James A. Ferretti

Rapid scan Fourier transform NMR spectroscopy

Abstract The theoretical basis for the use of cross correlation to extract an undistorted spectrum from a rapid scan response (as first suggested by Dadok) is examined in detail. It is shown that the desired information can be obtained either by cross correlating the rapid scan responses of the unknown and a reference consisting of only a single sharp NMR line or alternatively by applying to the Fourier transformed response an appropriate analytical function. Practical considerations in the use of rapid scan FT-NMR are explored, and illustrative examples are given. The method compares favorably with pulse FT methods in sensitivity and, in some instances, has distinct advantages over the pulse technique.

UEAITR: A NEW COMPUTER PROGRAM FOR ANALYSIS OF NMR SPECTRA: ANALYSIS OF THE PROTON SPECTRUM OF TRIISOPROPYLPHOSPHINE.

Abstract A new computer program for the analysis of NMR spectra is reported. It adds iterative evaluation of input parameters to an earlier program (UEANMR II) which makes use of magnetic equivalence factoring to reduce the size of certain matrices. The present program, by iterating on line frequencies rather than energy levels, and by using magnetic equivalence factoring, offers advantages of speed and convenience over previously described computer programs for NMR analysis. The analysis of the proton spectrum of triisopropylphosphine is described as an example and the trend of 2 J H - P with increased alkyl substitution is found to be opposite to that of the trend of 2 J H - X for all other hetero-atoms, X , reported in the literature.

The choice of optimal parameters for measurement of spin-lattice relaxation times. II. Comparison of saturation recovery, inversion recovery, and fast inversion recovery experiments

Abstract Using the theory developed in Part I of this series (1) we determine the optimum pulse spacings using saturation recovery, inversion recovery, fast inversion recovery, and Freeman-Hill techniques for the measurement of spin-lattice relaxation times, T1, for conditions of ideal 90° and 180° pulses. We also compare the performance of these techniques, using as the criterion the total experimental time required to reach a specified precision in the estimate of T1. The important assumptions made are (1) that an interval of uncertainty is known for the value of T1, i.e., that the experimenter has a priori knowledge of an interval (TA, TB) in which T1 lies, and (2) that both the equilibrium magnetization M(∞) and T1 are unknown. For the conditions that we consider we find that fast inversion recovery is the most efficient technique in obtaining a fixed precision in the estimate of T1. For any interval of uncertainty the waiting time W = 2 TB proves to be optimal or near optimal. For the ideal 90° and 180° pulse situation, we find that over a wide range of uncertainty intervals the inversion recovery technique is always quicker than saturation recovery in obtaining a fixed precision in the estimate of T1. Analyses that make preliminary estimates of the equilibrium magnetization are found to be slower than those that determine T1 and M(∞) simultaneously from a set of data.

A modified fast inversion-recovery technique for spin-lattice relaxation measurements☆

Abstract A modified version of the fast inversion-recovery (FIR) method for measuring spin-lattice relaxation times is proposed. Whereas the FIR method employs a fixed time W between successive 180°, τ, 90°, sequences, the modified FIR method uses a fixed value of δ = W + τ , so that W decreases as τ increases. Both FIR and modified FIR methods are shown to be capable of nearly equal precision under conditions of ideal 180 and 90° pulses. With imperfect pulses, however, the modified FIR method circumvents certain systematic errors inherent in the FIR method. Conditions under which such errors might become significant are discussed.

The choice of optimal parameters for measurement of spin-lattice relaxation times. I. Mathematical formulation

Abstract This paper, the first in a series on optimization of T 1 measurements, describes the mathematical procedures for the analysis. Detailed results are presented in papers to follow. We assume here that a least-squares analysis is used to process magnetization data and that a first-order analysis is sufficient for practical values of the signal-to-noise ratio. In contrast to earlier analyses, we assume an initial uncertainty in the a priori knowledge of T 1 , specifically that T 1 is known to be between T A and T B . We then calculate σ( T 2 ) T 1 , i.e., the dimensionless standard deviation of the estimate of T 1 . The optimum set of measurement times τ 1 , τ 2 , …, τ 3 ., is then defined to be that which minimizes the maximum of σ( T 1 ) T 1 for T 1 in the interval ( T A , T B ). The overall objective is that of obtaining maximum precision in the shortest time. We show how to calculate the optimum number of experiments required to achieve a specified precision and how to calculate the associated total spectrometer time. Finally, we calculate σ(T 1 ) T 1 , for experiments in which separate measurements are made of the equilibrium magnetization, M (∞).

