| 1. |
- Almqvist, Jonas, et al.
(författare)
-
Docking and homology modeling explain inhibition of the human vesicular glutamate transporters
- 2007
-
Ingår i: Protein Science. - 0961-8368 .- 1469-896X. ; 16:9, s. 1819-1829
-
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.
|
|
| 2. |
- Bauer, Mikael, et al.
(författare)
-
Zn2+ binding to human calbindin D(28k) and the role of histidine residues.
- 2008
-
Ingår i: Protein Science. - : Wiley. - 1469-896X .- 0961-8368. ; 17:4, s. 760-767
-
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.
|
|
| 3. |
- Berbalk, Christoph, et al.
(författare)
-
Accuracy analysis of multiple structure alignments
- 2009
-
Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 18:10, s. 2027-2035
-
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.
|
|
| 4. |
- Berglund, Lisa, et al.
(författare)
-
The epitope space of the human proteome
- 2008
-
Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 17:4, s. 606-613
-
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.
|
|
| 5. |
- Carey, Jannette, et al.
(författare)
-
Protein reconstitution and three-dimensional domain swapping: Benefits and constraints of covalency
- 2007
-
Ingår i: Protein Science. - : Wiley. - 1469-896X .- 0961-8368. ; 16:11, s. 2317-2333
-
Forskningsöversikt (refereegranskat)abstract
- The phenomena of protein reconstitution and three-dimensional domain swapping reveal that highly similar structures can be obtained whether a protein is comprised of one or more polypeptide chains. In this review, we use protein reconstitution as a lens through which to examine the range of protein tolerance to chain interruptions and the roles of the primary structure in related features of protein structure and folding, including circular permutation, natively unfolded proteins, allostery, and amyloid fibril formation. The results imply that noncovalent interactions in a protein are sufficient to specify its structure under the constraints imposed by the covalent backbone.
|
|
| 6. |
- Carey, Jannette, et al.
(författare)
-
WrbA bridges bacterial flavodoxins and eukaryotic NAD(P)H: quinone oxidoreductases
- 2007
-
Ingår i: Protein Science. - : Wiley. - 1469-896X .- 0961-8368. ; 16:10, s. 2301-2305
-
Tidskriftsartikel (refereegranskat)abstract
- The crystal structure of the flavodoxin-like protein WrbA with oxidized FMN bound reveals a close relationship to mammalian NAD(P) H:quinone oxidoreductase, Nqo1. Structural comparison of WrbA, flavodoxin, and Nqo1 indicates how the twisted open-sheet fold of flavodoxins is elaborated to form multimers that extend catalytic function from one-electron transfer between protein partners using FMN to two-electron reduction of xenobiotics using FAD. The structure suggests a novel physiological role for WrbA and Nqo1.
|
|
| 7. |
- Carnrot, Cecilia, et al.
(författare)
-
Mechanisms of substrate selectivity for Bacillus anthracis thymidylate kinase
- 2008
-
Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 17:9, s. 1486-1493
-
Tidskriftsartikel (refereegranskat)abstract
- Bacillus anthracis is well known in connection with biological warfare. The search for new drug targets and antibiotics is highly motivated because of upcoming multiresistant strains. Thymidylate kinase is an ideal target since this enzyme is at the junction of the de novo and salvage synthesis of dTTP, an essential precursor for DNA synthesis. Here the expression and characterization of thymidylate kinase from B. anthracis (Ba-TMPK) is presented. The enzyme phosphorylated deoxythymidine-5'-monophosphate (dTMP) efficiently with K-m and V-max values of 33 mu M and 48 mu mol mg(-1) min(-1), respectively. The efficiency of deoxyuridine-5'-monophosphate phosphorylation was; similar to 10% of that of dTMP. Several dTMP analogs were tested, and D-FMAUMP (2'-fluoroarabinosyl-5-methyldeoxyuridine-5'- monophosphate) was selectively phosphorylated with an efficiency of 172% of that of D-dTMP, but L-FMAUMP was a poor substrate as were 5-fluorodeoxyuridine-5'-monophosphate (5FdUMP) and 2',3'-dideoxy-2',3'-didehydrothymidine-5'-monophosphate (d4TMP). No activity could be detected with 3'-azidothymidine-5'-monophosphate (AZTMP). The corresponding nucleosides known as efficient anticancer and antiviral compounds were also tested, and D-FMAU was a strong inhibitor with an IC50 value of 10 mu M, while other nucleosides-L-FMAU, dThd, 5-FdUrd, d4T, and AZT, and 2'-arabinosylthymidine-were poor inhibitors. A structure model was built for Ba-TMPK based on the Staphylococcus aureus TMPK structure. Docking with various substrates suggested mechanisms explaining the differences in substrate selectivity of the human and the bacterial TMPKs. These results may serve as a start point for development of new antibacterial agents.
