Karin Kloiber

Direct observation of the dynamic process underlying allosteric signal transmission.

Allosteric regulation is an effective mechanism of control in biological processes. In allosteric proteins a signal originating at one site in the molecule is communicated through the protein structure to trigger a specific response at a remote site. Using NMR relaxation dispersion techniques we directly observe the dynamic process through which the KIX domain of CREB binding protein communicates allosteric information between binding sites. KIX mediates cooperativity between pairs of transcription factors through binding to two distinct interaction surfaces in an allosteric manner. We show that binding the activation domain of the mixed lineage leukemia (MLL) transcription factor to KIX induces a redistribution of the relative populations of KIX conformations toward a high-energy state in which the allosterically activated second binding site is already preformed, consistent with the Monod-Wyman-Changeux (WMC) model of allostery. The structural rearrangement process that links the two conformers and by which allosteric information is communicated occurs with a time constant of 3 ms at 27 degrees C. Our dynamic NMR data reveal that an evolutionarily conserved network of hydrophobic amino acids constitutes the pathway through which information is transmitted.

Synthesis of a 13C-methyl-group-labeled methionine precursor as a useful tool for simplifying protein structural analysis by NMR spectroscopy.

In the past decade NMR spectroscopy has emerged as one of the most powerful tools for the determination of protein structure and dynamics. The introduction of uniform N,C labeling, coupled with the development of triple-resonance experiments has permitted structural and dynamic studies of systems that are up to 20 kDa in molecular mass. The characterization of larger macromolecules requires techniques that maximize both spectral resolution and sensitivity due to chemical-shift overlap and increasing linewidth in high molecular-weight systems. Consequently, research has focused on correlation spectroscopy of side-chain methyl groups. New labeling techniques have been established by using a-ketoacids as biosynthetic precursors for the production of isotope-labeled proteins. Attention has therefore been turned towards the synthesis of a-ketobutyric and a-ketovaleric acid, biosynthetic precursors for the aliphatic amino acids isoleucine, as well as leucine and valine, respectively. NMR investigations of the trimeric E. coli enzyme IIA (34 kDa), the monomeric 82 kDa enzyme malate synthase G from E. coli, or studies of the protein p21-KID within a 45 kDa binary complex (p21-KID/Cdk2) are applications of these ACHTUNGTRENNUNGdevelopments. Furthermore, new methods for quantifying the amplitudes of motion of methyl-containing side chains have been developed, which are essential for a better understanding of dynamics and its relationship to function. Here we report a novel synthetic route towards a precursor of the S-C-methyl-bearing amino acid, methionine. The synthesis sequence is outlined in Scheme 1. Starting from easily accessible tert-butyl a-bromomethacrylate (1), homoallylic alcohol 2 was obtained through an indium mediated aqueous Barbier-type reaction between 1 and formaldehyde. The highest yields were obtained by using 4.5 mmol allyl ester (1), 9 mmol indium powder, and 36 mmol depolymerized paraformaldehyde in a mixture of THF and water (5:1) after 16 h of sonication. Primary alcohol 2 was converted to the corresponding tosylate 3 by using standard procedures. Treatment of 3 with potassium thioacetate in acetone gave thioacetyl-compound 4 after 6 h of sonication in excellent yield. Subsequent ozonolysis of the double bond at 78 8C in dichloromethane afforded 2-oxo-derivate 5. The a-keto functionality of 5 was further transformed into its N,N’-dimethylhydrazone 6. Although the thioester of 5 could have been cleaved by N,N’-dimethylhydrazine, the obviously greater reactivity of the a-keto functionality allowed for selective protection in high yield. All attempts to generate a thiol moiety starting from ester 6 by using a ZemplBn saponification protocol with sodium methanolate in dry methanol failed. Compound 7 was finally obtained by treatment of thioester 6 with ethylamine and silver triflate at 20 8C, followed by methylation of the thiolate with CH3I in THF. To prevent disulfide formation, silver triflate was applied in equimolar amounts; this pushed the reaction towards product formation due to silver iodide precipitation. Subsequently, the hydrazone of 7 was cleaved by using an aqueous HCl solution (1m) and THF to obtain a-ketoester 8. Hydrolysis of the tert-butyl ester 8 was performed in a mixture of an aqueous HCl solution (0.025m) and acetonitrile to yield the target precursor compound 9 in 32% overall yield (starting from 1). [a] M. Fischer, Prof. J. H usler, Prof. W. Schmid Institute of Organic Chemistry, University of Vienna W hringer Strasse 38, 1090 Vienna (Austria) Fax: (+43)1-4277-9 521 E-mail : walther.schmid@univie.ac.at

Automated NMR determination of protein backbone dihedral angles from cross-correlated spin relaxation

The simultaneous interpretation of a suite of dipole-dipole and dipole-CSA cross-correlation rates involving the backbone nuclei 13Cα, 1Hα,13CO, 15N and 1HN can be used to resolve the ambiguities associated with each individual cross-correlation rate. The method is based on the transformation of experimental cross-correlation rates via calculated values based on standard peptide plane geometry and solid-state 13CO CSA parameters into a dihedral angle probability surface. Triple resonance NMR experiments with improved sensitivity have been devised for the quantification of relaxation interference between 1Hα(i)-13Cα(i)/15N(i)-1HN(i) and 1Hα(i−1)-13Cα(i−1)/15N(i)-1HN(i) dipole-dipole mechanisms in 15N,13C-labeled proteins. The approach is illustrated with an application to 13C,15N-labeled ubiquitin.

