An efficient on-column expressed protein ligation strategy: application to segmental triple labeling of human apolipoprotein E3

Expressed protein ligation (EPL) is an intein-based approach that has been used for protein engineering and biophysical studies of protein structures. One major problem of the EPL is the low yield of final ligation product, primarily due to the complex procedure of the EPL, preventing EPL from gaining popularity in the research community. Here we report an efficient on-column EPL strategy, which focuses on enhancing the expression level of the intein-fusion protein that generates thioester for the EPL. We applied this EPL strategy to human apolipoprotein E (apoE) and routinely obtained 25-30 mg segmental, triple-labeled apoE from 1-L cell culture. The approaches reported here are general approaches that are not specific for apoE, thus providing a general strategy for a highly efficient EPL. In addition, we also report an isotopic labeling scheme that double-labels one domain and keeps the other domain of apoE deuterated. Such an isotopic labeling scheme can only be achieved using the EPL strategy. Our data indicated that the segmental triple-labeled apoEs using this labeling scheme produced high-quality, simplified NMR spectra, facilitating NMR spectral assignment. For large proteins, such as apoE, perdeuterated protein samples have to be used to reduce the linewidth of NMR signals, causing a major problem for the NOE-based NMR method, since perdeuterated proteins lack protons for NOE measurement. The new labeling strategy solves this problem and provides (13)C/(15)N double-labeled, protonated protein domains, allowing for determination of high-resolution NMR structure of these large proteins.     

Abstracts: Albany 2005, The 14th Conversation

Abstract

We report a cost efficient approach for amino-acid-type selective isotope labeling of proteins expressed in Leishmania tarentolae. The method provides an economically advantageous alternative to recently established protocol for isotopic labeling using expensive synthetic media. The method is based on cultivation of the L. tarentolae expression strain in a cheap complex medium supplemented with labeled amino acid(s). In this protocol, a labeled amino acid is deliberately diluted in the medium of undefined composition, which leads to a low-level isotope enrichment upon protein over-expression. The economic advantage of the protocol is achieved by avoiding large volumes of expensive synthetic medium. Decreased sensitivity of a NMR experiment due to low-level isotope enrichment is compensated by a five- to seven-fold increase of the yield of the recombinant protein in complex medium as compared to that in the synthetic medium. In addition, the decreased sensitivity can be compensated by using a higher magnetic field, cryo-detection system or higher number of transients during the NMR data acquisition. We show that enrichment as low as 5% does not compromise a NMR experiment and makes preparation of the recombinant proteins over- expressed in L. tarentolae economically viable. The method is demonstrated by selective labeling of the ～27 kDa enhanced green fluorescent protein (EGFP) with ¹⁵N-labeled valine.     

Review of methods to assign the nuclear magnetic resonance peaks of reductively methylated proteins

Reductive methylation of lysyl side-chain amines has been a successful tool in the advancement of high-resolution structural biology. The utility of this method has continuously gained ground as a protein chemical modification, first as a tool to aid protein crystallization and later as a probe in protein nuclear magnetic resonance (NMR) spectroscopy. As an isotope-labeling strategy for NMR studies, reductive methylation has contributed to the study of protein–protein interactions and global conformational changes. Although more detailed structural studies using this labeling strategy are possible, the hurdle of assigning the NMR peaks to the corresponding reductively methylated amine hinders its use. In this review, we discuss and compare strategies used to assign the NMR peaks of reductively methylated protein amines.     

Isotope labeling strategies for the study of high-molecular-weight proteins by solution NMR spectroscopy

The development of isotope labeling methodology has had a significant impact on NMR studies of high-molecular-weight proteins and macromolecular complexes. Here we review some of this methodology that has been developed and used in our laboratory. In particular, experimental protocols are described for the production of highly deuterated, uniformly 15N- and 13C-labeled samples of large proteins, with optional incorporation of selective isotope labels into methyl groups of isoleucine, leucine and valine residues. Various types of methyl labeling schemes are assessed, and the utility of different methyl labeling strategies is highlighted for studies ranging from protein structure determination to the investigation of side-chain dynamics. In the case of malate synthase G (MSG), the time frame of the whole preparation, including the protein refolding step, is about 70 h.     

