ISSN:1399-0047    

DOI:10.1107/S0907444994004592

出版商:International Union of Crystallography    

年代:1994

数据来源: WILEY

摘要:

This paper gives an overview of the science of crystals of biological macromolecules. The historical background of the field is outlined and the main achievements and open problems are discussed from both biological and physical–chemical viewpoints. Selected results, including data from the authors, illustrate this overview. The perspectives of crystallogenesis for structural biology, but also more general trends, are presente

ISSN:1399-0047    

DOI:10.1107/S0907444994001344

出版商:International Union of Crystallography    

年代:1994

数据来源: WILEY

摘要:

It is of considerable interest to separate the processes of viral infectivity and virion assembly. Until recently this has only been possible with viruses that could be disassembled and reassembledin vitro. Even in these cases it was difficult to establish the authenticity of reassembled capsid protein because of possible irreversible damage that may have occurred to the protein during disassembly. An ideal method for the study of virus assembly is a protein expression system in which conditions are appropriate for spontaneous particle formation from freshly synthesized polypeptides. The baculovirus expression system has proven to be an excellent means to this end. Recently, this approach has been used to study theT= 3 Flock House insect virus and it has been demonstrated that subunits with the wild‐type protein sequence, and with site‐specific mutations that prevent particle maturation, will assemble and crystallize. This same approach has now been used at Purdue to study theT= 4Nudaurelia ω capensisinsect virus. There is no cell culture system currently available for the study of NωV, thus the expression system provides the first opportunity to study assembly under controlled condi

ISSN:1399-0047    

DOI:10.1107/S090744499400123X

出版商:International Union of Crystallography    

年代:1994

数据来源: WILEY

摘要:

The early stages of the crystallization process of porcine pancreatic α‐amylase were investigated by quasi‐elastic light scattering. It is shown that at 288 and 293 K the diffusion coefficient does not monotonically change with increasing protein concentration but passes through a maximum at 10 mg ml−1. In supersaturated solutions, prior to nucleation, the protein is strictly monodisperse. Nucleation induces the formation of aggregates and a polydispersity of, for example, 18% for an initial supersaturationC/Ce= 5.8. Monodispersity is restored after the nuclei have grown and partially consumed the solute. On the other hand, polydispersity increases up to 20% at 298 K if the protein concentration decreases to 3–4 mg ml−1, values at which the solutions are under‐saturated. When the protein concentration exceeds 5–6 mg ml−1the protein becomes monodisperse again. These results, confirmed by those of another system we are studying (bovine pancreatic trypsin inhibitor), are at variance with the statements that supersaturation is always at the origin of aggregation and polydispersity, and that in undersaturated solutions the diffusion coefficient should remain constant for obtaining crystals once

ISSN:1399-0047    

DOI:10.1107/S0907444993014416

出版商:International Union of Crystallography    

年代:1994

数据来源: WILEY

摘要:

A dilute solution parameter obtained from static light‐scattering measurements is proposed as a predictor for protein crystallization experiments. The osmotic second virial coefficients,B22, have been measured for a variety of proteins in solvents that are known to promote crystallization and the values forB22were found to lie within a fairly narrow range which we refer to as a crystallization slot. Solution conditions which were known not to favor crystallization of the proteins resulted inB22values well outside the crystallization slo

ISSN:1399-0047    

DOI:10.1107/S0907444994001216

出版商:International Union of Crystallography    

年代:1994

数据来源: WILEY

摘要:

Lysozyme, which is known to crystallize readily in the presence of many salts, has never been crystallized by salting out with ammonium sulfate. In the present study, lysozyme was first completely desalted by treatment with strong cation‐ (H+form) and anion‐ (OH−form) exchange resins. This leads to a protein solution with only H+and OH−as counterions, corresponding to its isoionic point. Addition of 2.5–3 molar equivalents of H2SO4to isoionic lysozyme decreases the pH value to 9–8 and allows crystallization to take place. The space group was found to beP43212, similar to the classical lysozyme crystals grown in the presence of NaCl at pH 4.5, with unit‐cell dimensionsa=b= 78.9,c= 38.5 Å. Tentative explanation of the sulfate/lysozyme interaction was addressed by mass spectrometry, and shows non‐covalent binding of the

ISSN:1399-0047    

DOI:10.1107/S0907444994001320

出版商:International Union of Crystallography    

年代:1994

数据来源: WILEY

摘要:

