Insights into conformation and dynamics of protein GB1 during folding and unfolding by NMR

Understanding protein stability requires characterization of structural determinants of the folded and unfolded states. Many proteins are capable of populating partially folded states under specific solution conditions. Occasionally, coexistence of the folded and an unfolded state under non- or mildly denaturing conditions can be observed by NMR, allowing us to structurally probe these states under identical conditions. Here we report on a destabilized mutant of the B1 domain of protein G (GB1) whose equilibrium unfolding was systematically investigated. Backbone amide residual dipolar couplings (RDCs), the tryptophan Nepsilon-H resonance and the amide nitrogen transverse relaxation rates (R2s) for varying pH values and different temperatures were measured. The backbone amide RDCs indicate that prior to complete unfolding, two melting hot spots are formed at the turn around T11, L12 and K13 and the N terminus of the helix at A24 and T25. The RDCs for the low pH, thermally unfolded state of GB1 are very small and do not indicate the presence of any native-like structure. Amide nitrogen transverse relaxation rates for GB1 in the folded state at different temperatures exhibit large contributions from exchange processes and the associated dynamics display considerable heterogeneity. Our data provide clear evidence for intermediate conformations and multi-state equilibrium un/folding for this GB1 variant.     

GENFOLD: a genetic algorithm for folding protein structures using NMR restraints.

We report the development and validation of the program GENFOLD, a genetic algorithm that calculates protein structures using restraints obtained from NMR, such as distances derived from nuclear Overhauser effects, and dihedral angles derived from coupling constants. The program has been tested on three proteins: the POU domain (a small three-helix DNA-binding protein), bovine pancreatic trypsin inhibitor (BPTI), and the starch-binding domain from Aspergillus niger glucoamylase I, a 108-residue beta-sheet protein. Structures were calculated for each protein using published NMR restraints. In addition, structures were calculated for BPTI using artificial restraints generated from a high-resolution crystal structure. In all cases the fittest calculated structures were close to the target structure, and could be refined to structures indistinguishable from the target structures by means of a low-temperature simulated annealing refinement. The effectiveness of the program is similar to that of distance geometry and simulated annealing methods, and it is capable of using a very wide range of restraints as input. It can thus be readily extended to the calculation of structures of large proteins, for which few NOE restraints may be available.     

Observing a late folding intermediate of Ubiquitin at atomic resolution by NMR

The study of intermediates in the protein folding pathway provides a wealth of information about the energy landscape. The intermediates also frequently initiate pathogenic fibril formations. While observing the intermediates is difficult due to their transient nature, extreme conditions can partially unfold the proteins and provide a glimpse of the intermediate states. Here, we observe the high resolution structure of a hydrophobic core mutant of Ubiquitin at an extreme acidic pH by nuclear magnetic resonance (NMR) spectroscopy. In the structure, the native secondary and tertiary structure is conserved for a major part of the protein. However, a long loop between the beta strands β3 and β5 is partially unfolded. The altered structure is supported by fluorescence data and the difference in free energies between the native state and the intermediate is reflected in the denaturant induced melting curves. The unfolded region includes amino acids that are critical for interaction with cofactors as well as for assembly of poly‐Ubiquitin chains. The structure at acidic pH resembles a late folding intermediate of Ubiquitin and indicates that upon stabilization of the protein's core, the long loop converges on the core in the final step of the folding process.     

Insights into the local residual entropy of proteins provided by NMR relaxation.

A simple model is used to illustrate the relationship between the dynamics measured by NMR relaxation methods and the local residual entropy of proteins. The expected local dynamic behavior of well-packed extended amino acid side chains are described by employing a one-dimensional vibrator that encapsulates both the spatial and temporal character of the motion. This model is then related to entropy and to the generalized order parameter of the popular "model-free" treatment often used in the analysis of NMR relaxation data. Simulations indicate that order parameters observed for the methyl symmetry axes in, for example, human ubiquitin correspond to significant local entropies. These observations have obvious significance for the issue of the physical basis of protein structure, dynamics, and stability.     

