Structural characterisation of the human alpha-lactalbumin molten globule at high temperature

Molten globules are partially folded forms of proteins thought to be general intermediates in protein folding. The 15N-1H HSQC NMR spectrum of the human alpha-lactalbumin (alpha-LA) molten globule at pH 2 and 20 degrees C is characterised by broad lines which make direct study by NMR methods difficult; this broadening arises from conformational fluctuations throughout the protein on a millisecond to microsecond timescale. Here, we find that an increase in temperature to 50 degrees C leads to a dramatic sharpening of peaks in the 15N-1H HSQC spectrum of human alpha-LA at pH 2. Far-UV CD and ANS fluorescence experiments demonstrate that under these conditions human alpha-LA maintains a high degree of helical secondary structure and the exposed hydrophobic surfaces that are characteristic of a molten globule. Analysis of the H(alpha), H(N) and 15N chemical shifts of the human alpha-LA molten globule at 50 degrees C leads to the identification of regions of native-like helix in the alpha-domain and of non-native helical propensity in the beta-domain. The latter may be responsible for the observed overshoot in ellipticity at 222 nm in kinetic refolding experiments.     

Iterative cloning, overexpression, purification and isotopic labeling of an engineered dimer of a Ca(2+)-binding protein of the βγ-crystallin superfamily from Methanosarcina acetivorans

βγ-Crystallins are a large superfamily of proteins found in vertebrate eye lens. They are hetero-dimers (linked in tandem by a specific peptide) and are shown to bind calcium. The monomers possess two β-strand rich greek-key motifs. Recently, a structurally closest member to the family of lens βγ-crystallins has been described, for the first time, from the archaea Methanosarcina acetivorans, which is named as M-crystallin. Unlike lens βγ-crystallins, M-crystallin exits as a monomer. Here, we synthesized a dimeric gene of M-crystallin in which two monomers are linked by a 10-amino acid residue coding sequence. The linker sequence in the target protein is long and flexible enough to reduce the proximity between the individual crystallins in the dimer. This methodology would be highly beneficial in designing polyproteins (two or more proteins linked in tandem to aid mechanical stretching studies) that are regularly used in single-molecule force spectroscopy. The dimer of M-crystallin was overexpressed in Escherichia coli BLR(DE3) strain. The overexpressed protein containing an N-terminal hexa-histidine tag was purified using nickel affinity chromatography and then by size-exclusion chromatography. Further, a method to purify isotopically ((15)N) labeled protein with high yield for NMR studies is reported. The uniformly (15)N-labeled M-crystallin dimer thus produced has been characterized by recording sensitivity enhanced 2D [(15)N-(1)H] HSQC and other optical spectroscopy techniques. Observation of only one set of peaks in the HSQC, along with the structural characterization using optical spectroscopy, suggests that the domains in the dimer possess similar structure as that of the monomer.     

Nuclear Magnetic Resonance Studies on Huwentoxin-XI from the Chinese Bird Spider Ornithoctonus huwena： ^15N Labeling and Sequence-specific ^1H, ^15N Nuclear Magnetic Resonance Assignments

Huwentoxin-XI purified from the Chinese bird spider Ornithoctonus huwena is a toxin with both antiprotease activity and potassium channel blocking activity. To determine its solution structure, huwentoxin-XI was expressed in a yeast eukaryotic expression system and studied by NMR. The ^15N labeling strategy was used to facilitate the process of resonance assignments. The nearly complete sequence-specific assignments of proton and nitrogen resonances were obtained by analyzing a series of two-dimensional （2D） and three-dimensional （3D） spectra, including DQF-COSY, TOCSY, NOESY, ^15N-^1H HSQC, ^15N-^1H HNHA, ^15N-^1H HNHB, ^15N-^1H TOCSY-HSQC and ^15N-^1H NOESY-HSQC spectra. Secondary structure analysis of huwentoxin-XI showed that it mainly contains an N-terminal 310-helix from Thr3 to Arg5 and a C-terminal α-helix from Gln45 to Cys52, plus a triple-stranded antiparallel β-sheet of Glu18-Asn23, Thr26-Ile31 and Asn40-Lys41. These studies provide a solid basis for the final structure determination of huwentoxin-XI.     

