Protein and Peptide Letters - Volume 18, Issue 4, 2011

It is well known that the primary sequence of a protein governs its three dimensional (3D) structure [1], on which, we suppose, molecular behavior and biological function of the protein are relied. It is more than 50 years since the first protein structure, hemoglobin, was solved in atomic resolution by x-ray crystallography [2]; people believe that the static structure of a protein is the fundamental for its unique function. With the advance of modern biophysical techniques, the efficiency for solving protein structures is accelerating dramatically to a speed beyond our expectation. Up to date, there are about 67, 000 structures deposited in the protein data bank (PDB) (http://www.rcsb.org/pdb). However, how a protein exerts its biological function is still a mystery to biologists. We have very limited information about the relationship between protein function and its structure, not even can we predict the function of a protein from its 3D structure. Protein structure is intrinsically dynamic in solution or solution-membrane interfaces [3]. For a protein, there are actually a large number of structures in solution [4]. We usually determine the time-averaged structure for a protein in solution by NMR [5] or a defined rigid structure in crystal by x-ray crystallography [2]. Simultaneously we often disregard the structural motion of a protein or its ensemble of conformations in solution, which may be critical to protein function and diversity. The dynamical structure is on a wide range of timescales [6], normally from pico- to kilo-seconds, reflecting respectively to the libration of chemical bonds, rotation of side-chains, fluctuation of backbones, reorientation of domains, and overall tumbling of global proteins. There is no doubt that protein structural motions relate to its biological function, such as protein-protein interaction and enzymatic catalysis [7]. This hot topic issue is aimed to provide a recent update on protein dynamics, which is the fundamental for protein functionality. With the advent of post-genomic era, a myriad of protein structures have been elucidated at atomic level, exploring their dynamical behaviors in solution is of great significance in understanding of the diverse functions of proteins [8]. The forthcoming review articles focus on the recent progresses in the dynamical aspects of proteins and their putative functional relationships including principles and methodology. Yan & Ji review the current research on the conformational dynamics and catalytic mechanism of 6-hydroxymethyl- 7,8-dihydropterin pyrophosphokinase, while Doucet discusses NMR relaxation dispersion method to characterize protein motion for enzyme engineering. The recently proposed models for the membrane-mediated protein interactions are reviewed by Armstrong et al. Many proteins are intrinsically flexible or partially disordered; the studies by Wang focus on the dynamics of amyloid β monomer by NMR techniques, while the flexible linkers of protein structures that may be pivotal to self-assembly of nanotubes are also reviewed by Buch et al. Some biophysical techniques have been developed to characterize the protein dynamics in different timescales. Among them, NMR spectroscopy is most versatile to study protein dynamics almost at all range of timescales. Sze & Lai review the recent progress in analyzing protein backbone dynamics by various NMR techniques, while Yang summarizes the methodology for side-chain dynamics of proteins by 13C NMR relaxation. Finally, I must reiterate that the fourth dimension, time, might be added to structural biology, and it is the time to explore the structure-dynamics-function relationships of proteins in all aspects of life sciences. I hope that the special issue will stimulate and inspire more structural biologists to be involved in this fascinating field of research, and will promote the research to expand our sights to the dynamical perspective of protein science [6,8,9].

Enzymatic catalysis has conflicting structural requirements of the enzyme. In order for the enzyme to form a Michaelis complex, the enzyme must be in an open conformation so that the substrate can get into its active center. On the other hand, in order to maximize the stabilization of the transition state of the enzymatic reaction, the enzyme must be in a closed conformation to maximize its interactions with the transition state. The conflicting structural requirements can be resolved by a flexible active center that can sample both open and closed conformational states. For a bisubstrate enzyme, the Michaelis complex consists of two substrates in addition to the enzyme. The enzyme must remain flexible upon the binding of the first substrate so that the second substrate can get into the active center. The active center is fully assembled and stabilized only when both substrates bind to the enzyme. However, the side-chain positions of the catalytic residues in the Michaelis complex are still not optimally aligned for the stabilization of the transition state, which lasts only approximately 10-13 s. The instantaneous and optimal alignment of catalytic groups for the transition state stabilization requires a dynamic enzyme, not an enzyme which undergoes a large scale of movements but an enzyme which permits at least a small scale of adjustment of catalytic group positions. This review will summarize the structure, catalytic mechanism, and dynamic properties of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase and examine the role of protein conformational dynamics in the catalysis of a bisubstrate enzymatic reaction.

