scholarly journals In-cell thermodynamics and a new role for protein surfaces

2016 ◽  
Vol 113 (7) ◽  
pp. 1725-1730 ◽  
Author(s):  
Austin E. Smith ◽  
Larry Z. Zhou ◽  
Annelise H. Gorensek ◽  
Michael Senske ◽  
Gary J. Pielak

There is abundant, physiologically relevant knowledge about protein cores; they are hydrophobic, exquisitely well packed, and nearly all hydrogen bonds are satisfied. An equivalent understanding of protein surfaces has remained elusive because proteins are almost exclusively studied in vitro in simple aqueous solutions. Here, we establish the essential physiological roles played by protein surfaces by measuring the equilibrium thermodynamics and kinetics of protein folding in the complex environment of living Escherichia coli cells, and under physiologically relevant in vitro conditions. Fluorine NMR data on the 7-kDa globular N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) show that charge–charge interactions are fundamental to protein stability and folding kinetics in cells. Our results contradict predictions from accepted theories of macromolecular crowding and show that cosolutes commonly used to mimic the cellular interior do not yield physiologically relevant information. As such, we provide the foundation for a complete picture of protein chemistry in cells.

2020 ◽  
Vol 295 (27) ◽  
pp. 8958-8971 ◽  
Author(s):  
Katarina Jurikova ◽  
Martin Gajarsky ◽  
Mona Hajikazemi ◽  
Jozef Nosek ◽  
Katarina Prochazkova ◽  
...  

The ends of eukaryotic chromosomes typically contain a 3′ ssDNA G-rich protrusion (G-overhang). This overhang must be protected against detrimental activities of nucleases and of the DNA damage response machinery and participates in the regulation of telomerase, a ribonucleoprotein complex that maintains telomere integrity. These functions are mediated by DNA-binding proteins, such as Cdc13 in Saccharomyces cerevisiae, and the propensity of G-rich sequences to form various non-B DNA structures. Using CD and NMR spectroscopies, we show here that G-overhangs of S. cerevisiae form distinct Hoogsteen pairing–based secondary structures, depending on their length. Whereas short telomeric oligonucleotides form a G-hairpin, their longer counterparts form parallel and/or antiparallel G-quadruplexes (G4s). Regardless of their topologies, non-B DNA structures exhibited impaired binding to Cdc13 in vitro as demonstrated by electrophoretic mobility shift assays. Importantly, whereas G4 structures formed relatively quickly, G-hairpins folded extremely slowly, indicating that short G-overhangs, which are typical for most of the cell cycle, are present predominantly as single-stranded oligonucleotides and are suitable substrates for Cdc13. Using ChIP, we show that the occurrence of G4 structures peaks at the late S phase, thus correlating with the accumulation of long G-overhangs. We present a model of how time- and length-dependent formation of non-B DNA structures at chromosomal termini participates in telomere maintenance.


Author(s):  
Beverly E. Maleeff ◽  
Timothy K. Hart ◽  
Stephen J. Wood ◽  
Ronald Wetzel

Alzheimer's disease is characterized post-mortem in part by abnormal extracellular neuritic plaques found in brain tissue. There appears to be a correlation between the severity of Alzheimer's dementia in vivo and the number of plaques found in particular areas of the brain. These plaques are known to be the deposition sites of fibrils of the protein β-amyloid. It is thought that if the assembly of these plaques could be inhibited, the severity of the disease would be decreased. The peptide fragment Aβ, a precursor of the p-amyloid protein, has a 40 amino acid sequence, and has been shown to be toxic to neuronal cells in culture after an aging process of several days. This toxicity corresponds to the kinetics of in vitro amyloid fibril formation. In this study, we report the biochemical and ultrastructural effects of pH and the inhibitory agent hexadecyl-N-methylpiperidinium (HMP) bromide, one of a class of ionic micellar detergents known to be capable of solubilizing hydrophobic peptides, on the in vitro assembly of the peptide fragment Aβ.


1977 ◽  
Vol 16 (04) ◽  
pp. 157-162 ◽  
Author(s):  
C. Schümichen ◽  
B. Mackenbrock ◽  
G. Hoffmann

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.