Amino acid-selective isotope labeling of proteins for nuclear magnetic resonance study: Proteins secreted by Brevibacillus choshinensis

Here we report the first application of amino acid-type selective (AATS) isotope labeling of a recombinant protein secreted by Brevibacillus choshinensis for a nuclear magnetic resonance (NMR) study. To prepare the ¹⁵N-AATS-labeled protein, the transformed B. choshinensis was cultured in ¹⁵N-labeled amino acid-containing C.H.L. medium, which is commonly used in the Escherichia coli expression system. The analyses of the ¹H-¹⁵N heteronuclear single quantum coherence (HSQC) spectra of the secreted proteins with a ¹⁵N-labeled amino acid demonstrated that alanine, arginine, asparagine, cysteine, glutamine, histidine, lysine, methionine, and valine are suitable for selective labeling, although acidic and aromatic amino acids are not suitable. The ¹⁵N labeling for glycine, isoleucine, leucine, serine, and threonine resulted in scrambling to specific amino acids. These results indicate that the B. choshinensis expression system is an alternative tool for AATS labeling of recombinant proteins, especially secretory proteins, for NMR analyses.     

Isotopic labeling of recombinant proteins expressed in the protozoan host Leishmania tarentolae

Isotope labeling of recombinant proteins is a prerequisite for application of nuclear magnetic resonance spectroscopy (NMR) for the characterization of the three-dimensional structures and dynamics of proteins. Overexpression of isotopically labeled proteins in bacterial or yeast host organisms has several drawbacks. In this work, we tested whether the recently described eukaryotic protein expression system based on the protozoa Leishmania tarentolae could be used for production of amino acid specific (15)N-labeled recombinant proteins. Using synthetic growth medium we were able to express in L. tarentolae and purify to homogeneity (15)N-valine labeled Enchanced Green Fluorescent Protein (EGFP) with the final yield of 5.7 mg/liter of suspension culture. NMR study of isolated EGFP illustrated the success of the labeling procedure allowing identification of all 18 valine residues of the protein in the HSQC spectrum. Our results demonstrate the suitability of the L. tarentolae expression system for production of isotopically labeled 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.     

Cyclic enterobacterial common antigen: Potential contaminant of bacterially expressed protein preparations

We have previously reported the identification of the cyclic enterobacterial common antigen (ECA(CYC)) polysaccharide in E. coli strains commonly used for heterologous protein expression (PJA Erbel et al., J. Bacteriol. 185 (2003): 1995). Following this initial report, interactions among several NMR groups established that characteristic N -acetyl signals of ECA(CYC) have been observed in (15)N-(1)H HSQC spectra of samples of various bacterially-expressed proteins suggesting that this water-soluble carbohydrate is a common contaminant. We provide NMR spectroscopic tools to recognize ECA(CYC) in protein samples, as well as several methods to remove this contaminant. Early recognition of ECA-based NMR signals will prevent time-consuming analyses of this copurifying carbohydrate.     

Expression, Purification, and Mass Spectrometric Analysis of (15)N, (13)C-Labeled RGD-Hirudin, Expressed in Pichia pastoris, for NMR Studies