Despite impressive progress in protein engineering and design, our ability to create new and efficient enzyme activities remains a laborious and time-consuming endeavor. In the past few years, intricate combinations of rational mutagenesis, directed evolution and computational methods have paved the way to exciting engineering examples and are now offering a new perspective on the structural requirements of enzyme activity. However, these structure-function analyses are usually guided by the time-averaged static models offered by enzyme crystal structures, which often fail to describe the functionally relevant ‘invisible states’ adopted by proteins in space and time. To alleviate such limitations, NMR relaxation dispersion experiments coupled to mutagenesis studies have recently been applied to the study of enzyme catalysis, effectively complementing ‘structure-function’ analyses with ‘flexibility-function’ investigation. In addition to offering quantitative, site-specific information to help characterize residue motion, these NMR methods are now being applied to enzyme engineering purposes, providing a powerful tool to help characterize the effects of controlling long-range networks of flexible residues affecting enzyme function. Recent advancements in this emerging field are presented here, with particular attention to mutagenesis reports highlighting the relevance of NMR relaxation dispersion tools in enzyme engineering.

In this article we review the current status of our understanding of membrane mediated interactions from theory and experiment. Phenomenological mean field and molecular models will be discussed and compared to recent experimental results from dynamical neutron scattering and atomic force microscopy.

Aβ is widely recognized as a key molecule in Alzheimer's disease, causing neurotoxicity through Aβ aggregates, Aβ oligomers and fibrils. Aβ40 and Aβ42, composed of 40 and 42 residues, respectively, are the major Aβ species in human brain. Aβ42 aggregates much faster than Aβ40 but the mechanism of such difference in aggregation propensity is poorly understood. Using NMR spin relaxation, we have shown that Aβ40 and Aβ42 monomers have different dynamics in both backbone and sidechain on the ps-ns time scale. Aβ42 is more rigid in C-terminus in both backbone and sidechain while Aβ40 has more rigid methyl groups in the central hydrophobic cluster (CHC: Aβ17-21). These observations are consistent with differences in the major conformations of Aβ40 and Aβ42 monomers derived from replica exchange MD (REMD). To further demonstrate the relevance of dynamics in aggregation mechanism, a perturbation was introduced to Aβ42 in the form of M35 oxidation. After M35 side chain oxidation to sulfoxide, Aβ42 experiences Aβ40-like changes in dynamics. At the same time, M35 oxidation causes dramatic reduction in Aβ42 aggregation rate. These data have thus established an important role for protein dynamics in the mechanism of Aβ aggregation.

We construct nanotubes using native protein structures and their native associations from structural databases. The construction is based on a shape-guided symmetric self-assembly concept. Our strategy involves fusing judiciouslyselected oligomerization domains via peptide linkers. Linkers are inherently flexible, hence their choice is critical: they should position the domains in three-dimensional space in the desired orientation while retaining their own natural conformational tendencies; however, at the same time, retain the construct stability. Here we outline a design scheme which accounts for linker flexibility considerations, and present two examples. The first is HIV-1 capsid protein, which in vitro self-assembles into nanotubes and conical capsids, and its linker exists as a short flexible loop. The second involves novel nanotubes construction based on antimicrobial homodimer Magainin 2, employing linkers of distinct lengths and flexibility levels. Our strategy utilizes the abundance of unique shapes and sizes of proteins and their building blocks which can assemble into a vast number of combinations, and consequently, nanotubes of distinct morphologies and diameters. Computational design and assessment methodologies can help reduce the number of candidates for experimental validation. This is an invited paper for a special issue on protein dynamics, here focusing on flexibility in nanotube design based on protein building blocks.

Proteins are dynamic molecules that often undergo conformational changes while performing their specific functions, such as target recognition, ligand binding and catalysis. NMR spectroscopy is uniquely suited to study protein dynamics, because site-specific information can be obtained for motions that span a broad range of time scales. The information obtained from NMR dynamics experiments has provided insights into specific structural changes or conformational energetics associated with molecular function. In the last decade, a number of new advancements in NMR methodologies have further extended our ability to characterize protein dynamics. Here, we present an overview of current NMR technology that is used to monitor the dynamic properties of proteins.

Protein side chain dynamics is associated with protein stability, folding, and intermolecular interactions. Detailed dynamics information is crucial for the understanding of protein function and biochemical and biophysical properties, which can be obtained using NMR relaxation techniques. In this review, 13C relaxation of methine, methylene and methyl groups with and without 1H decoupling are described briefly for a better understanding of how spin relaxation is associated with motional (dynamics) parameters. Developments in the measurement and interpretation of 13C autorelaxation and cross-correlated relaxation data are presented too. Finally, recent progress in the use of 13C relaxation to probe the dynamics of protein side chains is detailed mainly for the dynamics of non-deuterated proteins on picosecondnanosecond timescales.