Germ-Line Mutation of NKX3.1 Cosegregates with Hereditary Prostate Cancer and Alters the Homeodomain Structure and Function

NKX3.1, a gene mapped to 8p21, is a member of the NK class of homeodomain proteins and is expressed primarily in the prostate. NKX3.1 exerts a growth-suppressive and differentiating effect on prostate epithelial cells. Because of its known functions and its location within a chromosomal region where evidence for prostate cancer linkage and somatic loss of heterozygosity is found, we hypothesize that sequence variants in the NKX3.1 gene increase prostate cancer risk. To address this, we first resequenced the NKX3.1 gene in 159 probands of hereditary prostate cancer families recruited at Johns Hopkins Hospital; each family has at least three first-degree relatives affected with prostate cancer. Twenty-one germ-line variants were identified in this analysis, including one previously described common nonsynonymous change (R52C), two novel rare nonsynonymous changes (A17T and T164A), and a novel common 18-bp deletion in the promoter. Overall, the germ-line variants were significantly linked to prostate cancer, with a peak heterogeneity logarithm of odds of 2.04 (P = 0.002) at the NKX3.1 gene. The rare nonsynonymous change, T164A, located in the homeobox domain of the gene, segregated with prostate cancer in a family with three affected brothers and one unaffected brother. Importantly, nuclear magnetic resonance solution structure analysis and circular dichroism studies showed this specific mutation to affect the stability of the homeodomain of the NKX3.1 protein and decreased binding to its cognate DNA recognition sequence. These results suggest that germ-line sequence variants in NKX3.1 may play a role in susceptibility to hereditary prostate cancer and underscore a role for NKX3.1 as a prostate cancer gatekeeper.

Conformational Analysis of the Tachykinins in Solution: Substance P and Physalaemin

A determination of the solution conformational behavior of two tachykinins, substance P and physalaemin, is described. Two-dimensional homonuclear Hartmann-Hahn (HOHAHA) and rotating-frame cross relaxation spectroscopy (ROESY) are used to obtain complete proton resonance assignments. Interproton distance restraints obtained from ROESY spectroscopy are used to characterize the conformational behavior. These data show that in solution both substance P and physalaemin exist in a mixture of conformational states, rather than as a single three-dimensional structure. In water both peptides prefer to be in an extended chain structure. In methanol, their behavior is described as a mixture of beta-turn conformations in dynamic equilibrium. Solvent titration data and chemical shift temperature coefficients complement the NMR estimate of interproton distances by locating hydrogen bonds and serving to identify predominant conformational states. The C-terminal tetrapeptide segment has the same conformational behavior for both substance P and physalaemin. In physalaemin, the midsegment of the peptide may also be constrained by formation of a salt bridge. The conformational behavior of substance P and physalaemin is discussed in relation to potency and receptor binding properties.

Role of interactions at the lipid-water interface for domain formation

The lipid-water interface is critical for the packing of lipid molecules in membranes. We have demonstrated that lateral phase separation in membranes can be driven by electrostatic interactions such as those involving charged lipid species and oppositely charged peptides, in addition to hydration effects at the lipid-water interface. By using nuclear magnetic resonance (NMR), circular dichroism and fluorescence spectroscopy we have shown that binding of a 21-amino acid peptide containing six positively charged arginine residues to mixed phosphatidylcholine (PC)/phosphatidylglycerol (PG) membranes results in a conformational change in the peptide from a random coil to a helical structure and causes the formation of domains of negatively charged PG. Binding of the peptide to PG membranes disorders the lipid hydrocarbon chains. The strength of lipid-peptide binding at the interface, the conformational change in the peptide, and domain formation with the negatively charged lipid are coupled energetically. The lipid-peptide association constant is lower for membranes containing 20 mol% PG in PC/PG mixtures than for 100% PG membranes. We suggest that one of the factors that lower the association constant in PC/PG membranes is entropic energy of formation of PG domains. Besides electrostatic interactions, hydration of lipids is important for domain formation. We have shown that dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylethanolamine separate under conditions of decreased water activity. Furthermore, water activity controls lipid packing stress in the hydrocarbon core and the headgroups of membranes as demonstrated by induction of an inverse-hexagonal-to-lamellar phase transition in dioleoylphosphatidylethanolamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Site-directed Mutations in the vnd/NK-2 Homeodomain BASIS OF VARIATIONS IN STRUCTURE AND SEQUENCE-SPECIFIC DNA BINDING

Secondary structures, DNA binding properties, and thermal denaturation behavior of six site-directed mutant homeodomains encoded by the vnd/NK-2 gene from Drosophila melanogaster are described. Three single site H52R, Y54M, and T56W mutations, two double site H52R/T56W and Y54M/T56W mutations, and one triple site H52R/Y54M/T56W mutation were investigated. These positions were chosen based on their variability across homeodomains displaying differences in secondary structure and DNA binding specificity. Multidimensional NMR, electrophoretic mobility shift assays, and circular dichroism spectropolarimetry studies were carried out on recombinant 80-amino acid residue proteins containing the homeodomain. Position 56, but more importantly position 56 in combination with position 52, plays an important role in determining the length of the recognition helix. The H52R mutation alone does not affect the length of this helix but does increase the thermal stability. Introduction of site mutations at positions 52 and 56 in vnd/NK-2 does not modify their high affinity binding to the 18-base pair DNA fragment containing the vnd/NK-2 consensus binding sequence, CAAGTG. Site mutations involving position 54 (Y54M, Y54M/T56W, and H52R/Y54M/T56W) all show a decrease of 1 order of magnitude in their binding affinity. The roles in structure and sequence specificity of individual atom-atom interactions are described.

Solid-phase synthesis of viscosin, a cyclic depsipeptide with antibacterial and antiviral properties

Viscosin, a cyclic depsipeptide (containing a peptide lactone) has been prepared by solid-phase chemistry using an Fmoc-protocol starting with an acid sensitive resin. Cyclization with the activating agent BOP-CI gave a product which was indistinguishable from natural viscosin thereby supporting the proposed structure.