A novel recombinant hirudin, RGD-hirudin, inhibits the activity of thrombin and the aggregation of platelets. Here, we successfully expressed (15)N, (13)C-labeled RGD-hirudin in Pichia pastoris in a fermenter. The protein was subsequently purified to yield sufficient quantities for structural and functional studies. The purified protein was characterized by HPLC and MALDI-TOF mass spectroscopy. Analysis revealed that the protein was pure and uniformly labeled with (15)N and (13)C. A bioassay showed that the anti-thrombin activity and the anti-platelet aggregation ability of the labeled protein were the same as those of unlabeled RGD-hirudin. Multidimensional heteronuclear NMR spectroscopy has been used to determine almost complete backbone (15)N, (13)C and (1)H resonance assignments of the r-RGD-Hirudin. The (15)N-(1)H HSQC spectrum of uniformly (15)N, (13)C-labeled RGD-hirudin allowed successful assignment of the signals. Examples of the quality of the data are provided for the (15)N-(l)H correlation spectrum, and by selected planes of the CBCA(CO)NH, CBCANH, and HNCO experiments. These results provide a basis for further studies on the structure-function relationship of RGD-hirudin with thrombin and platelets.     

Human SLC26A4/Pendrin STAS domain is a nucleotide-binding protein: Refolding and characterization for structural studies

Mutations in the human SLC26A4/Pendrin polypeptide (hPDS) cause Pendred Syndrome /DFNB4, syndromic deafness with enlargement of the vestibular aqueduct and low-penetrance goiter. Here we present data on cloning, protein overexpression and purification, refolding, and biophysical characterization of the recombinant hPDS STAS domain lacking its intrinsic variable sequence (STAS-ΔIVS). We report a reproducible protein refolding protocol enabling milligram scale expression and purification of uniformly ¹⁵N- and ¹³C^/15N-enriched hPDS STAS-ΔIVS domain suitable for structural characterization by solution NMR. Circular dichroism, one-dimensional ¹H, two-dimensional ¹H–¹⁵N HSQC, and ¹H–¹³C HSQC NMR spectra confirmed the well-folded state of purified hPDS STAS-ΔIVS in solution. Heteronuclear NMR chemical shift perturbation of select STAS-ΔIVS residues by GDP was observed at fast-to-intermediate NMR time scales. Intrinsic tryptophan fluorescence quench experiments demonstrated GDP binding to hPDS STAS-ΔIVS with K_d of 178 μM. These results are useful for structure/function characterization of hPDS STAS, the cytoplasmic subdomain of the congenital deafness protein, pendrin, as well as for studies of other mammalian STAS domains.     

Complete resonance assignment for the polypeptide backbone of interleukin 1 beta using three-dimensional heteronuclear NMR spectroscopy

The complete sequence-specific assignment of the 15N and 1H backbone resonances of the NMR spectrum of recombinant human interleukin 1 beta (153 residues, Mr = 17,400) has been obtained by using primarily 15N-1H heteronuclear three-dimensional (3D) NMR techniques in combination with 15N-1H heteronuclear and 1H homonuclear two-dimensional NMR. The fingerprint region of the spectrum was analyzed by using a combination of 3D heteronuclear 1H Hartmann-Hahn 15N-1H multiple quantum coherence (3D HOHAHA-HMQC) and 3D heteronuclear 1H nuclear Overhauser 15N-1H multiple quantum coherence (3D NOESY-HMQC) spectroscopies. We show that the problems of amide NH and C alpha H chemical shift degeneracy that are prevalent for proteins of this size are readily overcome by using the 3D heteronuclear NMR technique. A doubling of some peaks in the spectrum was found to be due to N-terminal heterogeneity of the 15N-labeled protein, corresponding to a mixture of wild-type and des-Ala-1-interleukin 1 beta. The complete list of 15N and 1H assignments is given for all the amide NH and C alpha H resonances of all non-proline residues, as well as the 1H assignments for some of the amino acid side chains. This first example of the sequence-specific assignment of a protein using heteronuclear 3D NMR provides a basis for further conformational and dynamic studies of interleukin 1 beta.     

