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Fluorinated aromatic amino acids (FAAs) are promising tools when studying protein structure and dynamics by NMR spectroscopy. The incorporation FAAs in mammalian expression systems has been introduced only recently. Here, we investigate the effects of FAAs incorporation in proteins expressed in human cells, focusing on the probability of incorporation and its consequences on the 19F NMR spectra. By combining 19F NMR, direct MS and x‐ray crystallography, we demonstrate that the probability of FAA incorporation is only a function of the FAA concentration in the expression medium and is a pure stochastic phenomenon. In contrast with the MS data, the x‐ray structures of carbonic anhydrase II reveal that while the 3D structure is not affected, certain positions lack fluorine, suggesting that crystallization selectively excludes protein molecules featuring subtle conformational modifications. This study offers a predictive model of the FAA incorporation efficiency and provides a framework for controlling protein fluorination in mammalian expression systems. [ABSTRACT FROM AUTHOR]

Several protein‐drug conjugates are currently being used in cancer therapy. These conjugates rely on cytotoxic organic compounds that are covalently attached to the carrier proteins or that interact with them via non‐covalent interactions. Human transthyretin (TTR), a physiological protein, has already been identified as a possible carrier protein for the delivery of cytotoxic drugs. Here we show the structure‐guided development of a new stable cytotoxic molecule based on a known strong binder of TTR and a well‐established anticancer drug. This example is used to demonstrate the importance of the integration of multiple biophysical and structural techniques, encompassing microscale thermophoresis, X‐ray crystallography and NMR. In particular, we show that solid‐state NMR has the ability to reveal effects caused by ligand binding which are more easily relatable to structural and dynamical alterations that impact the stability of macromolecular complexes. [ABSTRACT FROM AUTHOR]

Metalloproteins constitute a significant fraction of the proteome of all organisms and their characterization is critical for both basic sciences and biomedical applications. A large portion of metalloproteins bind paramagnetic metal ions, and paramagnetic NMR spectroscopy has been widely used in their structural characterization. However, the signals of nuclei in the immediate vicinity of the metal center are often broadened beyond detection. In this work, we show that it is possible to determine the coordination environment of the paramagnetic metal in the protein at a resolution inaccessible to other techniques. Taking the structure of a diamagnetic analogue as a starting point, a geometry optimization is carried out by fitting the pseudocontact shifts obtained from first principles quantum chemical calculations to the experimental ones. [ABSTRACT FROM AUTHOR]

This article describes the approach used to solve the structure of human IBA57 in‐house by 5‐amino‐2,4,6‐triiodoisophthalic acid (I3C) high‐energy‐remote single‐wavelength anomalous dispersion (SAD) phasing. Multiple orientations of the same triclinic crystal were exploited to acquire sufficient real data multiplicity for phasing. How the collection of an in‐house native data set and its joint use with the I3C derivative through a SIRAS approach decreases the data multiplicity needed by almost 50% is described. Furthermore, it is illustrated that there is a clear data‐multiplicity threshold value for success and failure in phasing, and how adding further data does not significantly affect substructure solution and model building. To our knowledge, this is the only structure present in the PDB that has been solved in‐house by remote SAD phasing in space group P1 using only one crystal. All of the raw data used, derived from the different orientations, have been uploaded to Zenodo in order to enable software developers to improve methods for data processing and structure solution, and for educational purposes. This article describes the methodological approach used to solve the structure of human IBA57; to our knowledge, this is the only structure present in the Protein Data Bank which has been solved in‐house by remote SAD phasing in space group P1 using only one crystal. [ABSTRACT FROM AUTHOR]

Resonance assignment and structural characterization of pharmacologically relevant proteins promise to improve understanding and safety of these proteins by rational design. However, the PEG coating that is used to evade the immune system also causes these molecules to "evade" the standard structural biology methodologies. We here demonstrate that it is possible to obtain the resonance assignment and a reliable structural model of large PEGylated proteins through an integrated approach encompassing NMR and X‐ray crystallography. Coating a protein with PEG may have structural consequences, but no single biophysical methodology can spot the possible modifications. This limitation has been overcome by the integration of solution and solid‐state NMR data, relaxometry, and X‐ray crystallography for the characterization of a PEGylated enzyme used in clinical practice. [ABSTRACT FROM AUTHOR]

The program REFMAC5 from CCP4 was modified to allow the simultaneous use of X-ray crystallographic data and paramagnetic NMR data (pseudocontact shifts and self- orientation residual dipolar couplings) and/or diamagnetic residual dipolar couplings. Incorporation of these long-range NMR restraints in REFMAC5 can reveal differences between solid-state and solution conformations of molecules or, in their absence, can be used together with X-ray crystallographic data for structural refinement. Since NMR and X-ray data are complementary, when a single structure is consistent with both sets of data and still maintains reasonably 'ideal' geometries, the reliability of the derived atomic model is expected to increase. The program was tested on five different proteins: the catalytic domain of matrix metalloproteinase 1, GB3, ubiquitin, free calmodulin and calmodulin complexed with a peptide. In some cases the joint re?nement produced a single model consistent with both sets of observations, while in other cases it indicated, outside the experimental uncertainty, the presence of different protein conformations in solution and in the solid state. [ABSTRACT FROM AUTHOR]

