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H H Klump

Publications and source records attributed to H H Klump.

At least 19 recordsLinked to original sources
PubMed2006

Exploring the energy landscape of the genetic code.

New insights into the arrangement of the genetic code table, based on the analysis of the physico-chemical properties of its molecular constituents, are reported in this paper. It will be demonstrated that the code has a twofold symmetry that is not apparent from the conventional code table, but becomes apparent when the codon-anticodon energies are listed for each triplet. The evolutionary development of the current code based on single base replacement mutations (transitions) from an 'iso-energetic' degenerated subset of 16 of the 64 codons is discussed. The energy landscape of all 64 codons is presented. A detailed analysis of the energy changes due to mutations in the 3rd, 1st or 2nd position of a codon reveals that the modern genetic code is highly robust. Changes come in small discrete steps that can be quantified in relation to the thermal noise of the system. The relation of the individual codon to its neighbours in the rearranged codon table can be completely understood based on thermodynamic considerations.

Biological Evolution↗
PubMed2006

Progress in designing nucleic acid structures and fine-tuning their interactions.

The prediction of the structure of biological macromolecules at the atomic level and the design of new meta-stable structures and secondary interactions are critical tests of our understanding of the structures and the inter-atomic forces that underlie molecular biology. The capacity to accurately predict and design new structures and interactions will allow us to create nucleic acid sequences that will fold in new and useful ways. Here, we present some results to demonstrate the progress we have made in designing and assembling new nucleic acid structures that will make an increasingly important contribution to biology and medicine. We call the reaction cycle that exemplifies our approach 'A handshake from a hairpin on the way to a double helix.'

Base Sequence↗
PubMed2002

Conformational energetics of stable and metastable states formed by DNA triplet repeat oligonucleotides: implications for triplet expansion diseases.

We have embedded the hexameric triplet repeats (CAG)(6) and (CTG)(6) between two (GC)(3) domains to produce two 30-mer hairpins with the sequences d[(GC)(3)(CAG)(6)(GC)(3)] and d[(GC)(3)(CTG)(6)(GC)(3)]. This construct reduces the conformational space available to these repetitive DNA sequences. We find that the (CAG)(6) and (CTG)(6) repeats form stable, ordered, single-stranded structures. These structures are stabilized at 62 degrees C by an average enthalpy per base of 1.38 kcal.mol(-1) for the CAG triplet and 2.87 kcal.mol(-1) for the CTG triplet, while being entropically destabilized by 3.50 cal.K(-1).mol(-1) for the CAG triplet and 7.6 cal.K(-1).mol(-1) for the CTG triplet. Remarkably, these values correspond, respectively, to 1/3 (for CAG) and 2/3 (for CTG) of the enthalpy and entropy per base values associated with Watson-Crick base pairs. We show that the presence of the loop structure kinetically inhibits duplex formation from the two complementary 30-mer hairpins, even though the duplex is the thermodynamically more stable state. Duplex formation, however, does occur at elevated temperatures. We propose that this thermally induced formation of a more stable duplex results from thermal disruption of the single-stranded order, thereby allowing the complementary domains to associate (perhaps via "kissing hairpins"). Our melting profiles show that, once duplex formation has occurred, the hairpin intermediate state cannot be reformed, consistent with our interpretation of kinetically trapped hairpin structures. The duplex formed by the two complementary oligonucleotides does not have any unusual optical or thermodynamic properties. By contrast, the very stable structures formed by the individual single-stranded triplet repeat sequences are thermally and thermodynamically unusual. We discuss this stable, triplet repeat, single-stranded structure and its interconversion with duplex in terms of triplet expansion diseases.

Base Sequence↗
PubMed2001

Impact of the third-strand orientation on the thermodynamic stability of the four-way DNA junction.