Preparation of amino-acid-type selective isotope labeling of protein expressed in Pichia pastoris

We report the culture conditions for successful amino-acid-type selective (AATS) isotope labeling of protein expressed in Pichia pastoris (P. pastoris). Rhodostomin (Rho), a six disulfide-bonded protein expressed in P. pastoris with the correct fold, was used to optimize the culture conditions. The concentrations of [alpha-15N] selective amino acid, nonlabeled amino acids, and ammonium chloride, as well as induction time, were optimized to avoid scrambling and to increase the incorporation rate and protein yield. The optimized protocol was successfully applied to produce AATS isotope-labeled Rho. The labeling of [alpha-15N]Cys has a 50% incorporation rate, and all 12 cysteine resonances were observed in HSQC spectrum. The labeling of [alpha-15N]Leu, -Lys, and -Met amino acids has an incorporation rate greater than 65%, and the expected number of resonances in the HSQC spectra were observed. In contrast, the labeling of [alpha-15N]Asp and -Gly amino acids has a low incorporation rate and the scrambling problem. In addition, the culture condition was successfully applied to label dendroaspin (Den), a four disulfide-bonded protein expressed in P. pastoris. Therefore, the described condition should be generally applicable to other proteins produced in the P. pastoris expression system. This is the first report to present a protocol for AATS isotope labeling of protein expressed in P. pastoris for NMR study.     

Uniform 13C isotope labeling of proteins with sodium acetate for NMR studies: application to human carbonic anhydrase II

Uniform double labeling of proteins for NMR studies can be prohibitively expensive, even with an efficient expression and purification scheme, due largely to the high cost of [13C6, 99%]glucose. We demonstrate here that uniformly (greater than 95%) 13C and 15N double-labeled proteins can be prepared for NMR structure/function studies by growing cells in defined media containing sodium [1,2-13C2, 99%]acetate as the sole carbon source and [15N, 99%]ammonium chloride as the sole nitrogen source. In addition, we demonstrate that this labeling scheme can be extended to include uniform carbon isotope labeling to any desired level (below 50%) by utilizing media containing equal amounts of sodium [1-13C, 99%]acetate and sodium [2-13C, 99%]acetate in conjunction with unlabeled sodium acetate. This technique is less labor intensive and more straightforward than labeling using isotope-enriched algal hydrolysates. These labeling schemes have been used to successfully prepare NMR quantities of isotopically enriched human carbonic anhydrase II. The activity and the 1H NMR spectra of the protein labeled by this technique are the same as those obtained from the protein produced from media containing labeled glucose; however, the cost of the sodium [1,2-13C2, 99%]acetate growth media is considerably less than the cost of the [13C6, 99%]glucose growth media. We report here the first published 13C and 15N NMR spectra of human carbonic anhydrase II as an important step leading to the assignment of this 29-kDa zinc metalloenzyme.     

A solution NMR investigation into the murine amelogenin splice-variant LRAP (Leucine-Rich Amelogenin Protein)

Amelogenins are the dominant proteins present in ameloblasts during the early stages of enamel biomineralization, making up > 90% of the matrix protein. Along with the full-length protein there are several splice-variant isoforms of amelogenin present including LRAP (Leucine-Rich Amelogenin Protein), a protein that consists of the first 33 and the last 26 residues of full-length amelogenin. Using solution-state NMR spectroscopy we have assigned the ¹H-¹⁵N HSQC spectrum of murine LRAP (rp(H)LRAP) in 2% acetic acid at pH 3.0 by making extensive use of previous chemical shift assignments for full-length murine amelogenin (rp(H)M180). This correlation was possible because LRAP, like the full-length protein, is intrinsically disordered under these solution conditions. The major difference between the ¹H-¹⁵N HSQC spectra of rp(H)M180 and rp(H)LRAP was an additional set of amide resonances for each of the seven non-proline residues between S12* and Y12 near the N-terminus of rp(H)LRAP indicating that the N-terminal region of LRAP exists in two different conformations. Analysis of the proline carbon chemical shifts suggests that the molecular basis for the two states is not a cis-trans isomerization of one or more of the proline residues in the N-terminal region. Starting from 2% acetic acid, where rp(H)LRAP was monomeric in solution, NaCl addition effected residue specific changes in molecular dynamics manifested by the reduction in intensity and disappearance of ¹H-¹⁵N HSQC cross peaks. As observed for the full-length protein, these perturbations may signal early events governing supramolecular self-assembly of rp(H)LRAP into nanospheres. However, the different patterns of ¹H-¹⁵N HSQC cross peak perturbation between rp(H)LRAP and rp(H)M180 in high salt suggest that the termini may behave differently in their respective nanospheres, and perhaps, these differences contribute to the cell signaling properties attributable to LRAP but not to the full-length protein.     

