A synergistic approach to protein crystallization: Combination of a fixed‐arm carrier with surface entropy reduction

Protein crystallographers are often confronted with recalcitrant proteins not readily crystallizable, or which crystallize in problematic forms. A variety of techniques have been used to surmount such obstacles: crystallization using carrier proteins or antibody complexes, chemical modification, surface entropy reduction, proteolytic digestion, and additive screening. Here we present a synergistic approach for successful crystallization of proteins that do not form diffraction quality crystals using conventional methods. This approach combines favorable aspects of carrier‐driven crystallization with surface entropy reduction. We have generated a series of maltose binding protein (MBP) fusion constructs containing different surface mutations designed to reduce surface entropy and encourage crystal lattice formation. The MBP advantageously increases protein expression and solubility, and provides a streamlined purification protocol. Using this technique, we have successfully solved the structures of three unrelated proteins that were previously unattainable. This crystallization technique represents a valuable rescue strategy for protein structure solution when conventional methods fail.     

The relationship between the solvent-accessible surface area of a protein and the number of native contacts in its structure

The degree of protein folding is characterized either by the solvent-accessible surface area (S _ASA ) or the actual number of native contacts (N _cont). Evidently, these values should correlate with each other, as a decrease in S _ASA caused by the change in protein conformation during its folding must be accompanied by the corresponding increase in the number of native contacts. It is shown that this correlation does exist and is very strong (the correlation coefficient exceeds 99%), which can be used for an accurate and rapid estimation of the protein surface area from the number of native contacts. Among the methods commonly used for calculating the native contacts, the atom-atom approach gives the best fit if hydrogen atoms are taken into account and the cutoff value for the distance between the centers of atoms is taken to be 8 Å. The latter means that two layers of surface atoms are required to shield the protein core from the solvent.     

Toward rational protein crystallization: A Web server for the design of crystallizable protein variants

Growing well-diffracting crystals constitutes a serious bottleneck in structural biology. A recently proposed crystallization methodology for "stubborn crystallizers" is to engineer surface sequence variants designed to form intermolecular contacts that could support a crystal lattice. This approach relies on the concept of surface entropy reduction (SER), i.e., the replacement of clusters of flexible, solvent-exposed residues with residues with lower conformational entropy. This strategy minimizes the loss of conformational entropy upon crystallization and renders crystallization thermodynamically favorable. The method has been successfully used to crystallize more than 15 novel proteins, all stubborn crystallizers. But the choice of suitable sites for mutagenesis is not trivial. Herein, we announce a Web server, the surface entropy reduction prediction server (SERp server), designed to identify mutations that may facilitate crystallization. Suggested mutations are predicted based on an algorithm incorporating a conformational entropy profile, a secondary structure prediction, and sequence conservation. Minor considerations include the nature of flanking residues and gaps between mutation candidates. While designed to be used with default values, the server has many user-controlled parameters allowing for considerable flexibility. Within, we discuss (1) the methodology of the server, (2) how to interpret the results, and (3) factors that must be considered when selecting mutations. We also attempt to benchmark the server by comparing the server's predictions with successful SER structures. In most cases, the structure yielding mutations were easily identified by the SERp server. The server can be accessed at http://www.doe-mbi.ucla.edu/Services/SER.     

Purification, crystallization and preliminary X-ray diffraction analysis of the phage T4 vertex protein gp24 and its mutant forms

The study of bacteriophage T4 assembly has revealed regulatory mechanisms pertinent not only to viruses but also to macromolecular complexes. The capsid of bacteriophage T4 is composed of the major capsid protein gp23, and a minor capsid protein gp24, which is arranged as pentamers at the vertices of the capsid. In this study the T4 capsid protein gp24 and its mutant forms were overexpressed and purified to homogeneity. The overexpression from plasmid vectors of all the constructs in Escherichia coli yields biologically active protein in vivo as determined by assembly of active virus following infection with inactivated gene 24 mutant viruses. The gp24 mutant was subjected to surface entropy reduction by mutagenesis and reductive alkylation in order to improve its crystallization properties and diffraction quality. To determine if surface mutagenesis targeting would result in diffractable crystals, two glutamate to alanine mutations (E89A,E90A) were introduced. We report here the biochemical observations and consequent mutagenesis experiment that resulted in improvements in the stability, crystallizability and crystal quality of gp24 without affecting the overall folding. Rational modification of the protein surface to achieve crystallization appears promising for improving crystallization behavior and crystal diffracting qualities. The crystal of gp24(E89A,E90A) diffracted to 2.6A resolution compared to wild-type gp24 at 3.80A resolution under the same experimental conditions. Surface mutation proved to be a better method than reductive methylation for improving diffraction quality of the gp24 crystals.     