Application of amino acid type-specific 1H- and 14N-labeling in a 2H-, 15N-labeled background to a 47 kDa homodimer: Potential for NMR structure determination of large proteins

NMR investigations of larger macromolecules (>20 kDa) are severely hindered by rapid 1H and 13C transverse relaxation. Replacement of non-exchangeable protons with deuterium removes many efficient 1H-1H and 1H-13C relaxation pathways. The main disadvantage of deuteration is that many of the protons which would normally be the source of NOE-based distance restraints are removed. We report the development of a novel labeling strategy which is based on specific protonation and 14N-labeling of the residues phenylalanine, tyrosine, threonine, isoleucine and valine in a fully deuterated, 15N-labeled background. This allows the application of heteronuclear half-filters, 15N-editing and 1H-TOCSY experiments to select for particular magnetization transfer pathways. Results from investigations of a 47 kDa dimeric protein labeled in this way demonstrated that the method provides useful information for the structure determination of large proteins.     

NMR assignments of sparsely labeled proteins using a genetic algorithm

Sparse isotopic labeling of proteins for NMR studies using single types of amino acid (¹⁵N or ¹³C enriched) has several advantages. Resolution is enhanced by reducing numbers of resonances for large proteins, and isotopic labeling becomes economically feasible for glycoproteins that must be expressed in mammalian cells. However, without access to the traditional triple resonance strategies that require uniform isotopic labeling, NMR assignment of crosspeaks in heteronuclear single quantum coherence (HSQC) spectra is challenging. We present an alternative strategy which combines readily accessible NMR data with known protein domain structures. Based on the structures, chemical shifts are predicted, NOE cross-peak lists are generated, and residual dipolar couplings (RDCs) are calculated for each labeled site. Simulated data are then compared to measured values for a trial set of assignments and scored. A genetic algorithm uses the scores to search for an optimal pairing of HSQC crosspeaks with labeled sites. While none of the individual data types can give a definitive assignment for a particular site, their combination can in most cases. Four test proteins previously assigned using triple resonance methods and a sparsely labeled glycosylated protein, Robo1, previously assigned by manual analysis, are used to validate the method and develop a criterion for identifying sites assigned with high confidence.     

Novel strategies to overcome expression problems encountered with toxic proteins: Application to the production of Lac repressor proteins for NMR studies

NMR studies of structural aspects of allosteric regulation by the Lac repressor requires overexpression and isotope labeling of the protein. The size of the repressor makes it a challenging target, putting constraints on both expression conditions and sample preparation methods to overcome problems associated with studies of larger proteins by NMR. We optimized protocols for the production of deuterated functionally active thermostable dimeric Lac repressor and its core domain mutants. The Lac repressor core domain has never been obtained as a recombinant protein, possibly due to the observed toxicity to the host cells. We overcame the core domain induced toxicity by co-expression of this domain with the full length Lac repressor, combined with a stringent control of culture conditions. Significant overexpression was only obtained if during all stages of pre-culturing the bacteria were kept in their exponential growth phase at low density. The sensitivity of NMR measurements is dramatically affected by buffer conditions; we therefore used a thermofluor buffer optimization screen to determine the optimal buffer conditions. The combined thermofluor and NMR screening method yielded thermostable fully functional Lac repressor domain samples suitable for high-resolution NMR studies. The optimized procedures to adapt Escherichia coli to growth in D₂O, to overcome toxicity, and to optimize protein sample conditions provides a broad range of universally applicable techniques for production of larger proteins for NMR spectroscopy.     

Segmental isotopic labeling by asparaginyl endopeptidase-mediated protein ligation

Segmental isotopic labeling can facilitate NMR studies of large proteins, multi-domain proteins, and proteins with repetitive sequences by alleviating NMR signal overlaps. Segmental isotopic labeling also allows us to investigate an individual domain in the context of a full-length protein by NMR. Several established methods are available for segmental isotopic labeling such as intein-mediated ligation, but each has specific requirements and limitations. Here, we report an enzymatic approach using bacterially produced asparagine endopeptidase from Oldenlandia affinis for segmental isotopic labeling of a protein with repetitive sequences, a designed armadillo repeat protein, by overcoming some of the shortcomings of enzymatic ligation for segmental isotopic labeling.     

Methyl labeling and TROSY NMR spectroscopy of proteins expressed in the eukaryote <Emphasis Type="Italic">Pichia pastoris</Emphasis>

¹³C Methyl TROSY NMR spectroscopy has emerged as a powerful method for studying the dynamics of large systems such as macromolecular assemblies and membrane proteins. Specific ¹³C labeling of aliphatic methyl groups and perdeuteration has been limited primarily to proteins expressed in E. coli, preventing studies of many eukaryotic proteins of physiological and biomedical significance. We demonstrate the feasibility of efficient ¹³C isoleucine δ1-methyl labeling in a deuterated background in an established eukaryotic expression host, Pichia pastoris, and show that this method can be used to label the eukaryotic protein actin, which cannot be expressed in bacteria. This approach will enable NMR studies of previously intractable targets.     