Construct optimization for protein NMR structure analysis using amide hydrogen/deuterium exchange mass spectrometry

Disordered or unstructured regions of proteins, while often very important biologically, can pose significant challenges for resonance assignment and three‐dimensional structure determination of the ordered regions of proteins by NMR methods. In this article, we demonstrate the application of ¹H/²H exchange mass spectrometry (DXMS) for the rapid identification of disordered segments of proteins and design of protein constructs that are more suitable for structural analysis by NMR. In this benchmark study, DXMS is applied to five NMR protein targets chosen from the Northeast Structural Genomics project. These data were then used to design optimized constructs for three partially disordered proteins. Truncated proteins obtained by deletion of disordered N‐ and C‐terminal tails were evaluated using ¹H‐¹⁵N HSQC and ¹H‐¹⁵N heteronuclear NOE NMR experiments to assess their structural integrity. These constructs provide significantly improved NMR spectra, with minimal structural perturbations to the ordered regions of the protein structure. As a representative example, we compare the solution structures of the full length and DXMS‐based truncated construct for a 77‐residue partially disordered DUF896 family protein YnzC from Bacillus subtilis, where deletion of the disordered residues (ca. 40% of the protein) does not affect the native structure. In addition, we demonstrate that throughput of the DXMS process can be increased by analyzing mixtures of up to four proteins without reducing the sequence coverage for each protein. Our results demonstrate that DXMS can serve as a central component of a process for optimizing protein constructs for NMR structure determination. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Refocusing revisited: An optimized,gradient-enhanced refocused HSQC and its applications in 2D and 3D NMR and in deuterium exchange experiments

Summary 2D ¹⁵N-¹H correlation spectra are ideal for measuring backbone amide populations to determine amide exchange protection factors in studies of protein folding or other structural features. Most protein NMR spectroscopists use HSQC, which has been shown to be generally superior to HMQC in both resolution and sensitivity. The refocused HSQC experiment is intrinsically less sensitive than the regular HSQC, due to T₂ relaxation during the refocusing delays. However, we show here that, when high ¹⁵N resolution is needed, an optimized refocused HSQC sequence that utilizes a semi-constant time evolution period and pulsed field gradients has better signal-to-noise ratio and resolution, and integrates more accurately, than a similar HSQC. The differences are demonstrated on a 20 kDa protein. The technique can also be applied to 3D NOESY experiments to eliminate strong NH₂ geminal peaks and their truncation artefacts at a modest cost in sensitivity.     

Production of large quantities of isotopically labeled protein in Pichia pastoris by fermentation

Heterologous expression in Pichia pastoris has many of the advantages of eukaryotic expression, proper folding and disulfide bond formation, glycosylation, and secretion. Contrary to other eukaryotic systems, protein production from P.pastoris occurs in simple minimal defined media making this system attractive for production of labeled proteins for NMR analysis. P.pastoris is therefore the expression system of choice for NMR of proteins that cannot be refolded from inclusion bodies or that require post-translational modifications for proper folding or function. The yield of expressed proteins from P.pastoris depends critically on growth conditions, and attainment of high cell densities by fermentation has been shown to improve protein yields by 10–100-fold. Unfortunately, the cost of the isotopically enriched fermentation media components, particularly 15NH4OH, is prohibitively high. We report fermentation methods that allow for both 15N- labeling from (15NH4)2SO4 and 13C-labeling from 13C-glucose or 13C-glycerol of proteins produced in Pichia pastoris. Expression of an 83 amino acid fragment of thrombomodulin with two N-linked glycosylation sites shows that fermentation is more cost effective than shake flask growth for isotopic enrichment.     