Fractal nature of protein surface roughness: a note on quantification of change of surface roughness in active sites,before and after binding

Year 2010 marked the 25th year since we came to know that roughness of a protein surface has fractal symmetry. Ever since the publication of Lewis and Rees' paper, hundreds of works from a spectrum of perspectives have established that fractal dimension (FD) can be considered as a reliable marker that describes roughness of protein surface objectively. In this article, we introduce readers to the fundamentals of fractals and present categorical biophysical and geometrical reasons as to why FD‐based constructs can describe protein surface roughness more accurately. We then review the commonality (and the lack of it) between numerous approaches that have attempted to investigate protein surface with fractal measures, before exploring the patterns in the results that they have produced. Apart from presenting the genealogy of approaches and results, we present an analysis that quantifies the difference in surface roughness in stretches of protein surface containing the active site, before and after binding to ligands, to underline the utility of FD‐based measures further. It has been found that surface stretches containing the active site, in general, undergo a significant increment in its roughness after binding. After presenting the entire repertoire of FD‐based surface roughness studies, we talk about two yet‐unexplored problems where application of FD‐based techniques can help in deciphering underlying patterns of surface interactions. Finally, we list the limitations of FD‐based constructs and put down several precautions that one must take while working with them. Copyright © 2013 John Wiley & Sons, Ltd.     

Oxidation of apolipoprotein(a) inhibits kringle-associated lysine binding: the loss of intrinsic protein fluorescence suggests a role for tryptophan residues in the lysine binding site.

Lipoprotein(a) [Lp(a)] is a low-density lipoprotein complex consisting of apolipoprotein(a) [apo(a)] disulfide-linked to apolipoprotein B-100. Lp(a) has been implicated in atherogenesis and thrombosis through the lysine binding site (LBS) affinity of its kringle domains. We have examined the oxidative effect of 2,2'-azobis-(amidinopropane) HCl (AAPH), a mild hydrophilic free radical initiator, upon the ability of Lp(a) and recombinant apo(a), r-apo(a), to bind through their LBS domains. AAPH treatment caused a time-dependent decrease in the number of functional Lp(a) or r-apo(a) molecules capable of binding to fibrin or lysine-Sepharose and in the intrinsic protein fluorescence of both Lp(a) and r-apo(a). The presence of a lysine analogue during the reaction prevented the loss of lysine binding and provided a partial protection from the loss of tryptophan fluorescence. The partial protection of fluorescence by lysine analogues was observed in other kringle-containing proteins, but not in proteins lacking kringles. No significant aggregation, fragmentation, or change in conformation of Lp(a) or r-apo(a) was observed as assessed by native or SDS-PAGE, light scattering, retention of antigenicity, and protein fluorescence emission spectra. Our results suggest that AAPH destroys amino acids in the kringles of apo(a) that are essential for lysine binding, including one or more tryptophan residues. The present study, therefore, raises the possibility that the biological roles of Lp(a) may be mediated by its state of oxidation, especially in light of our previous study showing that the reductive properties of sulfhydryl-containing compounds increase the LBS affinity of Lp(a) for fibrin.     

Cadmium-induced crystallization of proteins: II. Crystallization of the Salmonella typhimurium histidine-binding protein in complex with L-histidine, L-arginine, or L-lysine.

To further investigate favorable effects of divalent cations on the formation of protein crystals, three complexes of Salmonella typhimurium histidine-binding protein were crystallized with varying concentrations of cadmium salts. For each of the three histidine-binding protein complexes, cadmium cations were found to promote or improve crystallization. The optimal cadmium concentration is ligand specific and falls within a narrow concentration range. In each case, crystals grown in the presence of cadmium diffract to better than 2.0 angstroms resolution and belong to the orthorhombic space group P2(1)2(1)2(1). From our results and from the analysis of cadmium sites in well-refined protein structures, we propose that cadmium addition provides a generally useful technique to modify crystal morphology and to improve diffraction quality.     

Use of a fusion protein to obtain crystals suitable for X-ray analysis: crystallization of a GST-fused protein containing the DNA-binding domain of DNA replication-related element-binding factor, DREF.