The physical properties of a triple-helical DNA four-way junction J(T2T4) have been characterized by means of UV spectroscopy, CD spectroscopy, and differential scanning calorimetry (DSC). J(T2T4) is another four-way junction that was designed in addition to J(T1T3) (N. Makube and H. H. Klump (2000) Arch. Biochem. Biophys. 377, 31-42) to study the effects of third strands on the stability of the four-way junction with triple-helical arms. The pH titration curves illustrate the sequential folding of single strands to double-helical four-way junctions and finally the binding of third strands to their respective W-C duplexes. CD measurements confirm triplex formation under appropriate pH and ionic strength conditions. The CD spectra also suggest different melting patterns for the triple-helical arms of J(T2T4). The melting temperature as a function of pH or ionic strength characterizes the effect of the third strands on the structural stability. Increased sodium concentration and low pH conditions enhances and stabilizes the overall structure of the junction. The results also indicate that all triplexes in J(T2T4) are formed in the absence of salt and at low pH; however, the junction may, under these conditions, assume a conformation different from the one assumed in the presence of salt. Through the deconvolution of DSC data, the calorimetric enthalpies associated with melting of arms of the junctions were determined. The loops are designed to have the same enthalpic effect on the different arms. The stabilizing effect of the loops is more pronounced when those loops are shifted from arms 1 and 3 in J(T1T3) to arms 2 and 4 in J(T2T4) without changing any of the sequences. Overall, J(T2T4) is slightly more stable than J(T1T3). The differences can be attributed to sequence effects rather than structural effects. All the results illustrate that binding of the third strand in either of the two orientations 5'5'3' (J(T2T4)) or 5'3'3' (J(T1T3)) stabilizes the underlying double-helical four-way junction and its triple-helical arms.

Base Sequence↗
PubMed2001

Counterion association with native and denatured nucleic acids: an experimental approach.

The melting temperature of the poly(dA) . poly(dT) double helix is exquisitely sensitive to salt concentration, and the helix-to-coil transition is sharp. Modern calorimetric instrumentation allows this transition to be detected and characterized with high precision at extremely low duplex concentrations. We have taken advantage of these properties to show that this duplex can be used as a sensitive probe to detect and to characterize the influence of other solutes on solution properties. We demonstrate how the temperature associated with poly(dA) . poly(dT) melting can be used to define the change in bulk solution cation concentration imparted by the presence of other duplex and triplex solutes, in both their native and denatured states. We use this information to critically evaluate features of counterion condensation theory, as well as to illustrate "crosstalk" between different, non-contacting solute molecules. Specifically, we probe the melting of a synthetic homopolymer, poly(dA) . poly(dT), in the presence of excess genomic salmon sperm DNA, or in the presence of one of two synthetic RNA polymers (the poly(rA) . poly(rU) duplex or the poly(rU) . poly(rA) . poly(rU) triplex). We find that these additions cause a shift in the melting temperature of poly(dA) . poly(dT), which is proportional to the concentration of the added polymer and dependent on its conformational state (B versus A, native versus denatured, and triplex versus duplex). To a first approximation, the magnitude of the observed tm shift does not depend significantly on whether the added polymer is RNA or DNA, but it does depend on the number of strands making up the helix of the added polymer. We ascribe the observed changes in melting temperature of poly(dA) . poly(dT) to the increase in ionic strength of the bulk solution brought about by the presence of the added nucleic acid and its associated counterions. We refer to this communication between non-contacting biopolymers in solution as solvent-mediated crosstalk. By comparison with a known standard curve of tm versus log[Na+] for poly(dA) . poly(dT), we estimate the magnitude of the apparent change in ionic strength resulting from the presence of the bulk nucleic acid, and we compare these results with predictions from theory. We find that current theoretical considerations correctly predict the direction of the t(m) shift (the melting temperature increases), while overestimating its magnitude. Specifically, we observe an apparent increase in ionic strength equal to 5% of the concentration of the added duplex DNA or RNA (in mol phosphate), and an additional apparent increase of about 9.5 % of the nucleic acid concentration (mol phosphate) upon denaturation of the added DNA or RNA, yielding a total apparent increase of 14.5 %. For the poly(rU) . poly(rA) . poly(rU) triplex, the total apparent increase in ionic strength corresponds to about 13.6% of the amount of added triplex (moles phosphate). The effect we observe is due to coupled equilibria between the solute molecules mediated by modulations in cation concentration induced by the presence and/or the transition of one of the solute molecules. We note that our results are general, so one can use a different solute probe sensitive to proton binding to characterize subtle changes in solution pH induced by the presence of another solute in solution. We discuss some of the broader implications of these measurements/results in terms of nucleic acid melting in multicomponent systems, in terms of probing counterion environments, and in terms of potential regulatory mechanisms.

