helices and their relative spatial arrangements are stabilized by a variety of structural motifs, which include medium-range and long-range hydrogen bonds and salt bridges. Detailed comparison with the Antennapedia homeodomain, and studies of the complex formation with an operator DNA half-site provided initial information on the DNA-binding mode of the LFB1/HNF1 homeodomain.

The Role of Side-chain Hydrogen Bonds in the Formation and Stabilization of Secondary Structure in Soluble Proteins

Intra-molecular side-chain:main-chain (sch:mch) and side-chain (sch:sch) hydrogen bonds observed in 44 well refined crystallographic protein structures with non-homologous sequences have been identified, classified and analysed to detect recurring structural patterns. Each observed bond was characterized by the position of its acceptor and donor groups relative to the N and C termini of the particular secondary structure in which they occur and according to their appearance within the same of sequentially separated secondary structures. The role of short-range hydrogen bonds in the formation and stabilization of a secondary structure and the importance of long-range hydrogen bonds as a cohesive force for different structural segments were also examined. It was found that the N terminus of α-helices is characterized by recurring sch:mch and sch:sch bonds with elements of the preceding coil segment, while at the C terminus a frequent intra-helix sch:mch hydrogen bond was frequently observed. The residues at or near the β-strand termini often cross-linked, through hydrogen-bonding, non-sequential coil segments. Coil structures were characterized by recurring, internal sch:mch hydrogen bonding involving small polar side-chain groups situated at or near their N termini (coil N-capping). The significance of hydrogen bonds as formers and stabilizers of a protein fold and the association of its secondary structural units was also considered through an examination of bond density and distribution throughout the protein tertiary structure.