Crystals of glutathione-S-transferase (GST)-fused protein containing the DNA-binding domain of DNA replication-related element-binding factor, DREF, were obtained under crystallization conditions similar to those for GST. Preliminary X-ray crystallographic analysis revealed that crystals of the GST-fused protein belong to space group P6(1)22 or P6(5)22 with unit cell dimensions a = b = 140.4 A, c = 93.5 A and gamma = 120 degrees, having one molecule in the crystallographic asymmetric unit. The crystals diffract to 2.5 A resolution. The cell dimensions are related to those of GST crystals thus far reported. Crystallization of the DNA-binding domain that was cleaved from the fused protein by thrombin was also carried out using several methods under numerous conditions, but efforts to produce well-ordered large crystals were unsuccessful. A possible application of GST-fusion proteins for small target proteins or domains to obtain crystals suitable for X-ray structure determination is proposed.     

Crystal structure of a Fab complex formed with PfMSP1-19, the C-terminal fragment of merozoite surface protein 1 from Plasmodium falciparum: a malaria vaccine candidate

Merozoite surface protein 1 (MSP1) is the major protein component on the surface of the merozoite, the erythrocyte-invasive form of the malaria parasite Plasmodium. Present in all species of Plasmodium, it undergoes two distinct proteolytic maturation steps during the course of merozoite development that are essential for invasion of the erythrocyte. Antibodies specific for the C-terminal maturation product, MSP1-19, can inhibit erythrocyte invasion and parasite growth. This polypeptide is therefore considered to be one of the more promising malaria vaccine candidates. We describe here the crystal structure of recombinant MSP1-19 from P.falciparum (PfMSP1-19), the most virulent species of the parasite in humans, as a complex with the Fab fragment of the monoclonal antibody G17.12. This antibody recognises a discontinuous epitope comprising 13 residues on the first epidermal growth factor (EGF)-like domain of PfMSP1-19. Although G17.12 was raised against the recombinant antigen expressed in an insect cell/baculovirus system, it binds uniformly to the surface of merozoites from the late schizont stage, showing that the cognate epitope is exposed on the naturally occurring MSP1 polypeptide complex. Although the epitope includes residues that have been mapped to regions recognised by invasion-inhibiting antibodies studied by other workers, G17.12 does not inhibit erythrocyte invasion or MSP1 processing.     

Cell surface of the fish pathogenic bacterium Aeromonas salmonicida: I. Relationship between autoagglutination and the presence of a major cell envelope protein

A comparison was made of membrane protein patterns of various Aeromonas salmonicida strains, initially isolated from different habitats with respect to fish species affected, pathological entity, and geographic location of the outbreak of the disease. A major protein with a molecular weight of 54 000 was found in all autoagglutinating strains, whereas this protein is present in low amounts, or not at all, in non-autoagglutinating strains. Evidence for a causal relationship between the presence of this protein and the phenomenon of autoagglutination came from the observation that a change of the growth medium led simultaneously to an almost complete loss of the additional cell envelope protein and the property of autoagglutination. As it has already been reported that autoagglutination is correlated with the presence of an additional cell surface layer, we hypothesize that the additional cell envelope protein is the (major) subunit of this layer. The application of the gel immuno radio assay, an immunological technique suited to detect antigens in a gel, revealed that the additional cell envelope proteins of all tested strains are immunologically related. The possibility to the use of this protein as a component of a vaccine against A. salmonicida infections is discussed.     

Merozoite surface protein 2 of Plasmodium falciparum: expression, structure, dynamics, and fibril formation of the conserved N-terminal domain

Merozoite surface protein 2 (MSP2) is a GPI-anchored protein on the surface of the merozoite stage of the malaria parasite Plasmodium falciparum. It is largely disordered in solution, but has a propensity to form amyloid-like fibrils under physiological conditions. The N-terminal conserved region (MSP2(1-25)) is part of the protease-resistant core of these fibrils. To investigate the structure and dynamics of this region, its ability to form fibrils, and the role of individual residues in these properties, we have developed a bacterial expression system that yields > or =10 mg of unlabeled or (15)N-labeled peptide per litre of culture. Two recombinant versions of MSP2(1-25), wild-type and a Y7A/Y16A mutant, have been produced. Detailed conformational analysis of the wild-type peptide and backbone (15)N relaxation data indicated that it contains beta-turn and nascent helical structures in the central and C-terminal regions. Residues 6-21 represent the most ordered region of the structure, although there is some flexibility around residues 8 and 9. The 10-residue sequence (MSP2(7-16)) (with two Tyr residues) was predicted to have a higher propensity for beta-aggregation than the 8-mer sequence (MSP2(8-15)), but there was no significant difference in conformation between MSP2(1-25) and [Y7A,Y16A]MSP2(1-25) and the rate of fibril formation was only slightly slower in the mutant. The peptide expression system described here will facilitate further mutational analyses to define the roles of individual residues in transient structural elements and fibril formation, and thus contribute to the further development of MSP2 as a malaria vaccine candidate.     