1981 ◽  
Vol 45 (03) ◽  
pp. 285-289 ◽  
Author(s):  
J P Allain ◽  
A Gaillandre ◽  
D Frommel

SummaryFactor VIII complex and its interaction with antibodies to factor VIII have been studied in 17 non-haemophilic patients with factor VIII inhibitor. Low VIII:C and high VIIIR.Ag levels were found in all patients. VIII:WF levels were 50% of those of VTIIRrAg, possibly related to an increase of poorly aggregated and electrophoretically fast moving VIIIR:Ag oligomers.Antibody function has been characterized by kinetics of VIII :C inactivation, saturability by normal plasma and the slope of the affinity curve. Two major patterns were observed:1) Antibodies from 6 patients behaved similarly to those from haemophiliacs by showing second order inhibition kinetics, easy saturability and steep affinity slope (> 1).2) Antibodies from other patients, usually with lower titres, inactivated VIII :C according to complex order kinetics, were not saturable, and had a less steep affinity slope (< 0.7). In native plasma, or after mixing with factor VIII concentrate, antibodies of the second group did not form immune complexes with the whole factor VIII molecular complex. However, dissociation procedures did release some antibodies from apparently low molecular weight complexes formed in vivo or in vitro. For appropriate management of non-haemophilic patients with factor VIII inhibitor, it is important to determine the functional properties of their antibodies to factor VIII.


1979 ◽  
Vol 42 (05) ◽  
pp. 1630-1633 ◽  
Author(s):  
A G Castle ◽  
N Crawford

SummaryBlood platelets contain microtubule proteins (tubulin and HMWs) which can be polymerised “in vitro” to form structures which resemble the microtubules seen in the intact platelet. Platelet tubulin is composed of two non-identical subunits a and p tubulin which have molecular weights around 55,000 but can be resolved in alkaline SDS-polyacrylamide gel electrophoresis. These subunits associate as dimers with sedimentation coefficients of about 5.7 S although it is not known whether the dimer protein is a homo- or hetero-dimer. The dimer tubulin binds the anti-mitotic drug colchicine and the kinetics of this binding are similar to those reported for neurotubulins. Platelet microtubules also contain two HMW proteins which appear to be essential and integral components of the fully assembled microtubule. These proteins have molecular weights greater than 200,000 daltons. Fluorescent labelled antibodies to platelet and brain tubulins stain long filamentous microtubular structures in bovine lens epithelial cells and this pattern of staining is prevented by exposing the cells to conditions known to cause depolymerisation of cell microtubules.


2013 ◽  
Vol 36 (6) ◽  
pp. 1200-1204
Author(s):  
Ling ZHANG ◽  
Wei-Dong ZHANG ◽  
Xiao-Ling ZHANG ◽  
Kai LIU ◽  
Qing-Li LI ◽  
...  
Keyword(s):  

2020 ◽  
Vol 17 (3) ◽  
pp. 259-266 ◽  
Author(s):  
Xuan Chen ◽  
Sumei Zhang ◽  
Peipei Shi ◽  
Yangli Su ◽  
Dong Zhang ◽  
...  

Objective: Ischemia-reperfusion (I/R) injury is a pathological feature of ischemic stroke. This study investigated the regulatory role of miR-485-5p in I/R injury. Methods: SH-SY5Y cells were induced with oxygen and glucose deprivation/reoxygenation (OGD/R) to mimic I/R injury in vitro. Cells were transfected with designated constructs (miR-485- 5p mimics, miR-485-5p inhibitor, lentiviral vectors overexpressing Rac1 or their corresponding controls). Cell viability was evaluated using the MTT assay. The concentrations of lactate dehydrogenase, malondialdehyde, and reactive oxygen species were detected to indicate the degree of oxidative stress. Flow cytometry and caspase-3 activity assay were used for apoptosis assessment. Dual-luciferase reporter assay was performed to confirm that Rac family small GTPase 1 (Rac1) was a downstream gene of miR-485-5p. Results: OGD/R resulted in decreased cell viability, elevated oxidative stress, increased apoptosis, and downregulated miR-485-5p expression in SH-SY5Y cells. MiR-485-5p upregulation alleviated I/R injury, evidenced by improved cell viability, decreased oxidative markers, and reduced apoptotic rate. OGD/R increased the levels of Rac1 and neurogenic locus notch homolog protein 2 (Notch2) signaling-related proteins in cells with normal miR-485-5p expression, whereas miR- 485-5p overexpression successfully suppressed OGD/R-induced upregulation of these proteins. Furthermore, the delivery of vectors overexpressing Rac1 in miR-485-5p mimics-transfected cells reversed the protective effect of miR-485-5p in cells with OGD/R-induced injury. Conclusion: This study showed that miR-485-5p protected cells following I/R injury via targeting Rac1/Notch2 signaling suggest that targeted upregulation of miR-485-5p might be a promising therapeutic option for the protection against I/R injury.


1999 ◽  
Vol 111 (3) ◽  
pp. 198-205 ◽  
Author(s):  
Gerald G. Krueger ◽  
Jeffery R. Morgan ◽  
Marta J. Petersen
Keyword(s):  

Sign in / Sign up

Export Citation Format

Share Document