DSF Method Optimization and Its Application in Predicting Protein Thermal Aggregation Kinetics

Shi, Shuai; Semple, Andrew; Cheung, Jason K.; Shameem, Mohammed

doi:10.1002/jps.23633

Cited by 30 publications

(16 citation statements)

References 30 publications

(88 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The α-β transition temperature shows an exponential decay as a function of the polypeptide concentration. A comparable concentration dependence of thermally-induced conformational changes has been reported for several systems 41 42 43 . For PLL, differences in the temperature-induced α-β transition depending on polypeptide concentration have been mentioned in earlier reports 37 44 45 46 , but, to the best of our knowledge, it has not been investigated in a systematic manner.…”

Section: Resultssupporting

confidence: 78%

External cavity-quantum cascade laser infrared spectroscopy for secondary structure analysis of proteins at low concentrations

Schwaighofer

Alcaráz

Araman

et al. 2016

Sci Rep

View full text Add to dashboard Cite

1Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopy are analytical techniques employed for the analysis of protein secondary structure. The use of CD spectroscopy is limited to low protein concentrations (<2 mg ml −1 ), while FTIR spectroscopy is commonly used in a higher concentration range (>5 mg ml −1 ). Here we introduce a quantum cascade laser (QCL)-based IR transmission setup for analysis of protein and polypeptide secondary structure at concentrations as low as 0.25 mg ml −1 in deuterated buffer solution. We present dynamic QCL-IR spectra of the temperature-induced α-helix to β-sheet transition of poly-L-lysine. The concentration dependence of the α-β transition temperature between 0.25 and 10 mg ml −1 was investigated by QCL-IR, FTIR and CD spectroscopy. By using QCL-IR spectroscopy it is possible to perform IR spectroscopic analysis in the same concentration range as CD spectroscopy, thus enabling a combined analysis of biomolecules secondary structure by CD and IR spectroscopy.Structural analysis of biomolecules is of great importance in biology and biochemistry for characterising folding properties of proteins and polypeptides, as well as monitoring dynamic changes upon perturbation. Analytical methods used for investigation of biomolecule secondary structure include X-ray crystallography, nuclear magnetic resonance (NMR), circular dichroism (CD), as well as Raman and Fourier transform infrared (FTIR) spectroscopy 1 . Both NMR spectroscopy and X-ray crystallography are capable of providing structural information at atomic levels of resolution. However, NMR spectroscopy is restricted to relatively small biomolecules (≤ 40 kDa) at high concentrations and X-ray crystallography requires the availability of high-quality crystals of proteins, which is particularly demanding for membrane proteins [2][3][4] . In contrast, CD, Raman and FTIR spectroscopy are considered as low resolution techniques that provide overall structural information. Due to straightforward sample preparation and fast acquisition time, these methods are routinely used for rapid determination of secondary structure of proteins and for monitoring dynamic changes of protein structure. Due to their respective characteristics, infrared (IR) spectroscopy provides more dependable estimates of antiparallel β -sheets, whereas CD spectroscopy gives more confinable predictions of α -helix structures 5 . Regarding the complementary information provided, joint application of both methods would deliver most reliable results 6,7 . CD spectroscopy is a technique based on the difference in the absorption of the left-and right-handed circularly polarized light when it is in contact with the optically active compounds, or chromophores, present in the sample. In proteins, the most relevant chromophore is the amide group which absorbs in the far-UV region (180-240 nm). Their electronic transitions (n→ π*, π → π*) give signals at 220 and 190 nm. The periodic alignments of the amide groups in the polypeptide backbone lead to exciton co...

show abstract

Section: Resultssupporting

confidence: 78%

External cavity-quantum cascade laser infrared spectroscopy for secondary structure analysis of proteins at low concentrations

Schwaighofer

Alcaráz

Araman

et al. 2016

Sci Rep

View full text Add to dashboard Cite

show abstract

“…Further investigations on the conformational stability were performed by DSF, a sensitive technique that provides information on the thermal denaturation transitions of proteins and detects stabilizing ligand interactions [ 35 , 36 ]. Remarkably, only the first, low T m transition was detected by DSF, whereas the second appears to be lost due to baseline decay and distortion associated with aggregation of the protein following the unfolding of the first domain ( Figures 3 C and 3 D) [ 37 ].…”

Section: Resultsmentioning

confidence: 99%

Arc is a flexible modular protein capable of reversible self-oligomerization

Myrum

Baumann

Bustad

et al. 2015

Biochemical Journal

View full text Add to dashboard Cite

The immediate early gene product Arc (activity-regulated cytoskeleton-associated protein) is posited as a master regulator of long-term synaptic plasticity and memory. However, the physicochemical and structural properties of Arc have not been elucidated. In the present study, we expressed and purified recombinant human Arc (hArc) and performed the first biochemical and biophysical analysis of hArc's structure and stability. Limited proteolysis assays and MS analysis indicate that hArc has two major domains on either side of a central more disordered linker region, consistent with in silico structure predictions. hArc's secondary structure was estimated using CD, and stability was analysed by CD-monitored thermal denaturation and differential scanning fluorimetry (DSF). Oligomerization states under different conditions were studied by dynamic light scattering (DLS) and visualized by AFM and EM. Biophysical analyses show that hArc is a modular protein with defined secondary structure and loose tertiary structure. hArc appears to be pyramid-shaped as a monomer and is capable of reversible self-association, forming large soluble oligomers. The N-terminal domain of hArc is highly basic, which may promote interaction with cytoskeletal structures or other polyanionic surfaces, whereas the C-terminal domain is acidic and stabilized by ionic conditions that promote oligomerization. Upon binding of presenilin-1 (PS1) peptide, hArc undergoes a large structural change. A non-synonymous genetic variant of hArc (V231G) showed properties similar to the wild-type (WT) protein. We conclude that hArc is a flexible multi-domain protein that exists in monomeric and oligomeric forms, compatible with a diverse, hub-like role in plasticity-related processes.

show abstract

“…The advantages of DSF compared with conventional μDSC are the high‐throughput design with small sample volumes and short analysis times, which renders this method essential for formulation development. μDSC is needed for the detailed thermodynamic characterization based on heat capacity data with high resolution of all unfolding events or when excipients like surfactants challenge the application of DSF, although the gaps for application are closing . Furthermore, DSF in combination with k D and

A_{2}^{*}

analysis at higher temperatures helps to understand the link between unfolding, intermolecular interactions, and aggregation upon heating.…”

Section: Resultsmentioning

confidence: 99%

Temperature-Ramped Studies on the Aggregation, Unfolding, and Interaction of a Therapeutic Monoclonal Antibody

Menzen

Frieß

2014

Journal of Pharmaceutical Sciences

View full text Add to dashboard Cite

DSF Method Optimization and Its Application in Predicting Protein Thermal Aggregation Kinetics

Cited by 30 publications

References 30 publications

External cavity-quantum cascade laser infrared spectroscopy for secondary structure analysis of proteins at low concentrations

External cavity-quantum cascade laser infrared spectroscopy for secondary structure analysis of proteins at low concentrations

Arc is a flexible modular protein capable of reversible self-oligomerization

Temperature-Ramped Studies on the Aggregation, Unfolding, and Interaction of a Therapeutic Monoclonal Antibody

Contact Info

Product

Resources

About