| 1. |
- Engstrom, Maria, et al.
(författare)
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Ab initio g-tensor calculations of hydrogen bond effects on a nitroxide spin label
- 2001
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Ingår i: Chemical Physics Letters. - 0009-2614 .- 1873-4448. ; 338:06-apr, s. 407-413
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Tidskriftsartikel (refereegranskat)abstract
- Hydrogen bonding effects on the electron paramagnetic resonance (EPR) g-tensor of a nitroxide spin label was investigated by quantum chemical calculations. The restricted open-shell Hartree-Fock (ROHF) linear response method with the atomic mean field approximation (AMFI) was used in the calculations. The results show that hydrogen bonding reduces the g-tensor component directed along the NO bond, g(xx). This decrease is traced to higher excitation energy and lower spin-orbit coupling and angular momentum matrix elements for the n-pi* excitation. The calculations show that the g-tensor is practically invariable when hydrogen bonding was modeled with methanol instead of water.
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| 2. |
- Hammarström, Per, et al.
(författare)
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High-resolution probing of local conformational changes in proteins by the use of multiple labeling : Unfolding and self-assembly of human carbonic anhydrase II monitored by spin, fluorescent, and chemical reactivity probes
- 2001
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Ingår i: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 80:6, s. 2867-2885
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Tidskriftsartikel (refereegranskat)abstract
- Two different spin labels, N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide (IPSL) and (1-oxyl-2.2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate (MTSSL), and two different fluorescent labels 5-((((2-iodoacetyl)amino)ethyl)amino)naphtalene-1 -sulfonic acid (IAEDANS) and 6-bromoacetyl-2-dimetylaminonaphtalene (BADAN), were attached to the introduced C79 in human carbonic anhydrase (HCA II) to probe local structural changes upon unfolding and aggregation, HCA II unfolds in a multi-step manner with an intermediate state populated between the native and unfolded states. The spin label IPSL and the fluorescent label IAEDANS reported on a substantial change in mobility and polarity at both unfolding transitions at a distance of 7.4-11.2 Angstrom from the backbone of position 79. The shorter and less flexible labels BADAN and MTSSL revealed less pronounced spectroscopic changes in the native-to-intermediate transition, 6.6-9.0 Angstrom from the backbone. At intermediate guanidine (Gu)-HCl concentrations the occurrence of soluble but irreversibly aggregated oligomeric protein was identified from refolding experiments. At similar to1 M Gu-HCl the aggregation was found to be essentially complete. The size and structure of the aggregates could be varied by changing the protein concentration. EPR measurements and line-shape simulations together with fluorescence lifetime and anisotropy measurements provided a picture of the self-assembled protein as a disordered protein structure with a representation of both compact as well as dynamic and polar environments at the site of the molecular labels. This suggests that a partially folded intermediate of HCA II self-assembles by both local unfolding and intermolecular docking of the intermediates vicinal to position 79. The aggregates were determined to be 40-90 Angstrom in diameter depending on the experimental conditions and spectroscopic technique used.
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| 3. |
- Hammarström, Per, 1972-, et al.
(författare)
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Protein substrate binding induces conformational changes in the chaperonin GroEL : A suggested mechanism for unfoldase activity
- 2000
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 275:30, s. 22832-22838
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Tidskriftsartikel (refereegranskat)abstract
- Chaperonins are molecules that assist proteins during folding and protect them from irreversible aggregation. We studied the chaperonin GroEL and its interaction with the enzyme human carbonic anhydrase II (HCA II), which induces unfolding of the enzyme. We focused on conformational changes that occur in GroEL during formation of the GroEL-HCA II complex. We measured the rate of GroEL cysteine reactivity toward iodo[2-(14)C]acetic acid and found that the cysteines become more accessible during binding of a cysteine free mutant of HCA II. Spin labeling of GroEL with N-(1-oxy1-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide revealed that this additional binding occurred because buried cysteine residues become accessible during HCA II binding. In addition, a GroEL variant labeled with 6-iodoacetamidofluorescein exhibited decreased fluorescence anisotropy upon HCA II binding, which resembles the effect of GroES/ATP binding. Furthermore, by producing cysteine-modified GroEL with the spin label N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)iodoacetamide and the fluorescent label 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid, we detected increases in spin-label mobility and fluorescence intensity in GroEL upon HCA II binding. Together, these results show that conformational changes occur in the chaperonin as a consequence of protein substrate binding. Together with previous results on the unfoldase activity of GroEL, we suggest that the chaperonin opens up as the substrate protein binds. This opening mechanism may induce stretching of the protein, which would account for reported unfoldase activity of GroEL and might explain how GroEL can actively chaperone proteins larger than HCA II.
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| 4. |
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| 5. |
- Lautner, Ronald, et al.