Molecular dynamics studies on the NMR structures of rabbit prion protein wild type and mutants: surface electrostatic charge distributions

Prion diseases are invariably fatal and highly infectious neurodegenerative diseases that affect a wide variety of mammalian species such as sheep and goats, cattle, deer and elk, and humans. But for rabbits, studies have shown that they have a low susceptibility to be infected by prion diseases. This paper does molecular dynamics (MD) studies of rabbit NMR structures (of the wild type and its two mutants of two surface residues), in order to understand the specific mechanism of rabbit prion proteins (RaPrP^C). Protein surface electrostatic charge distributions are specially focused to analyze the MD trajectories. This paper can conclude that surface electrostatic charge distributions indeed contribute to the structural stability of wild-type RaPrP^C; this may be useful for the medicinal treatment of prion diseases.     

Studies on the maximization of recombinant Helicobacter pylori neutrophil-activating protein production in Escherichia coli: application of Taguchi robust design and response surface methodology for process optimization

The neutrophil-activating protein of Helicobacter pylori (HP-NAP) is a major antigen responsible for the generation of immune response in an infected individual. The cloning and expression of the gene corresponding to neutrophil-activating protein (NAP) were followed by process development for enhanced production and purification. The production process was developed in two parts. In the first part, some of the cultivation medium components (viz. carbon to nitrogen ratio, concentrations of sodium polyphosphate and magnesium sulphate) were optimized using the Taguchi robust experimental design. The intracellular NAP production level after 24 h of cultivation was considered as the target function or the dependent variable. There was a 76.8% increase in the NAP production level. Using this optimal medium composition obtained in the first part, the temperature of cultivation and the pH of cultivation medium were optimized in the second part. The NAP production level at hour 30 of cultivation was considered as the target function or the dependent variable. The optimal values for these two independent variables were 37.2 °C and 6.3 respectively. At this combination of temperature and pH, the theoretical maximum NAP production level was 1280 mg l^–1. This optimal combination was verified experimentally and the NAP production level was found to be 1261 mg l^–1. The optimization of the cultivation conditions resulted in a 61.5% increase in NAP production level. About a 2.91-fold overall increase in NAP production level at hour 24 of cultivation was achieved through process optimization.     

Litomosoides sigmodontis: vaccine-induced immune responses against Wolbachia surface protein can enhance the survival of filarial nematodes during primary infection

Wolbachia are bacteria present within the tissues of most filarial nematodes. Filarial nematode survival is known to be affected by immune responses generated during filarial nematode infection and immune responses to Wolbachia can be found in different species harbouring filarial nematode infections, including humans. Using the rodent filarial model Litomosoides sigmodontis, we show that pre-exposure to wolbachia surface protein in a Th1 context (but not in a Th2-context) enhances worm survival on subsequent challenge. This study suggests that despite abundant evidence that pro-inflammatory reactions to the endosymbiont have detrimental effects on the both the nematode and mammalian host, they may under some circumstances be beneficial to the nematode.     

Giardia duodenalis: direct experimental evidence for the absence of a glycosylphosphatidylinositol anchor in a variant surface protein

The trophozoites of Giardia duodenalis express variant surface proteins (VSPs) that cover the entire surface of the cell and can be altered by antigenic variation. In the present study, a VSP (VSPH7) expressed by the Giardia GS isolate was purified using Triton-X-114 extraction/phase partitioning and a combination of column chromatography methods. The purified VSP was typed by mass spectrometric fingerprint mapping and peptide sequencing and found to share 58-99.8% peptide identity with the VSPH7 protein sequence previously deduced from the cloned cDNA. Carbohydrate compositional analyses consistently showed the presence of galactose in the VSP preparations but a direct association of carbohydrate with the VSPH7 could not be established. Analysis of the C-terminal part of the purified VSPH7 by off-blot myo-inositol analysis provided for the first time direct experimental evidence that this protein is not modified via a GPI lipid.