Quantitation of rapid proton-deuteron amide exchange using hadamard spectroscopy

The rates of amide proton exchange in protein backbones are very useful reporters of accessibility and structural stability of specific residues and secondary structure elements. Measurement by monitoring changes in intensity of cross-peaks in standard (15)N-(1)H HSQC spectra as protons are replaced by solvent deuterons has become widely accepted. However, these methods are limited to relatively slow rates due to time limitations of the conventional 2D HSQC experiment. Here we show that a Hadamard encoded version of the HSQC, which relies on a multiplexed, frequency selective, excitation in the (15)N dimension, extends application to rates that are as much as an order of magnitude faster than those previously accessible.     

Secondary structure and calcium-induced folding of the Clostridium thermocellum dockerin domain determined by NMR spectroscopy

Assembly of the cellulosome, a large, extracellular cellulase complex, depends upon docking of a myriad of enzymatic subunits to homologous receptors, or cohesin domains, arranged in tandem along a noncatalytic scaffolding protein. Docking to the cohesin domains is mediated by a highly conserved domain, dockerin (DS), borne by each enzymatic subunit. DS consists of two 22-amino-acid duplicated sequences, each bearing homology to the EF-hand calcium-binding loop. To compare the DS structure with that of the EF-hand helix-loop-helix motif, we analyzed the solution secondary structure of the DS from the cellobiohydrolase CelS subunit of the Clostridium thermocellum cellulosome using multidimensional heteronuclear NMR spectroscopy. The effect of Ca(2+)-binding on the DS structure was first investigated by using 2D (15)N-(1)H HSQC NMR spectroscopy. Changes in the spectra during Ca(2+) titration revealed that Ca(2+) induces folding of DS into its tertiary structure. This Ca(2+)-induced protein folding distinguishes DS from typical EF-hand-containing proteins. Sequential backbone assignments were determined for 63 of 69 residues. Analysis of the NOE connectivities and H(alpha) chemical shifts revealed that each half of the dockerin contains just one alpha-helix, comparable to the F-helix of the EF-hand motif. Thus, the structure of the DS Ca(2+)-binding subdomain deviates from that of the canonical EF-hand motif.     

Observing selected domains in multi-domain proteins via sortase-mediated ligation and NMR spectroscopy

NMR spectroscopy has distinct advantages for providing insight into protein structures, but faces significant resolution challenges as protein size increases. To alleviate such resonance overlap issues, the ability to produce segmentally labeled proteins is beneficial. Here we show that the S. aureus transpeptidase sortase A can be used to catalyze the ligation of two separately expressed domains of the same protein, MecA (B. subtilis). The yield of purified, segmentally labeled MecA protein conjugate is ~40%. The resultant HSQC spectrum obtained from this domain-labeled conjugate demonstrates successful application of sortase A for segmental labeling of multi-domain proteins for solution NMR study.     

Intracellular pH modulates quinary structure

NMR spectroscopy can provide information about proteins in living cells. pH is an important characteristic of the intracellular environment because it modulates key protein properties such as net charge and stability. Here, we show that pH modulates quinary interactions, the weak, ubiquitous interactions between proteins and other cellular macromolecules. We use the K10H variant of the B domain of protein G (GB1, 6.2 kDa) as a pH reporter in Escherichia coli cells. By controlling the intracellular pH, we show that quinary interactions influence the quality of in‐cell ¹⁵N–¹H HSQC NMR spectra. At low pH, the quality is degraded because the increase in attractive interactions between E. coli proteins and GB1 slows GB1 tumbling and broadens its crosspeaks. The results demonstrate the importance of quinary interactions for furthering our understanding of protein chemistry in living cells.