A Novel Paramagnetic Relaxation Enhancement Tag for Nucleic Acids: A Tool to Study Structure and Dynamics of RNA

In this work, we present a novel 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) radical phosphoramidite building block, which can be attached to the 5′-terminus of nucleic acids. To investigate the paramagnetic relaxation enhancement (PRE) emanating from this radical center, we incorporated the TEMPO label into various types of RNAs. We measured proton PREs for selectively 13C-isotope labeled nucleotides to derive long-range distance restraints in a short 15 nucleotide stem–loop model system, underscoring the potential of the 5′-TEMPO tag to determine long-range distance restraints for solution structure determination. We subsequently applied the distance-dependent relaxation enhancement induced by the nitroxide radical to discern two folding states in a bistable RNA. Finally, we investigated the fast conformational sampling of the HIV-1 TAR RNA, a paradigm for structural flexibility in nucleic acids. With PRE NMR in combination with molecular dynamics simulations, the structural plasticity of this RNA was analyzed in the absence and presence of the ligand l-argininamide.

Autocorrelation Analysis of NOESY Data Provides Residue Compactness for Folded and Unfolded Proteins

A novel spectral entropy interpretation for protein NOESY data is presented for the investigation of the spatial distribution of residues in protein structures without the requirement of NOE cross peak assignments. In this approach individual traces S(i)(omega) from a 3D (15)N NOESY-HSQC taken at frequency positions corresponding to different amide groups (residue position i) are subjected to a self-convolution procedure thus leading to the autocorrelation function C(i)(omega) of the NOESY-trace for a particular backbone residue position. The characteristic spatial surrounding of a particular residue position is reflected in the corresponding autocorrelation function and can be quantified by taking the (spectral) entropy S(nu) as an information measure. The feasibility of this novel approach is demonstrated with applications to the proteins Cyclophilin D and Osteopontin and the protein complex between the lipocalin Q83 and the bacterial siderophore Enterobactin. Typically, large entropy values were found for residues located in structurally loosely defined regions, whereas small entropy values were found for residues in hydrophobic core regions of the protein with tightly interacting side chains and distinct chemical shift patterns. The applications to the unfolded Osteopontin and the Q83/Enterobactin protein complex indicated that both local compaction of the polypeptide chain due to transiently formed structural elements and subtle changes in side-chain packing can be efficiently probed by this novel approach.

Measurement of the protein backbone dihedral angle phi based on quantification of remote CSA/DD interference in inter-residue 13C'(i - 1)-13Calpha(i) multiple-quantum coherences.

A novel triple-resonance NMR method is presented for the measurement of the protein backbone dihedral angle φ based on differential multiple-quantum relaxation induced by relaxation interference between 1Hα(i)-13Cα(i) dipolar and 13C′(i−1) (carbonyl) chemical shift anisotropy mechanisms. The method employs a simultaneous transfer of 15N magnetization to the inter- and intra-residue 13Cα carbons as well as the directly attached carbonyl carbon 13C′. Results obtained on 13C,15N-labeled ubiquitin demonstrate the potential of the method.

Pharmacophore mapping via cross-relaxation during adiabatic fast passage.

A novel NMR method is demonstrated for the investigation of protein ligand interactions. In this approach an adiabatic fast passage pulse, i.e. a long, weak pulse with a linear frequency sweep, is used to probe (1)H-(1)H NOEs. During the adiabatic fast passage the effective rotating-frame NOE is a weighted average of transverse and longitudinal cross-relaxation contributions that can be tuned by pulse power and frequency sweep rate. It is demonstrated that the occurrence of spin diffusion processes leads to sizable deviations from the theoretical relationship between effective relaxation rate and effective tilt angle in the spin lock frame and can be used to probe protein-ligand binding. This methodology comprises high sensitivity and ease of implementation. The feasibility of this technique is demonstrated with two protein complexes, vanillic acid bound to the quail lipocalin Q83 and NAD(+) and AMP binding to alcohol dehydrogenase (ADH).

