Practical Methods for Solid-State NMR Distance Measurements on Large Biomolecules: Constant-Time Rotational Resonance

Simple modifications of the rotational resonance experiment substantially reduce the total experimental time needed to measure weak homonuclear dipolar couplings, a critical factor for achieving routine internuclear distance measurements in large biomolecular systems. These modifications also address several problems cited in the literature. Here we introduce a constant-time rotational resonance experiment that eliminates the need for control spectra to correct for effects from variable RF heating, particularly critical for accurate long-distance measurements. This reduces the total number of experiments needed by as much as a factor of 2. Other improvements incorporated include achieving selective inversion with a delay rather than a weak pulse (P. R. Costa et al., J. Am. Chem. Soc. 119, 10487-10493, 1997), which we observe results in the elimination of oscillations in peak intensities for short mixing time points. This reduces the total experiment time in two ways. First, there is no longer a need to average different "zero"-time points (S. O. Smith et al., Biochemistry 33, 6334-6341, 1994) to correct for intensity variations. Second, short-mixing-time lineshape differences observed in large membrane-bound proteins only appear with the weak-pulse inversion and not when using the delay inversion. Consistent lineshapes between short and long mixing times permit the use of a single spectrum for subtraction of natural abundance background signals from all labeled-protein time points. Elimination of these effects improves the accuracy and efficiency of rotational resonance internuclear distance measurements.     

Determination of Multiple φ-Torsion Angles in Proteins by Selective and Extensive C Labeling and Two-Dimensional Solid-State NMR

We describe an approach to efficiently determine the backbone conformation of solid proteins that utilizes selective and extensive ¹³C labeling in conjunction with two-dimensional magic-angle-spinning NMR. The selective ¹³C labeling approach aims to reduce line broadening and other multispin complications encountered in solid-state NMR of uniformly labeled proteins while still enhancing the sensitivity of NMR spectra. It is achieved by using specifically labeled glucose or glycerol as the sole carbon source in the protein expression medium. For amino acids synthesized in the linear part of the biosynthetic pathways, [1-¹³C]glucose preferentially labels the ends of the side chains, while [2-¹³C]glycerol labels the C^α of these residues. Amino acids produced from the citric-acid cycle are labeled in a more complex manner. Information on the secondary structure of such a labeled protein was obtained by measuring multiple backbone torsion angles φ simultaneously, using an isotropic–anisotropic 2D correlation technique, the HNCH experiment. Initial experiments for resonance assignment of a selectively ¹³C labeled protein were performed using ¹⁵N–¹³C 2D correlation spectroscopy. From the time dependence of the ¹⁵N–¹³C dipolar coherence transfer, both intraresidue and interresidue connectivities can be observed, thus yielding partial sequential assignment. We demonstrate the selective ¹³C labeling and these 2D NMR experiments on a 8.5-kDa model protein, ubiquitin. This isotope-edited NMR approach is expected to facilitate the structure determination of proteins in the solid state.     

Protein‐Affinitive Polydopamine Nanoparticles as an Efficient Surface Modification Strategy for Versatile Porous Scaffolds Enhancing Tissue Regeneration

Porous scaffolds for tissue regeneration are often functionalized with extracellular matrix proteins to enhance surface/cell interactions and tissue regeneration. However, continuous coatings produced by commonly used surface modification strategies may preclude cells from contacting and sensing the chemical and physical cues of the scaffold. Here, it is shown that polydopamine nanoparticles (PDA‐NPs) tightly adhere on various scaffolds to form nanostructures, and the coverage can be finely tuned. Furthermore, the PDA‐NPs have good affinity to a variety of proteins and peptides. Thus, the PDA‐NPs act as an anchor to immobilize signal biomolecules on scaffolds, and consequently promote cell activity and tissue regeneration. β‐Tricalcium phosphate (TCP) scaffolds decorated with PDA‐NPs demonstrate excellent osteoinductivity and bone‐regeneration performance due to the protein affinity of PDA‐NPs and the intrinsic bioactive characteristics of TCP scaffolds. In summary, PDA‐NPs with excellent affinity for protein adhesion represent a versatile platform to modify porous scaffolds while not compromising the biological functions of the scaffolds, and might have potential applications in tissue regeneration.     

Surface‐enhanced Raman scattering study of monolayers formed from mixtures of 4–mercaptobenzoic acid and various aromatic mercapto‐derivative bases

Monolayers formed from aromatic compounds have many potential applications, for example in construction of bioelectronic elements having high efficiency of electron transfer. In this paper, the composition of monolayers formed on silver surfaces from mixtures of 4‐mercaptobenzoic acid (MBA) and four model (stable and easily available) aromatic thiols with strong base properties: 4,6‐diamino‐2‐mercaptopyrimidine (APY), 1H‐1,2,4‐triazole‐3‐thiol (HTR), 4‐methyl‐4H‐1,2,4‐triazole‐3‐thiol (MTR) and 3‐amino‐1,2,4‐triazole‐5‐thiol (ATR), were determined from surface‐enhanced Raman scattering (SERS) measurements. Our investigations showed that among studied aromatic bases, APY is the most promising candidate for the formation of mixed monolayers with MBA. In the whole pH range studied (2–12.5), mixed MBA + APY monolayers with similar surface concentration of both components are formed during the adsorption from the 0.5 mM MBA + 0.5 mM APY aqueous solution. Desorption of MBA and APY from the mixed monolayer is, however, significantly different. During immersion in water, surface concentration of APY decreases significantly faster than MBA (a significant part of the adsorbed MBA molecules is present on the silver surface even after 2 h of soaking in water). Presence of chlorides, bovine serum albumin or laccase in the surrounding solution does not observably influence the structure of MBA + APY monolayers. The properties of monolayers formed from MBA and substituted triazoles were found to be significantly different than those of MBA + APY monolayers. Copyright © 2009 John Wiley & Sons, Ltd.     