Animals↗
PubMed2001

Communication between noncontacting macromolecules.

We present a quantitative experimental demonstration of solvent-mediated communication between noncontacting biopolymers. We show that changes in the activity of a solvent component brought about by a conformational change in one biopolymer can result in changes in the physical properties of a second noncontacting biopolymer present in solution. Specifically, we show that the release of protons on denaturation of a donor polymer (in this case, a four-stranded DNA tetraplex, iDNA) modulates the melting temperature of a noncontacting, acceptor polymer [in this case poly(A)]. In addition to such proton-mediated cross talk, we also demonstrate counterion-mediated cross talk between noncontacting biopolymers. Specifically, we show that counterion association/release on denaturation of native salmon sperm DNA (the donor polymer) can modulate the melting temperature of poly(dA) x poly(dT) (the acceptor polymer). Taken together, these two examples demonstrate how poly(A) and poly(dA) x poly(dT) can serve as molecular probes that report the pH and free salt concentrations in solution, respectively. Further, we demonstrate how such through-solvent dialogue between biopolymers that do not directly interact can be used to evaluate (in a model-free manner) association/dissociation reactions of solvent components (e.g., protons, sodium cations) with one of the two biopolymers. We propose that such through-solution dialogue is a general property of all biopolymers. As a result, such solvent-mediated cross talk should be considered when assessing reactions of multicomponent systems such as those that exist in essentially all biological processes.

Animals↗
PubMed2000

A four-way junction with triple-helical arms: design, characterization, and stability.

The formation of the four-way junction containing four triple-helical arms has been demonstrated using chemical methods (polyacrylamide gel electrophoresis and chemical footprinting using OsO(4) as a probe) and physical methods (UV absorbance melting and DSC). The junction J(T1T3) was assembled from two 20-mer purine strands and two 44-mer pyrimidine strands. To determine the contribution of the different arms to the stability of the complete structure of J(T1T3), the junction was compared to two simplified substructures, J(T1) and J(T3), respectively. Common to these complexes is the underlying double-helical four-way junction Js. Addition of Na(+) had a profound effect on stabilizing and subsequently folding the junctions into the stacked X-structures. The following results support the structure present: (i) The native polyacrylamide electrophoresis exhibits only a single band(s) corresponding to one species present when all four single strands are mixed in equal amounts. (ii) OsO(4) modifications were investigated at pH 5.0 and in the presence of 10 mM Mg(2+) and 100 mM Na(+). There is no cleavage of thymine residues at the branch point and throughout the structure. (iii) The thermal unfolding of J(T1) and J(T3) illustrates that the triple-helical arms are more stable than the double-helical arms which are contained in these junctions and that J(T1T3) with four triple-helical arms is slightly more stable than J(T1) and J(T3). (iv) The calorimetric transition enthalpies determined for the arms of J(T1T3) are comparable to those associated with the unfolding of its corresponding arms in J(T1) and J(T3). The results also illustrate that the formation of the junctions is not restricted by the pH, [Na(+)], sequence composition of the arms, and/or the loop position.

Base Sequence↗
PubMed1998

Protein thermostability above 100 degreesC: a key role for ionic interactions.

The discovery of hyperthermophilic microorganisms and the analysis of hyperthermostable enzymes has established the fact that multisubunit enzymes can survive for prolonged periods at temperatures above 100 degreesC. We have carried out homology-based modeling and direct structure comparison on the hexameric glutamate dehydrogenases from the hyperthermophiles Pyrococcus furiosus and Thermococcus litoralis whose optimal growth temperatures are 100 degreesC and 88 degreesC, respectively, to determine key stabilizing features. These enzymes, which are 87% homologous, differ 16-fold in thermal stability at 104 degreesC. We observed that an intersubunit ion-pair network was substantially reduced in the less stable enzyme from T. litoralis, and two residues were then altered to restore these interactions. The single mutations both had adverse effects on the thermostability of the protein. However, with both mutations in place, we observed a fourfold improvement of stability at 104 degreesC over the wild-type enzyme. The catalytic properties of the enzymes were unaffected by the mutations. These results suggest that extensive ion-pair networks may provide a general strategy for manipulating enzyme thermostability of multisubunit enzymes. However, this study emphasizes the importance of the exact local environment of a residue in determining its effects on stability.