A new galactose-specific lectin, named BBL, was purified from seeds of Bauhinia bauhinioides by precipitation with ammonium sulfate, followed by two steps of ion exchange chromatography. BBL haemagglutinated rabbit erythrocytes (native and treated with proteolytic enzymes) showing stability even after exposure to 60 °C for one hour. The lectin haemagglutinating activity was optimum between pH 8.0 and 9.0 and inhibited after incubation with D-galactose and its derivatives, especially α-methyl-D-galactopyranoside. The pure protein possessed a molecular mass of 31 kDa by SDS-PAGE and 28.310 Da by mass spectrometry. The lectin pro-inflammatory activity was also evaluated. The s.c. injection of BBL into rats induced a dose-dependent paw edema, an effect that occurred via carbohydrate site interaction and was significantly reduced by L-NAME, suggesting an important participation of nitric oxide in the late phase of the edema. These findings indicate that BBL can be used as a tool to better understand the mechanisms involved in inflammatory responses.

Neuropeptide FF (NPFF) belongs to an opioid-modulating peptide family. NPFF has been reported to play important roles in the control of pain and analgesia through interactions with the opioid system. However, very few studies examined the effect of supraspinal NPFF system on analgesia induced by opiates administered at the peripheral level. In the present study, intracerebroventricular (i.c.v.) injection of NPFF (1, 3 and 10 nmol) dose-dependently inhibited systemic morphine (0.12 mg, i.p.) analgesia in the mouse tail flick test. Similarly, i.c.v. administration of dNPA and NPVF, two agonists highly selective for NPFF2 and NPFF1 receptors, respectively, decreased analgesia induced by i.p. morphine in mice. Furthermore, these anti-opioid activities of NPFF and related peptides were blocked by pretreatment with the NPFF receptors selective antagonist RF9 (10 nmol, i.c.v.). These results demonstrate that activation of central NPFF1 and NPFF2 receptors has the similar anti-opioid actions on the antinociceptive effect of systemic morphine.

Water-soluble, biological-compatible, and excellent fluorescent CdSe/CdS quantum dots (QDs) with L-cysteine as capping agent were synthesized in aqueous medium. Fluorescence (FL) spectra, absorption spectra, and transmission electron microscopy (TEM) were employed to investigate the quality of the products. The interactions between QDs and bovine serum albumin (BSA) were studied by absorption and FL titration experiments. With addition of QDs, the FL intensity of BSA was significantly quenched which can be explained by static mechanism in nature. When BSA was added to the solution of QDs, FL intensity of QDs was faintly quenched. Fluorescent imaging suggests that QDs can be designed as a probe to label the Escherchia coli (E. coli) cells. These results indicate CdSe/CdS/L-cysteine QDs can be used as a probe for labeling biological molecule and bacteria cells.

Allergic reactions to hymenoptera stings are one of the major reasons for IgE-mediated anaphylaxis. However, proper diagnosis using venom extracts is severely affected by molecular cross-reactivity. In this study recombinant honeybee venom major allergen phospholipase A2 (Api m 1) was produced for the first time in insect cells. Using baculovirus infection of different insect cell lines allergen versions providing a varying degree of cross-reactive carbohydrate determinants as well as a non glycosylated variant could be obtained as secreted soluble proteins in high yields. The resulting molecules were analyzed for their glycosylation and proved to show advantageous properties regarding cross-reactivity in sIgE-based assays. Additionally, in contrast to the enzymatically active native protein the inactivated allergen did not induce IgE-independent effector cell activation. Thus, insect cell-derived recombinant Api m 1 with defined CCD phenotypes might provide further insights into hymenoptera venom IgE reactivities and contribute to an improved diagnosis of hymenoptera venom allergy.

The advent of sugar-immobilized gold nano-particles (SGNPs), lipid-based nanoparticles, nanochromatography and nano-electrophoresis has revolutionized the methodology for proteins purification and proteomic research. This review provides an overview on the effective method developed for fast purification of proteins from extracts using SGNPs. In addition, the current application of microfluidic systems for analytical purposes in biochemistry will also be explored that include the micro total analysis systems (μ-TAS) and lab-on-a-chip (LOC) analyses which are capable of isolation and detection of proteins at the nanogram level. Finally, we describe why the lipid-based nano-particles (LBNPs) can enable the analysis in microchip electroseparation and how anionic and cationic LBNPs can be used for proteins separation.