超高压处理对荔枝果肉中两种酶和可溶性蛋白的影响

 为了研究超高压处理对荔枝果肉中过氧化物酶（POD）、果胶甲基酯酶（PME）及可溶性蛋白含量的影响,将荔枝（“淮枝”品种）果肉在100~400 MPa压力、10 ℃温度条件下处理30 min,采用分光光度法测定果肉中POD、PME的活性,用天然凝胶电泳法以及活性染色法测定POD、PME同工酶的变化,用SDS凝胶电泳法测定果肉中可溶性蛋白含量。结果表明：100~200 MPa超高压处理使荔枝果肉中POD活性上升且出现新的同工酶,300~400 MPa超高压处理则使其活性降低且新出现的同工酶消失;100 MPa压力处理使荔枝果肉中PME活性上升且出现新的同工酶,200~400 MPa超高压处理则使其活性降低且新出现的同工酶消失;100 MPa超高压处理使荔枝果肉中可溶性蛋白含量有所增加,而在200~400 MPa压力处理下其含量持续下降。     

Intrinsic Fluorescence of Actin

In this work actin is used to illustrate connection of protein fluorescence characteristics with its structure. On one hand, it has been demonstrated what kind of information about the contribution of each tryptophan residues to the bulk fluorescence spectrum can be obtained from the special analysis of protein three-dimensional structure. On the other hand, potentials of intrinsic fluorescence for elucidation of proteins structure, dynamics and processes of folding-unfolding are shown. In particular, using this method a new essentially unfolded kinetic intermediate state of actin was detected and characterized, and the place of inactivated actin and its kinetic predecessor in the process of folding-unfolding was determined. It has been revealed that inactivated actin is not intermediate state between the native and completely unfolded states, as it has been accepted before, but a result of protein misfolding. On the basis of the obtained data a new model of actin folding-unfolding pathway has been proposed.     

Bubbles in DNA by random force

We model single strand binding (SSB) proteins as agents exerting randomly oriented force on the bonds in DNA unzipping. The fluctuating force is found to unzip the double stranded DNA (dsDNA) via opening of bubbles along the chain.     

残留偶极耦合及其在蛋白质结构研究中的应用

近年来溶液中残留偶极耦合常数被用来获取生物大分子化学键之间相对取向等长程构象约束条件,用于计算或优化蛋白质及其复合物的三维空间结构. 介绍了用异核多维NMR技术测量残留偶极耦合常数的方法,及其在蛋白质结构计算中的一些应用：优化蛋白质溶液结构,评价蛋白质结构质量,确定蛋白质结构域取向,获取有关配体的构象和取向的信息,在缺乏NOE数据时构建蛋白质结构等.      

Scanning probe microscopy characterization of gold-chemisorbed poplar plastocyanin mutants

Two poplar plastocyanin mutants adsorbed onto gold electrodes have been characterized at single molecule level by scanning probe microscopy. Immobilization of the two redox metalloprotein mutants on Au(1 1 1) surface was achieved by either a disulphide bridge (PCSS) or a single thiol (PCSH), both the anchoring groups having been introduced by site-directed mutagenesis. Scanning tunneling microscopy (STM) and atomic force microscopy (AFM) analysis gives evidence of a stable and robust binding of both mutants to gold. The lateral dimensions, as estimated by STM, and the height above the gold substrate, as evaluated by AFM, of the two mutants well agree with crystallographic sizes. A narrower height distribution is observed for PCSS compared to PCSH, corresponding to a more homogeneous orientation of the former mutant adsorbed onto gold. Major differences between the mutants are observed by electrochemical STM. In particular, the image contrast of adsorbed PCSS is affected by tuning the external electrochemical potential to the redox levels of the mutant, consistent with some involvement of copper active site in the tunneling process. On the contrary, no contrast variation is observed in electrochemical STM of adsorbed PCSH. Moreover, scanning tunneling spectroscopy experiments reveal asymmetric I–V characteristics for single PCSS proteins, reminiscent of a rectifying-like behaviour, whereas an almost symmetric I–V relation is observed for PCSH.     

Powder diffraction from a continuous microjet of submicrometer protein crystals

Atomic‐resolution structures from small proteins have recently been determined from high‐quality powder diffraction patterns using a combination of stereochemical restraints and Rietveld refinement [Von Dreele (2007), J. Appl. Cryst. 40 , 133–143; Margiolaki et al. (2007), J. Am. Chem. Soc. 129 , 11865–11871]. While powder diffraction data have been obtained from batch samples of small crystal‐suspensions, which are exposed to X‐rays for long periods of time and undergo significant radiation damage, the proof‐of‐concept that protein powder diffraction data from nanocrystals of a membrane protein can be obtained using a continuous microjet is shown. This flow‐focusing aerojet has been developed to deliver a solution of hydrated protein nanocrystals to an X‐ray beam for diffraction analysis. This method requires neither the crushing of larger polycrystalline samples nor any techniques to avoid radiation damage such as cryocooling. Apparatus to record protein powder diffraction in this manner has been commissioned, and in this paper the first powder diffraction patterns from a membrane protein, photosystem I, with crystallite sizes of less than 500 nm are presented. These preliminary patterns show the lowest‐order reflections, which agree quantitatively with theoretical calculations of the powder profile. The results also serve to test our aerojet injector system, with future application to femtosecond diffraction in free‐electron X‐ray laser schemes, and for serial crystallography using a single‐file beam of aligned hydrated molecules.