Crystals of bovine cellular retinoic acid‐binding protein (CRABPI) have been grown from protein expressed inE. coli. Two different crystal forms were obtained. Crystals containing protein with the ligand all‐transretinoic acid belong to space groupP41orP43,a=b= 41.36,c= 202.71 Å and diffract to 2.5 Å. Crystals of CRABP with the synthetic retinoid analogue Am80 diffract to 1.9 Å with space groupP21and cell dimensionsa= 37.03,b= 105.93,c= 40.31 Å, β = 110.28°. Considerations in the crystallization of proteins with light‐sensitive li

ISSN:1399-0047    

DOI:10.1107/S0907444994001204

出版商:International Union of Crystallography    

年代:1994

数据来源: WILEY

摘要:

A novel strategy is presented for the crystallization of membrane proteins or other proteins with low solubility and/or stability. The method is illustrated with the lactose permease fromEscherichia coli, in which a fusion is constructed between the permease and a `carrier' protein. The carrier is a soluble, stable protein with its C and N termini close together in space at the surface of the protein, so that the carrier can be introduced into an internal position of the target protein. The carrier is chosen with convenient spectral or enzymatic properties, making the fusion protein easier to handle than the native molecule. Data are presented for the successful construction, expression and purification of a fusion product between lactose permease and cytochromeb562fromE. coli. The lactose transport activity of the fusion protein is similar to that of wild‐type lactose permease, and the fusion product has an absorption spectrum in the visible range which is essentially identical to that of cytochromeb562. The fusion protein has a higher proportional polar surface area than wild‐type permease, and should have better possibilities of forming the strong directional intermolecular contacts required of a crystal latt

ISSN:1399-0047    

DOI:10.1107/S0907444993014301

出版商:International Union of Crystallography    

年代:1994

数据来源: WILEY

摘要:

Enzymatic deglycosylation has been used in attempts to crystallize several glycoproteins with the aim of overcoming the problems resulting from heterogeneity and flexibility of the attached glycan chains. An endoglycosidase preparation fromFlavobacterium meningosepticum, comprising the enzymes endo F and PNGase‐F, was used in experiments on the mammalian binding proteins lactoferrin and haemopexin. Significant differences were found in the susceptibility of different proteins to deglycosylation. For human lactoferrin (Lf) and its recombinant N‐terminal half‐molecule (LfN), deglycosylation was rapid and complete, and was essential for obtaining high‐quality crystals of both apo‐Lf and LfN; for bovine Lf, however, complete deglycosylation did not occur. Similarly, for rabbit haemopexin the carbohydrate chain on the C‐terminal domain was easily removed, but the three chains on the N‐terminal domain proved more resistant and their removal led to some fragmentation of the protein. Nevertheless, this approach provided the only means of crystallizing the C‐terminal domain and is likely to be useful for othe

ISSN:1399-0047    

DOI:10.1107/S0907444993013435

出版商:International Union of Crystallography    

年代:1994

数据来源: WILEY

摘要:

Quasi‐elastic light scattering (QELS) was used to investigate quantitatively the mechanisms of nucleation, postnucleation growth, and dissolution in ensembles of both crystalline and amorphous aggregates of satellite tobacco mosaic virus (STMV), ferritin, apoferritin and pumpkin seed globulin. At low supersaturation conditions, as described previously for small molecule crystallization, the metastable region was obtained. Under these conditions aggregation took place, but crystallization did not proceed and critical nuclei did not form over a long period of time. The critical solution supersaturation necessary to obtain crystals, σ = ln(c/s) wherecandsare concentration and solubility of protein, varied from ∼0.1 for pumpkin seed globulin to ∼0.9 for STMV. For higher supersaturation conditions when aggregation processes leading to formation of crystals are not established immediately but after a certain induction period, the supersaturation‐dependent critical nuclear size,Rc, for different macromolecular systems was estimated from time‐dependent size‐distribution analyses to be in the range of ∼103for proteins such as pumpkin globulin to approximately 10 for virus particles. From the same data, the molar interfacial free energy was deduced to be 3.3–9.2 kJ mol−1. These are believed to be among the first estimates for macromolecular crystals. Under conditions of moderate supersaturation where induction periods preceded the appearance of critical nuclei, the potential barriers for formation were estimated to be in the range 8.3–50 kJ mol−1. Growth and dissolution kinetics for pumpkin seed globulin were investigated. These experiments allowed determination of protein solubilityversussolution temperature, protein and precipitant concentrations. Aggregation patterns which lead to crystal formation are distinctly different to those which produce an amorphous precipitate. The results provide additional evidence that QELS can be used to find general criteria that allow one to discriminate between conditions for a given protein system leading to crystalline or amorphous states at early sta

ISSN:1399-0047    

DOI:10.1107/S0907444993013319

出版商:International Union of Crystallography    

年代:1994

数据来源: WILEY