Amino Acid Sequence↗
PubMed1996

Triple helical structures involving inosine: there is a penalty for promiscuity.

Inosine has the ability to act as a "wild-card" binding nonspecifically to both A.T and G.C base pairs. This has obvious implications for the design of oligonucleotide site-directed probes. In this paper we present a series of oligonucleotides with a 5'pur9-pyr9-pyr9 motif which are designed to fold up sequentially into intramolecular triple helices. One or more inosines are incorporated into the Hoogsteen strands in place of T's and/or C's. Once folded into the triplex, the inosine-containing third strand is incorporated in parallel orientation to the purine strand of the duplex. The influence of inosine on the triplex-duplex equilibrium, characterized by the melting temperature (Tm) and on the phase boundaries, as a function of pH and/or ionic strength, has been assessed by means of UV and CD spectroscopy. There are two distinguishable influences of third-strand inosines which affect binding, namely, backbone distortion due to bulkiness (I for T and I for C+) and/or loss of intramolecular ion pairs between protonated cytosines and the backbone phosphates (I for C+). A single thymine replacement drops the Tm by 25.0 (+/- 2.1) degrees C, and replacing a single protonated cytosine drops the Tm by 32.1 (+/- 1.0) degrees C at pH 6.0. On introducing two inosines in place of thymines, the Tm at pH 6.0 of the triple helix to hairpin transition is lowered by 35.5 (+/- 1.4) degrees C; on introducing two inosines in place of cytosines, the Tm drops by 44.5 (+/- 1.0) degree C, and on replacing a cytosine and a neighboring thymine with inosines, the Tm of the same transition is lowered by 29.2 (+/- 1.6) degrees C. Replacing more than two thymines or cytosines, respectively, eliminates the binding of the Hoogsteen strand at room temperature altogether. Under no circumstances does inosine replacement stabilize the triplex helix: it is a poor substitute and its role as a wild-card is limited.

Base Composition↗
PubMed1995

Thermodynamic characterization of a triple-helical three-way junction containing a Hoogsteen branch point.

We have designed a Hoogsteen (HG) triple-helical three-way junction (ternary complex) constructed from three 33-mer oligonucleotides based on the same subset of sequences used for the Watson-Crick (WC) triple-helical three-way junction, characterized previously (P. L. Hüsler and H. H. Klump (1994) Arch. Biochem. Biophys., 313, 29-38). The junction differs primarily in the assembly of the branch point and the ends of the arms. The three oligonucleotides can each fold into a WC hairpin, linked by a four-member cytosine loop, each containing a homo-pyrimidine 10-mer single-strand extension. On lowering the pH (between 6 and 4), the extensions mutually associate to one of the other hairpins via Hoogsteen (HG) hydrogen bonding. Collectively, this process results in the formation of the branch point and the triple-helical arms. The HG triple-helical three-way junction is characterized by gel electrophoresis, circular dichroism, uv melting, and differential scanning calorimetry. The junction undergoes thermal unfolding in two distinct temperature regions. In the temperature range 15 to 50 degrees C loss of HG base pairing results in the dissociation of the three-way junction. Between 55 and 95 degrees C the resulting hairpins undergo further successive unfolding. The overall calorimetric unfolding enthalpy and entropy changes associated with the loss of HG base pairing are approximately equal to the sum of the enthalpy and entropy changes associated with the dissociation of the HG base pairing in the isolated arms (170.6 kcal.mol-1; 540.1 cal.mol-1.K-1). It is apparent from these results that in the proximity of the branch point the structure is not perturb or strain. This result is contrary to the results obtained for the WC triple-helical three-way and for three-way junctions constructed from canonical double-helical DNA. Complete folding of the junction requires either high Na+ (600 mM) ion concentrations or 40-60 mM Mg2+.

Base Sequence↗
PubMed1995

Prediction of pH-dependent properties of DNA triple helices.

The thermodynamic properties of two triple helices were investigated by uv thermal denaturation, differential scanning calorimetry, and pH titrations. Starting from the grand partition function and using matrix methods we present a formalism that describes pH effects on the thermal stability of triple helices. The formalism can be used over a wide pH range and is not restricted to the limiting case where the pH is larger or smaller than the pK alpha of cytosine. Furthermore, it covers nearest neighbor electrostatic effects of closely spaced cytosines in the Hoogsteen strand which can shift the pK alpha of cytosine to lower pH values. A procedure is employed to predict enthalpy and entropy changes for triplex formation. These values are in accordance with the results obtained by differential scanning calorimetry.