(författare)
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Apolipoprotein e genotype and the diagnostic accuracy of cerebrospinal fluid biomarkers for Alzheimer disease.
- 2014
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Ingår i: JAMA psychiatry. - : American Medical Association (AMA). - 2168-6238 .- 2168-622X. ; 71:10, s. 1183-91
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Tidskriftsartikel (refereegranskat)abstract
- Several studies suggest that the apolipoprotein E (APOE) ε4 allele modulates cerebrospinal fluid (CSF) levels of β-amyloid 42 (Aβ42). Whether this effect is secondary to the association of the APOE ε4 allele with cortical Aβ deposition or whether APOE ε4 directly influences CSF levels of Aβ42 independently of Aβ pathology remains unknown.
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| 6. |
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| 7. |
- Osterlund, Maria, et al.
(författare)
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Probing inhibitor-induced conformational changes along the interface between tissue factor and factor VIIa
- 2001
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 40:31, s. 9324-9328
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Tidskriftsartikel (refereegranskat)abstract
- Upon injury of a blood vessel, activated factor VII (FVIIa) forms a high-affinity complex with its allosteric regulator, tissue factor (TF), and initiates blood clotting. Active site-inhibited factor VIIa (FVIIai) binds to TF with even higher affinity. We compared the interactions of FVIIai and FVIIa with soluble TF (sTF). Six residues in sTF were individually selected for mutagenesis and site-directed labeling. The residues are distributed along the extensive binding interface, and were chosen because they are known to interact with the different domains of FVIIa. Fluorescent and spin probes were attached to engineered Cys residues to monitor local changes in hydrophobicity, accessibility, and rigidity in the sTF-FVIIa complex upon occupation of the active site of FVIIa. The results show that inhibition of FVIIa caused the structures around the positions in sTF that interact with the protease domain of FVIIa to become more rigid and less accessible to solvent. Thus, the presence of an active site inhibitor renders the interface in this region less flexible and more compact, whereas the interface between sTF and the light chain of FVIIa is unaffected by active site occupancy.
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| 8. |
- Osterlund, Marie, et al.
(författare)
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Spectroscopic probing of the influence of calcium and the Gla domain on the interaction between the first EGF domain in factor VIla and tissue factor
- 2000
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 267:20, s. 6204-6211
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Tidskriftsartikel (refereegranskat)abstract
- The binding of factor VIIa (FVIIa) to tissue factor (TF) initiates blood coagulation. The binary complex is dependent on Ca2+ binding to several sites in FVIIa and is maintained by multiple contacts distributed throughout the various domains. Although the contributions from various residues and domains, including the Ca2+ coordination, to the global binding energy have been characterized, their importance for specific local interactions is virtually unknown. To address this aspect, we have attached four spectroscopic probes to an engineered Cys residue replacing Phe140 in soluble TF (sTF). This allows the monitoring of local changes in hydrophobicity and rigidity upon complex formation at the interface between the first epidermal growth factor-like (EGF1) domain of FVIIa and sTF. The fluorescent labels used sense a more hydrophobic environment and the spin labels are dramatically immobilized when FVIIa binds sTF. The results obtained with a 4-carboxy-glutamic acid (Gla)-domainless derivative of FVIIa indicate that the Gla domain has no or minimal influence on the interaction between EGF1 and sTF. However, there is a difference in local Ca2+ dependence between Gla-domainless and full-length FVIIa.
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| 9. |
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| 10. |
- Owenius, Rikard, et al.
(författare)
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GroEL-induced stretching of a substrate protein: An EPR/SDSL study in BIOPHYSICAL JOURNAL, vol 88, issue 1, pp 562A-562A
- 2005
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Ingår i: BIOPHYSICAL JOURNAL. - : Elsevier (Cell Press) / Biophysical Society. ; , s. 562A-562A
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Konferensbidrag (refereegranskat)abstract
- The Hsp60-type chaperonin GroEL assists in the folding of the enzyme Human Carbonic Anhydrase II (HCA II) and protects it from aggregation.It is still a controversy whether the action of GroEL is an active or passive process. Single- and double-cysteine mutants were specificallyspin labeled at a topological breakpoint in the β-core of HCA II. X-band electron paramagnetic resonance (EPR) was used at physiologicaltemperatures to monitor the GroEL-induced structural changes in this region of HCA II. Inter-residue distance calculations based on dipolarinteraction show that the proximity of the labeled positions F147 and K213 in the native state of HCA II is ~11±2 Å, and that it is virtuallyintact in the thermally-induced molten-globule state that binds to GroEL. However, upon interaction with GroEL a spin-spin distance increaseto ~22±3 Å indicates a conformational change in HCA II that is part of the GroEL-induced substrate stretch that enables structural rearrangementof a misfolded substrate protein.
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