TOUCHSTONEX: protein structure prediction with sparse NMR data

TOUCHSTONEX, a new method for folding proteins that uses a small number of long-range contact restraints derived from NMR experimental NOE (nuclear Overhauser enhancement) data, is described. The method employs a new lattice-based, reduced model of proteins that explicitly represents C(alpha), C(beta), and the sidechain centers of mass. The force field consists of knowledge-based terms to produce protein-like behavior, including various short-range interactions, hydrogen bonding, and one-body, pairwise, and multibody long-range interactions. Contact restraints were incorporated into the force field as an NOE-specific pairwise potential. We evaluated the algorithm using a set of 125 proteins of various secondary structure types and lengths up to 174 residues. Using N/8 simulated, long-range sidechain contact restraints, where N is the number of residues, 108 proteins were folded to a C(alpha)-root-mean-square deviation (RMSD) from native below 6.5 A. The average RMSD of the lowest RMSD structures for all 125 proteins (folded and unfolded) was 4.4 A. The algorithm was also applied to limited experimental NOE data generated for three proteins. Using very few experimental sidechain contact restraints, and a small number of sidechain-main chain and main chain-main chain contact restraints, we folded all three proteins to low-to-medium resolution structures. The algorithm can be applied to the NMR structure determination process or other experimental methods that can provide tertiary restraint information, especially in the early stage of structure determination, when only limited data are available.     

Building native protein conformation from NMR backbone chemical shifts using Monte Carlo fragment assembly

We have been analyzing the extent to which protein secondary structure determines protein tertiary structure in simple protein folds. An earlier paper demonstrated that three-dimensional structure can be obtained successfully using only highly approximate backbone torsion angles for every residue. Here, the initial information is further diluted by introducing a realistic degree of experimental uncertainty into this process. In particular, we tackle the practical problem of determining three-dimensional structure solely from backbone chemical shifts, which can be measured directly by NMR and are known to be correlated with a protein's backbone torsion angles. Extending our previous algorithm to incorporate these experimentally determined data, clusters of structures compatible with the experimentally determined chemical shifts were generated by fragment assembly Monte Carlo. The cluster that corresponds to the native conformation was then identified based on four energy terms: steric clash, solvent-squeezing, hydrogen-bonding, and hydrophobic contact. Currently, the method has been applied successfully to five small proteins with simple topology. Although still under development, this approach offers promise for high-throughput NMR structure determination.     

NMR insights into protein allostery

Allosterism is one of nature's principal methods for regulating protein function. Allosterism utilizes ligand binding at one site to regulate the function of the protein by modulating the structure and dynamics of a distant binding site. In this review, we first survey solution NMR techniques and how they may be applied to the study of allostery. Subsequently, we describe several examples of application of NMR to protein allostery and highlight the unique insight provided by this experimental technique.     

Structural insights for designed alanine-rich helices: comparing NMR helicity measures and conformational ensembles from molecular dynamics simulation

The temperature dependence of helical propensities for the peptides Ac-ZGG-(KAAAA)(3)X-NH(2) (Z = Y or G, X = A, K, and D-Arg) were studied both experimentally and by MD simulations. Good agreement is observed in both the absolute helical propensities as well as relative helical content along the sequence; the global minimum on the calculated free energy landscape corresponds to a single alpha-helical conformation running from K4 to A18 with some terminal fraying, particularly at the C-terminus. Energy component analysis shows that the single helix state has favorable intramolecular electrostatic energy due to hydrogen bonds, and that less-favorable two-helix globular states have favorable solvation energy. The central lysine residues do not appear to increase helicity; however, both experimental and simulation studies show increasing helicity in the series X = Ala --> Lys --> D-Arg. This C-capping preference was also experimentally confirmed in Ac-(KAAAA)(3)X-GY-NH(2) and (KAAAA)(3)X-GY-NH(2) sequences. The roles of the C-capping groups, and of lysines throughout the sequence, in the MD-derived ensembles are analyzed in detail.     