Base Sequence↗
PubMed1994

Electrostatic effects in DNA triple helices.

Electrostatic effects dominate many aspects of nucleic acid behavior in a sequence independent manner. Sequence dependent electrostatic effects are introduced when a polypyrimidine, which contains one or more protonated cytosines, binds in the major groove (Hoogsteen side) of a complementary Watson-Crick double helix. Depending on the number of cytosines in the third strand (global effect) and on their relative position (local effect), the cytosines either enhance or decrease the binding affinity of the third strand, because adjacent protonated cytosines destabilize the third strand binding compared to cytosines separated by intervening thymines. This local effect (crowding) can reverse the effect of global composition. To investigate the extent of the local and global electrostatic effects further, two families of oligonucleotides have been synthesized. They share as a common design feature that they all fold sequentially into isosterical intramolecular triple helices by way of hairpin intermediates. This is confirmed by P1 nuclease probing, CD spectroscopy, and UV spectroscopy. The thermal stability of these conformations depends on the sequences, pH, and the ionic strength and can be summarized as follows: The energy of third strand binding depends on the protonated cytosine content in the Hoogsteen strand. It increases with increasing cytosine content (global composition) below pH 7.1 (150 mM Na+), decreases above pH 7.1, and is independent of the cytosine content at pH 7.1. At pH 6.75 the energy of binding increases with increasing cytosine content below 400 mM Na+, decreases above 400 mM Na+, and is independent of the global composition at 400 mM Na+.

Animals↗
PubMed1994

Unfolding of a branched double-helical DNA three-way junction with triple-helical ends.

We have designed three oligonucleotides (33 mers) which when mixed in a 1:1:1 ratio form double-helical DNA three-way junctions with triple helical ends in the pH interval pH 4 to 5.5. The triplex to coil transition is initiated by raising the temperature and was recorded by temperature gradient gel electrophoresis, uv melting, and differential scanning calorimetry. The transitions can be deconvoluted into three subtransitions representing the independent thermal denaturation of each of the arms. We have proposed a model for the unfolding pathway and give the thermodynamic parameters for each step as calculated using the formalism outlined in the appendix.

Base Sequence↗
PubMed1993

Characterization, cloning, and in vitro expression of the extremely thermostable glutamate dehydrogenase from the hyperthermophilic Archaeon, ES4.

Glutamate dehydrogenase (GDH) from the hyperthermophilic Archaeon ES4 (optimal growth temperature 98 degrees C and maximum growth temperature 110 degrees C) was purified to homogeneity. The purified native enzyme had an M(r) of 270,000 +/- 5,000 and was shown by gel filtration and SDS-polyacrylamide gel electrophoresis to be a hexamer with identical subunits of M(r) = 46,000 +/- 3,000. The hexameric subunit composition was also evident from electron micrographs, which show a triangular antiprism structure very similar to that of bovine GDH. The enzyme is exceptionally thermostable, with a half-time of inactivation of 3.5 h at 105 degrees C. Differential scanning calorimetry revealed a tm for denaturation of 113 degrees C, and a tm for activation at 60 degrees C. Antigenic cross-reaction with ES4 GDH was observed with the purified GDH from the thermophilic Archaea, Pyrococcus furiosus and Thermococcus litoralis as well as with bovine and yeast GDHs. The genome of ES4 was shown to contain a single copy of the gdhA gene, and this was cloned and sequenced. The deduced amino acid sequence of the GDH from ES4 corresponded to the NH2-terminal amino acid sequence obtained from the pure protein. From the nucleotide sequence the ES4 protein is composed of 420 residues. It has a relatively high hydrophobicity and a low number of sulfur-containing residues compared with mesophilic GDHs. Relatively high homology (52%) exists between the deduced amino acid sequence of ES4 GDH and Clostridium difficile GDH. Of the two distinct families of GDH sequences known, ES4 GDH belongs to the same family as vertebrates, C. difficile, and other Archaea. The gdhA gene of ES4 was expressed in vitro in a rabbit reticulocyte cell-free lysate, thus providing a system for structural studies of the mechanisms of thermostability in hyper-thermophilic proteins.