Abstract: Pseudocontact shifts (pcs) and paramagnetic residual dipolar couplings (rdc) provide structural information that can be used to assess the adequacy of a crystallographic structure to represent the solution structure of a protein. This can be done by attaching a lanthanide binding tag to the protein. There are cases in which only local rearrangements are sufficient to match the NMR data and cases where significant secondary structure or domain rearrangements from the solid state to the solution state are needed. We show that the two cases are easily distinguishable. Whereas the use of solution restraints in the latter case is described in the literature, here we deal with how to obtain a better model of the solution structure in a case (the catalytic domain of the matrix metalloproteinase MMP-1) of the former class. [Copyright &y& Elsevier]

TEM-72, a class A β-lactamase identified in isolates of Enterobacteriaceae, is a quadruple mutant of TEM-1 (Q39K, M182T, G238S and E240K) and shows extended-spectrum β-lactamase (ESBL) properties arising from the G238S and E240K substitutions. Although many structures of TEM variants have been published, they do not include an enzyme with the simultaneous presence of both of the ESBL-conferring G238S and E240K substitutions. Furthermore, the structure shows the presence of a citrate anion bound to the TEM-72 active site, where it interacts with all of the conserved residues of class A β-lactamases. The present structure supports the use of polycarboxylates as a scaffold for the design of broad-spectrum inhibitors of serine β-lactamases. [ABSTRACT FROM AUTHOR]

There is presently an increasing variety of choice in the software for macromolecular phasing and automated model building. In addition to its positive features, this variety poses the problem of which software to use in a specific crystallographic case. Moreover, it must be decided whether a sequence of programs should be used to achieve structure solution more accurately and more rapidly, taking into account the features of the different programs: some software is more suitable for dealing with low-symmetry rather than high-symmetry space groups in the detection of heavy-atom sites, while others can give better estimates of the figures of merit on phases in certain cases (which is crucial in dealing with maximum likelihood) and others are more suitable for chain tracing at low/medium-low resolution than at high resolution or vice versa. The `integrated' choice of different software has become popular among crystallographers, especially when facing crystallographic cases that are not straightforward. A few examples will be presented on the use of different programs to achieve the goal of structure solution and the associated practicalities that can make the difference between solving or not solving a structure. [ABSTRACT FROM AUTHOR]

The smallest extrinsic polypeptide of the water-oxidizing complex (PsbQ) was extracted and purified from spinach (Spinacia oleracea) photosystem II (PSII) membranes. It was then crystallized in the presence of Zn2+ and its structure was determined by X-ray diffraction at 1.95-Å resolution using the multiwavelength anomalous diffraction method, with the zinc as the anomalous scatterer. The crystal structure shows that the core of the protein is a four-helix bundle, whereas the amino-terminal portion, which possibly interacts with the photosystem core, is not visible in the crystal. The distribution of positive and negative charges on the protein surface might explain the ability of PsbQ to increase the binding of Cl- and Ca2+ and make them available to PSII. [ABSTRACT FROM AUTHOR]

The crystallographic structure of the complex between human aldose reductase (AR2) and one of its inhibitors, 1DD384, has been solved at 1.7 Å resolution from crystals obtained at pH 5.0. This structure shows that the binding of the inhibitor's hydrophilic head to the catalytic residues Tyr48 and His110 differs from that found previously with porcine AR2. The difference is attributed to a change in the protonation state of the inhibitor (pKa = 4.52) when soaked with crystals of human (at pH 5.0) or pig lens AR2 (at pH 6.2). This work demonstrates how strongly the detailed binding of the inhibitor's polar head depends on its protonation state. [ABSTRACT FROM AUTHOR]

The class B non-specific acid phosphatase AphA from Escherichia coli has been expressed in E. coli and purified following a new protocol. ESI mass speatroscopy shows that the purified enzyme solution contains two polypeptides with molecular weights differing by 185 Da corresponding to two different cleavage sites of the signal peptide from the AphA E. coli precursor, Despite the solution heterogeneity, X-ray quality crystals have been obtained However, the crystals have a tendency to give polymorphs and to lose long-range order with time while maintaining an intact crystal habit Crystals have been grown in space groups I222 and C2 with three different unit cells and different asymmetric unit contents. Diffraction data to 1.6 Å resolution have been collected with synchrotron radiation at ESRF and DESY. [ABSTRACT FROM AUTHOR]

A copper-containing amine oxidase extracted from bovine serum (BSAO) and purified to homogeneity has been deglycosylated and crystallized. The crystals obtained belong to space group P2[sub 1]2[sub 1]2[sub 1], with unit-cell parameters a = 77.68, b = 131.19, c = 134.00 Å, and diffract to at least 2.4 Å resolution. BSAO is the first mammalian amine oxidase to be crystallized. [ABSTRACT FROM AUTHOR]