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| 2. |
- Allison, Timothy M., et al.
(författare)
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Complementing machine learning‐based structure predictions with native mass spectrometry
- 2022
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Ingår i: Protein Science. - : John Wiley & Sons. - 0961-8368 .- 1469-896X. ; 31:6
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Tidskriftsartikel (refereegranskat)abstract
- The advent of machine learning-based structure prediction algorithms such as AlphaFold2 (AF2) and RoseTTa Fold have moved the generation of accurate structural models for the entire cellular protein machinery into the reach of the scientific community. However, structure predictions of protein complexes are based on user-provided input and may require experimental validation. Mass spectrometry (MS) is a versatile, time-effective tool that provides information on post-translational modifications, ligand interactions, conformational changes, and higher-order oligomerization. Using three protein systems, we show that native MS experiments can uncover structural features of ligand interactions, homology models, and point mutations that are undetectable by AF2 alone. We conclude that machine learning can be complemented with MS to yield more accurate structural models on a small and large scale.
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| 3. |
- Almqvist, Jonas, et al.
(författare)
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Docking and homology modeling explain inhibition of the human vesicular glutamate transporters
- 2007
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Ingår i: Protein Science. - 0961-8368 .- 1469-896X. ; 16:9, s. 1819-1829
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Tidskriftsartikel (refereegranskat)abstract
- As membrane transporter proteins, VGLUT1-3 mediate the uptake of glutamate into synaptic vesicles at presynaptic nerve terminals of excitatory neural cells. This function is crucial for exocytosis and the role of glutamate as the major excitatory neurotransmitter in the central nervous system. The three transporters, sharing 76% amino acid sequence identity in humans, are highly homologous but differ in regional expression in the brain. Although little is known regarding their three- dimensional structures, hydropathy analysis on these proteins predicts 12 transmembrane segments connected by loops, a topology similar to other members in the major facilitator superfamily, where VGLUT1-3 have been phylogenetically classified. In this work, we present a three- dimensional model for the human VGLUT1 protein based on its distant bacterial homolog in the same superfamily, the glycerol- 3-phosphate transporter from Escherichia coli. This structural model, stable during molecular dynamics simulations in phospholipid bilayers solvated by water, reveals amino acid residues that face its pore and are likely to affect substrate translocation. Docking of VGLUT1 substrates to this pore localizes two different binding sites, to which inhibitors also bind with an overall trend in binding affinity that is in agreement with previously published experimental data.
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| 5. |
- Andrésen, Cecilia, et al.
(författare)
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Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance
- 2010
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Ingår i: Protein Science. - : Cold Spring Harbor Laboratory Press. - 0961-8368 .- 1469-896X. ; 19:4, s. 680-692
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Tidskriftsartikel (refereegranskat)abstract
- The self-assembling MexA-MexB-OprM efflux pump system, encoded by the mexO operon, contributes to facile resistance of Pseudomonas aeruginosa by actively extruding multiple antimicrobials. MexR negatively regulates the mexO operon, comprising two adjacent MexR binding sites, and is as such highly targeted by mutations that confer multidrug resistance (MDR). To understand how MDR mutations impair MexR function, we studied MexR-wt as well as a selected set of MDR single mutants distant from the proposed DNA-binding helix. Although DNA affinity and MexA-MexB-OprM repression were both drastically impaired in the selected MexR-MDR mutants, MexR-wt bound its two binding sites in the mexO with high affinity as a dimer. In the MexR-MDR mutants, secondary structure content and oligomerization properties were very similar to MexR-wt despite their lack of DNA binding. Despite this, the MexR-MDR mutants showed highly varying stabilities compared with MexR-wt, suggesting disturbed critical interdomain contacts, because mutations in the DNA-binding domains affected the stability of the dimer region and vice versa. Furthermore, significant ANS binding to MexR-wt in both free and DNA-bound states, together with increased ANS binding in all studied mutants, suggest that a hydrophobic cavity in the dimer region already shown to be involved in regulatory binding is enlarged by MDR mutations. Taken together, we propose that the biophysical MexR properties that are targeted by MDR mutations stability, domain interactions, and internal hydrophobic surfaces are also critical for the regulation of MexR DNA binding.
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| 6. |
- Bauer, Mikael, et al.
(författare)
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Zn2+ binding to human calbindin D(28k) and the role of histidine residues.
- 2008
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Ingår i: Protein Science. - : Wiley. - 1469-896X .- 0961-8368. ; 17:4, s. 760-767
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Tidskriftsartikel (refereegranskat)abstract
- We have studied the binding of Zn2+ to the hexa EF-hand protein, calbindin D(28k)-a strong Ca2+-binder involved in apoptosis regulation-which is highly expressed in brain tissue. By use of radioblots, isothermal titration calorimetry, and competition with a fluorescent Zn2+ chelator, we find that calbindin D(28k) binds Zn2+ to three rather strong sites with dissociation constants in the low micromolar range. Furthermore, we conclude based on spectroscopic investigations that the Zn2+-bound state is structurally distinct from the Ca2+-bound state and that the two forms are incompatible, yielding negative allosteric interaction between the zinc- and calcium-binding events. ANS titrations reveal a change in hydrophobicity upon binding Zn2+. The binding of Zn2+ is compatible with the ability of calbindin to activate myo-inositol monophosphatase, one of the known targets of calbindin. Through site-directed mutagenesis, we address the role of cysteine and histidine residues in the binding of Zn2+. Mutation of all five cysteines into serines has no effect on Zn2+-binding affinity or stoichiometry. However, mutating histidine 80 into a glutamine reduces the binding affinity of the strongest Zn2+ site, indicating that this residue is involved in coordinating the Zn2+ ion in this site. Mutating histidines 5, 22, or 114 has significantly smaller effects on Zn2+-binding affinity.