Amino Acid Sequence↗
PubMed1993

Energetics of Z-DNA formation in poly d(A-T), poly d(G-C), and poly d(A-C) poly d(G-T).

The conformational change for the alternating purine-pyrimidine polydeoxyribonucleotides i.e. poly d(A-T), poly d(G-C), and poly d(A-C) poly d(G-T) from a right-handed conformation at room temperature to the left-handed Z-DNA like double helix at elevated temperatures has been studied by UV spectroscopy, Raman spectroscopy, and by adiabatic differential scanning microcalorimetry (DSC) in the presence of Na+ and Mg2+ or Ni2+ respectively as counterions. The differential UV spectra reveal through a hyperchromic shift at around 280nm and a hypochromic shift at 260nm that a conformational change to the left-handed conformation occurs. The Raman spectra clearly show characteristic changes, a drastic decrease of the band at 680cm-1 and the appearance of a new band at 628cm-1, due to the change of the purine bases to the syn conformation upon inversion of the helix-handedness. The course of the transition as function of temperature can be followed quantitatively by plotting the change in the excess heat capacity vs. temperature. The transition enthalpy delta H for the B- to Z-DNA transition per mole base pairs (mbp) amounts to 2.0 +/- 0.2kcal for poly d(G-C), to 4.0 +/- 0.4kcal for poly d(A-T), and to 3.1 +/- 0.3kcal for poly d(A-C) poly d(G-T). The enthalpy change due to the Z-DNA to coil transitions (per mole base pairs) amounts to 11kcal for poly d(G-C), 10.5kcal for poly d(A-T) and 11.3kcal for poly d(A-C) poly d(G-T).

Calorimetry, Differential Scanning↗
PubMed1993

Energetics of a stable intramolecular DNA triple helix formation.

We have designed and synthesized by conventional chemical techniques a 38mer oligonucleotide consisting of a 5'd(Pu)10d(C)4d(Py)10d(T)4d(Py)10(3') sequence. This oligonucleotide assumes a randomly coiled conformation at pH 12. At pH 8.0 a hairpin helix forms between its 5' purine decamer sequence and the consecutive pyrimidine decamer leaving the second pyrimidine decamer as a dangling disordered 3' extension. On reducing the pH to 4.5 this second pyrimidine decamer folds back onto the major groove of the hairpin helix resulting in an intramolecular triple-stranded stem-loop structure. We have used a variety of biochemical (gel mobility, P1 nuclease digestion) and biophysical (ultraviolet light and circular dichroism spectroscopy, fluorimetry, microcalorimetry) techniques to characterize the different conformers, their stability and the folding pathway into an intramolecular triple helix. The thermodynamic properties of this intramolecular triple strand in 100 mM-Na+ are: tm, 71 degrees C; delta HvH, 119.4(+/- 11.9) kcal mol-1; delta Hcal, 121.9 (+/- 6.1) kcal mol-1 at pH 4.5; those of the hairpin are: tm, 63 degrees C; delta HvH, 71.7(+/- 4.0) kcal mol-1; delta Hcal, 69.9(+/- 3.5) kcal mol-1 at pH 8.0. At intermediate pH values, the triplex to coil transition breaks up into its component triplex to hairpin and hairpin to coil transitions with thermodynamic properties: tm, 41 degrees C; delta HvH, 58.7(+/- 4.2) kcal mol-1; delta Hcal, 39.8(+/- 2.0) kcal mol-1; and tm, 63 degrees C; delta HvH, 71.7(+/- 4.0) kcal mol-1; delta Hcal, 69.6(+/- 3.5) kcal mol-1 at pH 6.7.

Base Sequence↗
PubMed1993

A novel nucleosome assembly procedure (with a little help from pectin).

The acidic polysaccharide pectin (alpha-1,4-polygalacturonic acid) has been introduced as a nucleosome assembly facilitator as a substitute for polyglutamic acid. The pectin-assembled nucleosomes were indistinguishable from polyglutamic acid-assembled nucleosomes by thermal denaturation and DNAse I digestion. Pectin had two major advantages over polyglutamic acid-the yield of assembled cores was approximately 50% higher and the pectin could be easily removed after completion of the reassembly procedure by dialysis following pectinase cleavage.

Animals↗