|
|
| 8. |
- Fast, Jonas, et al.
(författare)
-
Stability of HAMLET--A kinetically trapped {alpha}-lactalbumin oleic acid complex.
- 2005
-
Ingår i: Protein Science. - : Wiley. - 1469-896X .- 0961-8368. ; 14:2, s. 329-340
-
Tidskriftsartikel (refereegranskat)abstract
- The stability toward thermal and urea denaturation was measured for HAMLET (human -lactalbumin made lethal to tumor cells) and -lactalbumin, using circular dichroism and fluorescence spectroscopy as well as differential scanning calorimetry. Under all conditions examined, HAMLET appears to have the same or lower stability than -lactalbumin. The largest difference is seen for thermal denaturation of the calcium free (apo) forms, where the temperature at the transition midpoint is 15°C lower for apo HAMLET than for apo -lactalbumin. The difference becomes progressively smaller as the calcium concentration increases. Denaturation of HAMLET was found to be irreversible. Samples of HAMLET that have been renatured after denaturation have lost the specific biological activity toward tumor cells. Three lines of evidence indicate that HAMLET is a kinetic trap: (1) It has lower stability than -lactalbumin, although it is a complex of -lactalbumin and oleic acid; (2) its denaturation is irreversible and HAMLET is lost after denaturation; (3) formation of HAMLET requires a specific conversion protocol.
|
|
| 9. |
|
|
| 10. |
- Forsgren, Nina, 1979-, et al.
(författare)
-
Crystal structure of the variable domain of the Streptococcus gordonii surface protein SspB
- 2009
-
Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 18:9, s. 1896-1905
-
Tidskriftsartikel (refereegranskat)abstract
- The Antigen I/II (AgI/II) family of proteins are cell wall anchored adhesins expressed on the surface of oral streptococci. The AgI/II proteins interact with molecules on other bacteria, on the surface of host cells, and with salivary proteins. Streptococcus gordonii is a commensal bacterium, and one of the primary colonizers that initiate the formation of the oral biofilm. S. gordonii expresses two AgI/II proteins, SspA and SspB that are closely related. One of the domains of SspB, called the variable (V-) domain, is significantly different from corresponding domains in SspA and all other AgI/II proteins. As a first step to elucidate the differences among these proteins, we have determined the crystal structure of the V-domain from S. gordonii SspB at 2.3 A resolution. The domain comprises a beta-supersandwich with a putative binding cleft stabilized by a metal ion. The overall structure of the SspB V-domain is similar to the previously reported V-domain of the Streptococcus mutans protein SpaP, despite their low sequence similarity. In spite of the conserved architecture of the binding cleft, the cavity is significantly smaller in SspB, which may provide clues about the difference in ligand specificity. We also verified that the metal in the binding cleft is a calcium ion, in concurrence with previous biological data. It was previously suggested that AgI/II V-domains are carbohydrate binding. However, we tested that hypothesis by screening the SspB V-domain for binding to over 400 glycoconjucates and found that the domain does not interact with any of the carbohydrates.
|
|
