NMR studies in solution of poly-L-proline

The isomerization of poly-L -proline in different solvents has been studied by NMR spectroscopy. Different resonance signals for the CH_α protons have been obtained for the two different helical conformations of thus compound, namely form I and form II.     

An infrared study on the conformation of poly-L-proline in concentrated aqueous salt solutions

The infrared absorption of poly-L -proline in concentrated aqueous salt solutions was measured in the fundamental region. Of primary interest were the carbonyl absorption of the peptide linkage and the methylene C–H bending absorption of the pyrrolidine ring. These spectral regions each show an additional component in the concentrated salt solutions. Using the position of the absorptions of poly-L -proline I (cis) and II (trans) as models, we conclude that both cis–trans linkages are present in the peptide in salt solutions. Increasing the temperature shifts the equilibrium slightly in favor of cis.     

Microcalorimetric study of polypeptides in the conformation of the left helix of the poly-L-proline II type

Microcalorimetry shows that polypeptide partial heat capacity changes linearly with temperature in the region of M-conformation existence and that there is a peculiarity in the transition region where this structure is exhausted, without cooperative heat absorption.     

Specific ion effects on macromolecular interactions in Escherichia coli extracts

Protein characterization in situ remains a major challenge for protein science. Here, the interactions of ΔTat‐GB1 in Escherichia coli cell extracts were investigated by NMR spectroscopy and size exclusion chromatography (SEC). ΔTat‐GB1 was found to participate in high molecular weight complexes that remain intact at physiologically‐relevant ionic strength. This observation helps to explain why ΔTat‐GB1 was not detected by in‐cell NMR spectroscopy. Extracts pre‐treated with RNase A had a different SEC elution profile indicating that ΔTat‐GB1 predominantly interacted with RNA. The roles of biological and laboratory ions in mediating macromolecular interactions were studied. Interestingly, the interactions of ΔTat‐GB1 could be disrupted by biologically‐relevant multivalent ions. The most effective shielding of interactions occurred in Mg²⁺‐containing buffers. Moreover, a combination of RNA digestion and Mg²⁺ greatly enhanced the NMR detection of ΔTat‐GB1 in cell extracts.     

Binding of alcohols to the peptide CO group of poly-L-proline in the I and II conformation. II. Binding constants from infrared measurements in solution

Trifluoroethanol, benzyl alcohol, and n-butanol bind to the peptide and acelyl CO groups of poly-O-acetyl-L -hydroxyproline in dichloromethane via hydrogen bonds. The binding aflinity decreases from trifhioroelhanol to n-buitanol. For the acelyl CO groups the binding does not depend on the conformation of the polymer but for the peptide CO groups the binding constants are larger by a factor of two to five time when it is in the helix II conformation (all peptide bonds trans) than when it assumes the helix I conformation (all peptide bonds cis). This preference is explained by the higher accessibility of the peptide CO groups in the II helix. The small additional energy which results from the preferential binding is sufficient, to induce a complete I → II transition because of the very high cooperativily of the system. The quantitative dependence of the equilibrium constant s for the propagation step of the transition on solvent composition (ratio of trifluoroethanol or benzyl alcohol to n-butanol) is derived from the binding data. It agrees satisfactorily with the empirical relation obtained from a best fit to transition curves of Ganseret al. The I ? II conversion of poly-L -proline is therefore an example of a conformational transition whose solvent dependence can be explained by a binding mechanism.     

Specific ion effects on the growth rates of Staphylococcus aureus and Pseudomonas aeruginosa

Motivated by recent advances in the physical and chemical basis of the Hofmeister effect, we measured the rate cell growth of S. aureus--a halophilic pathogenic bacterium--and of P. aeruginosa, an opportunistic pathogen, in the presence of different aqueous salt solutions at different concentrations (0.2, 0.6 and 0.9 M). Microorganism growth rates depend strongly on the kind of anion in the growth medium. In the case of S. aureus, chloride provides a favorable growth medium, while both kosmotropes (water structure makers) and chaotropes (water structure breakers) reduce the microorganism growth. In the case of P. aeruginosa, all ions affect adversely the bacterial survival. In both cases, the trends parallel the specific ion, or Hofmeister, sequences observed in a wide range of physico-chemical systems. The correspondence with specific ion effect obtained in other studies, on the activities of a DNA restriction enzyme, of horseradish peroxidase, and of Lipase A (Aspergillus niger) is particularly striking. This work provides compelling evidence for Hofmeister effects, physical chemistry in action, in these organisms.     

Effect of sodium ion concentration on transfer RNA conformation in solution studied by Rayleigh light scattering

The sodium ion concentration dependent conformational changes of transfer RNA (unfractionated tRNA from baker's yeast) have been studied in unbuffered aqueous solutions by Rayleigh light scattering. Changes of the optical parameters of the molecule indicated the following conformational changes of tRNA with increasing NaCl concentration: in salt-free solution tRNA molecules have an irregular hairpin loop-like structure in which the orientation of base rings is not correlated. Upon addition of a small amount of NaCl (0.005 M) an increasing ordering of this structure is observed. In 0.1 M-NaCl the molecule has an extended structure with ordered regions (arms). Further increase of sodium ion concentration up to 2 M results in folding of the extended structure and formation of a compact and rigid conformation in which most of the bases are nearly perpendicular to the symmetry axis of the molecule.     