NMR resonance assignments for sparsely <Superscript>15</Superscript>N labeled proteins

For larger proteins, and proteins not amenable to expression in bacterial hosts, it is difficult to deduce structures using NMR methods based on uniform ¹³C, ¹⁵N isotopic labeling and observation of just nuclear Overhauser effects (NOEs). In these cases, sparse labeling with selected ¹⁵N enriched amino acids and extraction of a wider variety of backbone-centered structural constraints is providing an alternate approach. A limitation, however, is the absence of resonance assignment strategies that work without uniform ¹⁵N, ¹³C labeling or preparation of numerous samples labeled with pairs of isotopically labeled amino acids. In this paper an approach applicable to a single sample prepared with sparse ¹⁵N labeling in selected amino acids is presented. It relies on correlation of amide proton exchange rates, measured from data on the intact protein and on digested and sequenced peptides. Application is illustrated using the carbohydrate binding protein, Galectin-3. Limitations and future applications are discussed.     

15N-metabolic labeling for comparative plasma membrane proteomics in Arabidopsis cells

An important goal for proteomic studies is the global comparison of proteomes from different genotypes, tissues, or physiological conditions. This has so far been mostly achieved by densitometric comparison of spot intensities after protein separation by 2-DE. However, the physicochemical properties of membrane proteins preclude the use of 2-DE. Here, we describe the use of in vivo labeling by the stable isotope 15N as an alternative approach for comparative membrane proteomic studies in plant cells. We confirm that 15N-metabolic labeling of proteins is possible and efficient in Arabidopsis suspension cells. Quantification of 14N versus 15N MS signals reflects the relative abundance of 14N and 15N proteins in the sample analyzed. We describe the use of 15N-metabolic labeling to perform a partial comparative analysis of Arabidopsis cells following cadmium exposure. By focusing our attention on plasma membrane proteins, we were able to confidently identify proteins showing up to 5-fold regulation compared to unexposed cells. This study provides a proof of principle that 15N-metabolic labeling is a useful technique for comparative membrane proteome studies.     

Structure determination of α-helical membrane proteins by solution-state NMR: Emphasis on retinal proteins

The biochemical processes of living cells involve a numerous series of reactions that work with exceptional specificity and efficiency. The tight control of this intricate reaction network stems from the architecture of the proteins that drive the chemical reactions and mediate protein–protein interactions. Indeed, the structure of these proteins will determine both their function and interaction partners. A detailed understanding of the proximity and orientation of pivotal functional groups can reveal the molecular mechanistic basis for the activity of a protein. Together with X-ray crystallography and electron microscopy, NMR spectroscopy plays an important role in solving three-dimensional structures of proteins at atomic resolution. In the challenging field of membrane proteins, retinal-binding proteins are often employed as model systems and prototypes to develop biophysical techniques for the study of structural and functional mechanistic aspects. The recent determination of two 3D structures of seven-helical trans-membrane retinal proteins by solution-state NMR spectroscopy highlights the potential of solution NMR techniques in contributing to our understanding of membrane proteins. This review summarizes the multiple strategies available for expression of isotopically labeled membrane proteins. Different environments for mimicking lipid bilayers will be presented, along with the most important NMR methods and labeling schemes used to generate high-quality NMR spectra. The article concludes with an overview of types of conformational restraints used for generation of high-resolution structures of membrane proteins. This article is part of a Special Issue entitled: Retinal Proteins — You can teach an old dog new tricks.     

What distinguishes GroEL substrates from other Escherichia coli proteins?

Experimental studies and theoretical considerations have shown that only a small subset of Escherichia coli proteins fold in vivo with the help of the GroE chaperone system. These proteins, termed GroE substrates, have been divided into three classes: (a) proteins that can fold independently, but are found to associate with GroEL; (b) proteins that require GroE when the cell is under stress; and (c) 'obligatory' proteins that require GroE assistance even under normal conditions. It remains unclear, however, why some proteins need GroE and others do not. Here, we review experimental and computational studies that addressed this question by comparing the sequences and structural, biophysical and evolutionary properties of GroE substrates with those of nonsubstrates. In general, obligatory substrates are found to have lower folding propensities and be more aggregation prone. GroE substrates are also more conserved than other proteins and tend to utilize more optimal codons, but this latter feature is less apparent for obligatory substrates. There is no evidence, however, for any specific sequence signatures although there is a tendency for sequence periodicity. Our review shows that reliable sequence- or structure-based predictions of GroE dependency remain a challenge. We suggest that the different classes of GroE substrates be studied separately and that proper control test sets (e.g. TIM barrel proteins that need GroE for folding versus TIM barrels that fold independently) be used more extensively in such studies.     