NMR and protein folding: equilibrium and stopped-flow studies.

NMR studies are now unraveling the structure of intermediates of protein folding using hydrogen-deuterium exchange methodologies. These studies provide information about the time dependence of formation of secondary structure. They require the ability to assign specific resonances in the NMR spectra to specific amide protons of a protein followed by experiments involving competition between folding and exchange reactions. Another approach is to use 19F-substituted amino acids to follow changes in side-chain environment upon folding. Current techniques of molecular biology allow assignments of 19F resonances to specific amino acids by site-directed mutagenesis. It is possible to follow changes and to analyze results from 19F spectra in real time using a stopped-flow device incorporated into the NMR spectrometer.     

Folding of a beta-sheet protein monitored by real-time NMR spectroscopy

At low ionic strength, apoplastocyanin forms an unfolded state under non-denaturing conditions. The refolding of this state is sufficiently slow to allow real-time NMR experiments to be performed. Folding of apoplastocyanin, initiated by the addition of salt and followed by real-time 2D 1H-15N heteronuclear single quantum coherence (HSQC) spectroscopy, is highly cooperative. A concomitant increase in the intensity of both sequential and long-range nuclear Overhauser effects (NOEs) between backbone amide protons in successive acquisitions of 1H-15N HSQC-NOESY-HSQC spectra provides the first direct observation of the development of structure-specific NOEs as a protein folds. Our results show that the local and long-range interactions in the native apoplastocyanin are formed simultaneously, consistent with highly cooperative formation of the native structure.     

登革病毒衣壳蛋白在大肠杆菌中的表达及其自组装活性的研究

采用高保真RT-PCR自登革2型病毒43株基因组RNA中扩增全长C基因及缺失羧基端Cv片段,分别构建可表达C及Cv的重组质粒pLEX—C和pLEX—Cv,转化E．coliGI724后用色氨酸诱导表达。经SDS—PAGE分析,表达的C及Cv蛋白相对分子质量分别约为12000和10000,分别约占菌体蛋白总量的19％和13％。Western印迹检测表明重组表达的C蛋白均可被特异识别登革病毒衣壳蛋白的单克隆抗体特异识别。表达的蛋白经过硫酸铵沉淀和蔗糖密度梯度离心后,通过琼脂糖凝胶电泳和负染电镜均未能检测到衣壳样颗粒的存在,说明登革病毒衣壳蛋白可能不具体外自组装活性。     

An engineered chaperonin caging a guest protein: Structural insights and potential as a protein expression tool

The structure of a chaperonin caging a substrate protein is not quite clear. We made engineered group II chaperonins fused with a guest protein and analyzed their structural and functional features. Thermococcus sp. KS-1 chaperonin alpha-subunit (TCP) which forms an eightfold symmetric double-ring structure was used. Expression plasmids were constructed which carried two or four TCP genes ligated head to tail in phase and a target protein gene at the 3' end of the linked TCP genes. Electron microscopy showed that the expressed gene products with the molecular sizes of ~120 kDa (di-TCP) and ~230 kDa (tetra-TCP) formed double-ring complexes similar to those of wild-type TCP. The tetra-TCP retained ATPase activity and its thermostability was significantly higher than that of the wild type. A 260-kDa fusion protein of tetra-TCP and green fluorescent protein (GFP, 27 kDa) was able to form the double-ring complexes with green fluorescence. Image analyses indicated that the GFP moiety of tetra-TCP/GFP fusion protein was accommodated in the central cavity, and tetra-TCP/GFP formed the closed-form similar to that crystallographically resolved in group II chaperonins. Furthermore, it was suggested that caging GFP expanded the cavity around the bottom. Using this tetra-TCP fusion strategy, two virus structural proteins (21-25 kDa) toxic to host cells or two antibody fragments (25-36 kDa) prone to aggregate were well expressed in the soluble fraction of Escherichia coli. These fusion products also assembled to double-ring complexes, suggesting encapsulation of the guest proteins. The antibody fragments liberated by site-specific protease digestion exhibited ligand-binding activities.     