Histone H1: Ultracentrifugation studies of the effects of ionic strength and denaturants on the solution conformation

The conformation of histone H1 has been examined under native and denaturing conditions in the absence of DNA or chromatin. Sedimentation coefficients were determined for Histone H1 in 0.1 m KCl and in 6 m guanidine hydrochloride solutions at pH 7.4. The influence of ionic strength on the conformation of histone H1 has been determined by measurement of the sedimentation coefficient in tetramethylammonium chloride solutions of up to 2.5 m and extrapolated to infinite ionic strength. Results from these experiments suggest that the native conformation of histone H1 is very asymmetric in shape. The molecule is best described as a prolate ellipsoid with axes of 312 Å (2a) and 16 Å (2b) in low ionic strength media and also as a prolate ellipsoid with axes of 202 Å (2a) and 20 Å (2b) at high ionic strength or when associated with polyanions, e.g., DNA. Denaturation of histone H1 by guanidine hydrochloride was found to be completely reversible. In 6 m guanidine hydrochloride, the H1 molecule collapses to a sphere but the original extended conformation of the protein is readily restored on dialysis. This suggests rigid conformational requirements for the H1 molecule as incorporated into chromatin. The shape and dimensions for the H1 molecule at high ionic strength are not sufficiently conclusive to locate H1 in the chromatin structure. It is proposed, however, that viable models for chromatin architecture must be consistent with the histone H1 solution dimensions obtained here.     

Structural flexibility and fast proton transfer reflected by the dielectric properties of poly-L-proline in aqueous solution

Dielectric dispersion curves for the helix-11 form of poly-L-proline in aqueous solution have been determined for various pH in the acid range of zwitterion formation. The results could be excellently described by means of a Cole-Cole dispersion function involving the three parameters Δ?⁰ (total dielectric increment), τ_r (effective rotational relaxation time) and h (characterizing the width of the dispersion region). The quantities τ_r, and h were found to be clearly independent of pH and added inert electrolyte. An analysis of the data permits an evaluation of the dipole moments and leads to the conclusion that the molecule cannot be considered to be a completely stretched rigid rod but must be more or less bent. Addition of formic acid slightly below pH 4 caused a distinct broadening of the experimental curves which could be quantitatively interpreted by a second dielectric relaxation process due to orientation of zwitterions by means of fast proton transfer.     

Specific inhibitory conformation of dipeptides for chymotrypsin

Based on the analyzed conformation of a chymotrypsin inhibitor H-delta Phe-Phe-OMe, we have designed a series of diastereomeric phenylalanylphenylalanine methyl esters and derivatives as possible inhibitors. Among the peptides synthesized, H-D-Phe-L-Phe-OMe was found to be very resistant to chymotrypsin in spite of its L-Phe-OMe structure at the C-terminus. It inhibited the enzyme fairly strongly and competitively with Ki = 9.0 x 10(-5) M in the assay using Ac-Tyr-OEt as a substrate. The measurements of the NOEs in high-resolution 1H-NMR analyses indicated the presence of the hydrophobic core built by the intramolecular interaction between the D-Phe-phenyl and ester-methyl groups. It was suggested that this core interacts with the chymotrypsin S2 site (Trp215) and Phe2 with the S1 site. The backbone structure of this dipeptide was assumed to be in an inhibitory conformation that fits the active center of the enzyme.     

蛋白质溶液构象的研究方法

蛋白质的结构决定其功能。研究蛋白质溶液构象的手段日新月异,文章介绍了近年来研究蛋白质在溶液中三级结构、二级结构和基团微环境的常用手段。     

Stereoelectronic effects on polyproline conformation

The polyproline type II (PPII) helix is a prevalent conformation in both folded and unfolded proteins, and is known to play important roles in a wide variety of biological processes. Polyproline itself can also form a type I (PPI) helix, which has a disparate conformation. Here, we use derivatives of polyproline, (Pro)10, (Hyp)10, (Flp)10, and (flp)10, where Hyp is (2S,4R)-4-hydroxyproline, Flp is (2S,4R)-4-fluoroproline, and flp is (2S,4S)-4-fluoroproline, to probe for a stereoelectronic effect on the conformation of polyproline. Circular dichroism spectral analyses show that 4R electron-with-drawing substituents stabilize a PPII helix relative to a PPI helix, even in a solvent that favors the PPI conformation, such as n-propanol. The stereochemistry at C4 ordains the relative stability of PPI and PPII helices, as (flp)10 forms a mixture of PPI and PPII helices in water and a PPI helix in n-propanol. The conformational preferences of (Pro)10 are intermediate between those of (Hyp)10/(Flp)10 and (flp)10. Interestingly, PPI helices of (flp)10 exhibit cold denaturation in n-propanol with a value of T(s) near 70 degrees C. Together, these data show that stereoelectronic effects can have a substantial impact on polyproline conformation and provide a rational means to stabilize a PPI or PPII helix.