特异性的蛋白小分子荧光探针及其标记技术

活体蛋白荧光标记技术已经被广泛应用于蛋白质功能的可视化研究中。荧光蛋白常被用来研究蛋白质在生物体内的表达和定位,但由于它本身体积比较大,往往会影响目标蛋白的生物活性。特异性的小分子荧光探针以其体积小、膜透性好、背景噪音低以及制备方便的优点成为蛋白质研究的一个有力工具。本文将简要介绍近几年来各类特异性小分子蛋白荧光探针的研究进展。     

Parallel screening and optimization of protein constructs for structural studies

A major challenge in structural biology remains the identification of protein constructs amenable to structural characterization. Here, we present a simple method for parallel expression, labeling, and purification of protein constructs (up to 80 kDa) combined with rapid evaluation by NMR spectroscopy. Our approach, which is equally applicable for manual or automated implementation, offers an efficient way to identify and optimize protein constructs for NMR or X‐ray crystallographic investigations.     

Catalysis, commitment and encapsulation during GroE-mediated folding.

The Escherichia coli GroE chaperones assist protein folding under conditions where no spontaneous folding occurs. To achieve this, the cooperation of GroEL and GroES, the two protein components of the chaperone system, is an essential requirement. While in many cases GroE simply suppresses unspecific aggregation of non-native proteins by encapsulation, there are examples where folding is accelerated by GroE.Using maltose-binding protein (MBP) as a substrate for GroE, it had been possible to define basic requirements for catalysis of folding. Here, we have analyzed key steps in the interaction of GroE and the MBP mutant Y283D during catalyzed folding. In addition to high temperature, high ionic strength was shown to be a restrictive condition for MBP Y283D folding. In both cases, the complete GroE system (GroEL, GroES and ATP) compensates the deceleration of MBP Y283D folding. Combining kinetic folding experiments and electron microscopy of GroE particles, we demonstrate that at elevated temperatures, symmetrical GroE particles with GroES bound to both ends of the GroEL cylinder play an important role in the efficient catalysis of MBP Y283D refolding. In principle, MBP Y283D folding can be catalyzed during one encapsulation cycle. However, because the commitment to reach the native state is low after only one cycle of ATP hydrolysis, several interaction cycles are required for catalyzed folding.     

Automated structure determination of proteins with the SAIL-FLYA NMR method

The labeling of proteins with stable isotopes enhances the NMR method for the determination of 3D protein structures in solution. Stereo-array isotope labeling (SAIL) provides an optimal stereospecific and regiospecific pattern of stable isotopes that yields sharpened lines, spectral simplification without loss of information, and the ability to collect rapidly and evaluate fully automatically the structural restraints required to solve a high-quality solution structure for proteins up to twice as large as those that can be analyzed using conventional methods. Here, we describe a protocol for the preparation of SAIL proteins by cell-free methods, including the preparation of S30 extract and their automated structure analysis using the FLYA algorithm and the program CYANA. Once efficient cell-free expression of the unlabeled or uniformly labeled target protein has been achieved, the NMR sample preparation of a SAIL protein can be accomplished in 3 d. A fully automated FLYA structure calculation can be completed in 1 d on a powerful computer system.     

Enzymatic labeling of arbitrary proteins.

This paper describes an enzymatic labeling technique (ELT), using transglutaminases. On the basis of the ELT, isotopic nuclei are easily incorporated into the gamma-carboxyamide groups of glutamine residues in arbitrary proteins, without changing their chemical structures. We have also shown that, by using ELT, protein aggregation was easily checked for NMR studies and that it can be applicable for the screening of weakly bound ligands for proteins. Owing to the simple preparation of the isotope-labeled proteins, ELT should be useful for speeding up various structural and functional analyses of arbitrary proteins.     

NMR resonance assignment of selectively labeled proteins by the use of paramagnetic ligands

Selective isotopic labeling of larger proteins greatly simplifies protein NMR spectra and reduces signal overlap, but selectively labeled proteins cannot be easily assigned since the sequential assignment method is not applicable. Here we describe a strategy for resonance assignment in selectively labeled proteins. Our approach involves a spin-labeled analog of a ligand of which the three-dimensional structure in complex with the target protein is known. Other methods for introduction of the spin label are possible. The paramagnetic center causes faster relaxation of all neighboring nuclei in a distance-dependent manner. Measurement of this effect allows to deduce distances between isotopically labeled residues and the paramagnetic center which can be used for resonance assignment. The method is demonstrated for the catalytic domain of Abl kinase in complex with the inhibitor, STI571.