Transient structure formation in unfolded acyl-coenzyme A-binding protein observed by site-directed spin labelling

Paramagnetic relaxation has been used to monitor the formation of structure in the folding peptide chain of guanidinium chloride-denatured acyl-coenzyme A-binding protein. The spin label (1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)methanesulfonate (MTSL) was covalently bound to a single cysteine residue introduced into five different positions in the amino acid sequence. It was shown that the formation of structure in the folding peptide chain at conditions where 95% of the sample is unfolded brings the relaxation probe close to a wide range of residues in the peptide chain, which are not affected in the native folded structure. It is suggested that the experiment is recording the formation of many discrete and transient structures in the polypeptide chain in the preface of protein folding. Analysis of secondary chemical shifts shows a high propensity for alpha-helix formation in the C-terminal part of the polypeptide chain, which forms an alpha-helix in the native structure and a high propensity for turn formation in two regions of the polypeptide that form turns in the native structure. The results contribute to the idea that native-like structural elements form transiently in the unfolded state, and that these may be of importance to the initiation of protein folding.     

In-cell protein NMR and protein leakage

In-cell nuclear magnetic resonance spectroscopy is a tool for studying proteins under physiologically relevant conditions. In some instances, however, protein signals from leaked protein are observed in the liquid surrounding the cells. Here, we examine the expression of four proteins in Escherichia coli. We describe the controls that should be used for in-cell NMR experiments and show that leakage is likely when the protein being studied exceeds ～20% of the total cellular protein.     

Solution NMR evidence for a cis Tyr-Ala peptide group in the structure of [Pro93Ala] bovine pancreatic ribonuclease A

Proline peptide group isomerization can result in kinetic barriers in protein folding. In particular, the cis proline peptide conformation at Tyr92-Pro93 of bovine pancreatic ribonuclease A (RNase A) has been proposed to be crucial for chain folding initiation. Mutation of this proline-93 to alanine results in an RNase A molecule, P93A, that exhibits unfolding/refolding kinetics consistent with a cis Tyr92-Ala93 peptide group conformation in the folded structure (Dodge RW, Scheraga HA, 1996, Biochemistry 35:1548-1559). Here, we describe the analysis of backbone proton resonance assignments for P93A together with nuclear Overhauser effect data that provide spectroscopic evidence for a type VI beta-bend conformation with a cis Tyr92-Ala93 peptide group in the folded structure. This is in contrast to the reported X-ray crystal structure of [Pro93Gly]-RNase A (Schultz LW, Hargraves SR, Klink TA, Raines RT, 1998, Protein Sci 7:1620-1625), in which Tyr92-Gly93 forms a type-II beta-bend with a trans peptide group conformation. While a glycine residue at position 93 accommodates a type-II bend (with a positive value of phi93), RNase A molecules with either proline or alanine residues at this position appear to require a cis peptide group with a type-VI beta-bend for proper folding. These results support the view that a cis Pro93 conformation is crucial for proper folding of wild-type RNase A.     

NMR of fd coat protein

The conformations of the major coat protein of a filamentous bacteriophage can be described by nuclear magnetic resonance spectroscopy of the protein and the virus. The NMR experiments involve detection of the ¹³C and ¹H nuclei of the coat protein. Both the ¹³C and ¹H nuclear magnetic resonance (NMR) spectra show that regions of the polypeptide chain have substantially more motion than a typical globular protein. The fd coat protein was purified by gel chromatography of the SDS solubilized virus. Natural abundance ¹³C NMR spectra at 38 MHz resolve all of the nonprotonated aromatic carbons from the three phenylalanines, two tyrosines, and one tryptophan of the coat protein. The α carbons of the coat protein show at least two different classes of relaxation behavior, indicative of substantial variation in the motion of the backbone carbons in contrast to the rigidity of the α carbons of globular proteins. The ¹H spectrum at 360 MHz shows all of the aromatic carbons and many of the amide protons. Titration of a ¹H spectra gives the pKas for the tyrosines.     