Mathematical treatment of adiabatic fast passage pulses for the computation of nuclear spin relaxation rates in proteins with conformational exchange

Although originally designed for broadband inversion and decoupling in NMR spectroscopy, recent methodological developments have introduced adiabatic fast passage (AFP) pulses into the field of protein dynamics. AFP pulses employ a frequency sweep, and have not only superior inversion properties with respect to offset effects, but they are also easily implemented into a pulse sequence. As magnetization is dragged from the +z to the −z direction, Larmor precession is impeded since magnetization becomes spin-locked, which is a potentially useful feature for the investigation of microsecond to millisecond dynamics. A major drawback of these pulses as theoretical prediction is concerned, however, results from their time-dependent offset: simulations of spin density matrices under the influence of a time-dependent Hamiltonian with non-commuting elements are costly in terms of computational time, rendering data analysis impracticable. In this paper we suggest several ways to reduce the computational time without compromising accuracy with respect to effects such as cross-correlated relaxation and modulation of the chemical shift.

Longitudinal exchange: an alternative strategy towards quantification of dynamics parameters in ZZ exchange spectroscopy

Longitudinal exchange experiments facilitate the quantification of the rates of interconversion between the exchanging species, along with their longitudinal relaxation rates, by analyzing the time-dependence of direct correlation and exchange cross peaks. Here we present a simple and robust alternative to this strategy, which is based on the combination of two complementary experiments, one with and one without resolving exchange cross peaks. We show that by combining the two data sets systematic errors that are caused by differential line-broadening of the exchanging species are avoided and reliable quantification of kinetic and relaxation parameters in the presence of additional conformational exchange on the ms–μs time scale is possible. The strategy is applied to a bistable DNA oligomer that displays different line-broadening in the two exchanging species.

Kinetics of DNA Refolding from Longitudinal Exchange NMR Spectroscopy

Nucleic acids have been attracting a lot of attention due to the discovery that they can fulfill tasks beyond transforming the genetic information that is stored in the genome into proteins. Such noncoding ribonucleic acids were discovered a few years ago, and their function in cellular processes is currently being addressed by a variety of experimental approaches. High-resolution structural studies of riboswitch RNAs, for example, have provided promising starting points for the development of new antibiotics. Notably, the gene regulation mechanisms of riboswitch RNAs are based on their propensity to populate alternative folds with (nearly degenerate) free energies that are modulated in response to external stimuli. Thus, the ability of a particular RNA sequence to specifically recognize small molecules and to populate multiple folds enables it to fulfill a gene regulatory function. Notwithstanding, DNA should be able to accomplish similar tasks as such RNA sequences. This hypothesis is supported by the fact that in vitroselected DNA enzymes and aptamers with similar or even identical functionalities as their RNA counterparts are known. Although substantial effort is put into the discovery of naturally occurring DNA catalysts and aptamers, so far no such species have been reported. Yet, such DNA-based systems are conceivable and could fulfill a biological function or, on the other hand, be implemented in a molecular biology toolbox for the control of gene expression. There are some examples of conformational heterogeneity of DNA. For example, human telomeric repeats are able to coexist in multiple G quadruplex forms, which potentially exert different functions depending on the conformational state of the quadruplex. In addition, during homologous recombination, DNA undergoes a strand exchange process to form a Holliday junction. Again, this four-way junction displays conformational heterogeneity with potential functional implications for the processing of the Holliday junction during recombination. While such conformational heterogeneity and secondary structure rearrangements have been investigated in detail for RNA, DNA secondary structural heterogeneity was addressed only recently by using NMR spectroscopic techniques. NMR spectroscopy has proven to be well suited for detecting and quantifying distinct folding states of nucleic acids, and for determining the kinetics and the thermodynamics of the refolding process. In this work we present a site-specific Cisotope labeling procedure for DNA and use it to characterize secondary structure heterogeneity of a bistable DNA sequence. We introduce isolated C-spin labels into the methyl groups of thymidines that can be employed to detect and quantify dynamic properties using relaxation-based NMR techniques for CH3-spin systems. [9] Our strategy involves the synthetic preparation of a CH3-modified thymidine phosphoramidite building block, which is subsequently inserted into DNA by using standard solid-phase synthesis. We prepared various C-methyl labeled variants of a bistable DNA model system to investigate the kinetics and thermodynamics of the refolding transition between the two folding states using C-longitudinal exchange (ZZ) NMR spectroscopy at various temperatures. The results are analyzed and discussed in relation to data from analogous bistable RNA systems. Two synthesis routes for the preparation of the CH3-thymidine labeled phosphoramidite building block 5 have been described. Both routes use a lithation step and subsequent treatment with C-methyl iodide to yield the key intermediate. We basically employed a slight modification of the more recent synthesis route of Bergstrom et al. to give the Cmodified thymidine nucleoside 3, which was then converted into the thymidine phosphoramidite 5 using standard procedures (Scheme 1). The synthesis route was started from commercially available 2’-deoxy uridine by treating the hydroxyl