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| 7. |
- Baumgartner, Stefan, et al.
(författare)
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The discoidin domain family revisited : new members from prokaryotes and a homology-based fold prediction
- 1998
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 7:7, s. 31-1626
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Tidskriftsartikel (refereegranskat)abstract
- Members of the discoidin (DS) domain family, which includes the C1 and C2 repeats of blood coagulation factors V and VIII, occur in a great variety of eukaryotic proteins, most of which have been implicated in cell-adhesion or developmental processes. So far, no three-dimensional structure of a known example of this extracellular module has been determined, limiting the usefulness of identifying a new sequence as member of this family. Here, we present results of a recent search of the protein sequence database for new DS domains using generalized profiles, a sensitive multiple alignment-based search technique. Several previously unrecognized DS domains could be identified by this method, including the first examples from prokaryotic species. More importantly, we present statistical, structural, and functional evidence that the D1 domain of galactose oxidase whose three-dimensional structure has been determined at 1.7 A resolution, is a distant member of this family. Taken together, these findings significantly expand the concept of the DS domain, by extending its taxonomic range and by implying a fold prediction for all its members. The proposed alignment with the galactose oxidase sequence makes it possible to construct homology-based three-dimensional models for the most interesting examples, as illustrated by an accompanying paper on the C1 and C2 domains of factor V.
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| 8. |
- Berbalk, Christoph, et al.
(författare)
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Accuracy analysis of multiple structure alignments
- 2009
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 18:10, s. 2027-2035
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Tidskriftsartikel (refereegranskat)abstract
- Protein structure alignment methods are essential for many different challenges in protein science, such as the determination of relations between proteins in the fold space or the analysis and prediction of their biological function. A number of different pairwise and multiple structure alignment (MStA) programs have been developed and provided to the community. Prior knowledge of the expected alignment accuracy is desirable for the user of such tools. To retrieve an estimate of the performance of current structure alignment methods, we compiled a test suite taken from literature and the SISYPHUS database consisting of proteins that are difficult to align. Subsequently, different MStA programs were evaluated regarding alignment correctness and general limitations. The analysis shows that there are large differences in the success between the methods in terms of applicability and correctness. The latter ranges from 44 to 75% correct core positions. Taking only the best method result per test case this number increases to 84%. We conclude that the methods available are applicable to difficult cases, but also that there is still room for improvements in both, practicability and alignment correctness. An approach that combines the currently available methods supported by a proper score would be useful. Until then, a user should not rely on just a single program.
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| 9. |
- Bergkvist, Anders, et al.
(författare)
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Surface interactions in the complex between cytochrome f and the E43Q/D44N and E59K/E60Q plastocyanin double mutants as determined by (1)H-NMR chemical shift analysis
- 2001
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Ingår i: Protein Science. - : John Wiley & Sons. - 0961-8368 .- 1469-896X. ; 10:12, s. 2623-2626
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Tidskriftsartikel (refereegranskat)abstract
- A combination of site-directed mutagenesis and NMR chemical shift perturbation analysis of backbone and side-chain protons has been used to characterize the transient complex of the photosynthetic redox proteins plastocyanin and cytochrome f. To elucidate the importance of charged residues on complex formation, the complex of cytochrome f and E43Q/D44N or E59K/E60Q spinach plastocyanin double mutants was studied by full analysis of the (1)H chemical shifts by use of two-dimensional homonuclear NMR spectra. Both mutants show a significant overall decrease in chemical shift perturbations compared with wild-type plastocyanin, in agreement with a large decrease in binding affinity. Qualitatively, the E43Q/D44N mutant showed a similar interaction surface as wild-type plastocyanin. The interaction surface in the E59K/E60Q mutant was distinctly different from wild type. It is concluded that all four charged residues contribute to the affinity and that residues E59 and E60 have an additional role in fine tuning the orientation of the proteins in the complex.
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| 10. |
- Berglund, Lisa, et al.
(författare)
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The epitope space of the human proteome
- 2008
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 17:4, s. 606-613
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Tidskriftsartikel (refereegranskat)abstract
- In the post-genome era, there is a great need for protein-specific affinity reagents to explore the human proteome. Antibodies are suitable as reagents, but generation of antibodies with low cross-reactivity to other human proteins requires careful selection of antigens. Here we show the results from a proteomewide effort to map linear epitopes based on uniqueness relative to the entire human proteome. The analysis was based on a sliding window sequence similarity search using short windows (8, 10, and 12 amino acid residues). A comparison of exact string matching (Hamming distance) and a heuristic method (BLAST) was performed, showing that the heuristic method combined with a grid strategy allows for whole proteome analysis with high accuracy and feasible run times. The analysis shows that it is possible to find unique antigens for a majority of the human proteins, with relatively strict rules involving low sequence identity of the possible linear epitopes. The implications for human antibody-based proteomics efforts are discussed.
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