The relation of the X-ray B-factor to protein dynamics: insights from recent dynamic solid-state NMR data

In addressing the potential use of B-factors derived from X-ray scattering data of proteins for the understanding the (functional) dynamics of proteins, we present a comparison of B-factors of five different proteins (SH3 domain, Crh, GB1, ubiquitin and thioredoxin) with data from recent solid-state nuclear magnetic resonance experiments reflecting true (rotational) dynamics on well-defined timescales. Apart from trivial correlations involving mobile loop regions and chain termini, we find no significant correlation of B-factors with the dynamic data on any of the investigated timescales, concluding that there is no unique and general correlation of B-factors with the internal reorientational dynamics of proteins.     

HIV-1 CAP2NC蛋白的表达及体外自组装

构建并表达HIV-1 CAP2NC蛋白,探索其体外自组装条件。通过PCR技术扩增HIV-1(NL4-3毒株)CAP2NC基因片段,并将其连接到原核表达载体pTO-T7,获得重组质粒pTO-T7-CAP2NC,然后转化至大肠杆菌BL21(DE3)菌株,经疏水层析纯化后获得重组蛋白CAP2NC。SDS-PAGE结果表明,重组蛋白CAP2NC可在大肠杆菌可溶高效表达,经纯化后纯度约为95%。ELISA检测表明重组蛋白CAP2NC可被HIV-1衣壳蛋白特异性单克隆抗体识别,具有较好反应活性。重组蛋白透析后在非原性SDS-PAGE中呈现为多种聚体形式。分子筛排阻层析分析CAP2NC蛋白透析后可进行组装,负染电镜进一步观察显示CAP2NC蛋白在RNA存在条件下,可形成空心管状颗粒,其形态结构与HIV-1病毒衣壳体外自组装形成的类似。上述结果表明HIV-1 CAP2NC蛋白具有体外自组装的性质,为进一步在体外研究非成熟病毒样颗粒结构奠定基础。     

Temperature-jump NMR study of protein folding: Ribonuclease A at low pH

Summary The kinetic process of folding of bovine pancreatic ribonuclease A in a²H₂O environment at pH 1.2 was examined by a recently developed temperature-jump NMR method (Akasaka et al., (1990) Rev. Sci. Instrum.61, 66–68). Upon temperature-jump down from 45°C to 29°C, which was attained within 6 s, the proton NMR spectral changes were followed consecutively in time intervals of seconds. There was a rapid spectral change, which was finished within the jump period, followed by a much slower process which lasted for a minute or longer. Rates of the slower process were measured at different positions of the polypeptide chain as intensity changes of individual His and Tyr proton signals of the folded conformer and as intensity changes of aliphatic and His protons of the unfolded conformer. Most of these rates coincided with each other within experimental error with an average value of 2.8×10^–2s^–1. The result gave clear experimental evidence that the slow folding of RNase A at low pH is a cooperative process involving most regions of the molecule, not only thermodynamically, but kinetically as well.     

New insights into structural determinants of prion protein folding and stability

Prions are the etiological agent of fatal neurodegenerative diseases called prion diseases or transmissible spongiform encephalopathies. These maladies can be sporadic, genetic or infectious disorders. Prions are due to post-translational modifications of the cellular prion protein leading to the formation of a β-sheet enriched conformer with altered biochemical properties. The molecular events causing prion formation in sporadic prion diseases are still elusive. Recently, we published a research elucidating the contribution of major structural determinants and environmental factors in prion protein folding and stability. Our study highlighted the crucial role of octarepeats in stabilizing prion protein; the presence of a highly enthalpically stable intermediate state in prion-susceptible species; and the role of disulfide bridge in preserving native fold thus avoiding the misfolding to a β-sheet enriched isoform. Taking advantage from these findings, in this work we present new insights into structural determinants of prion protein folding and stability.