Protein characterization

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The protein characterization includes biochemical and biophysical methods for determining the properties of a protein or for displaying a proteome .

properties

To determine their properties, proteins are usually released through cell disruption and separated from the other components of the starting material by means of protein purification .

ID

The amino acid sequence can be obtained by an Edman degradation , a Schlack-Kumpf degradation or mass spectrometry by a de novo sequencing by in-gel digestion are determined. Post-translational modifications can be determined by Western blot or mass spectrometry.

A protein can also be identified with specific antibodies by immunolabeling in a Western blot or by affinity chromatography . Proof can also be provided by measuring its function, in the case of enzymes using enzyme kinetics . Clearly identifiable parts of a protein can be detected by mass spectrometry ( MALDI-TOF , ESI - MS / MS , by LC -MS / MS), via a peptide mass fingerprint or de novo sequencing after in-gel digestion , which is then detected via databases how to identify mascot .

The transposon tagging , the TILLING or untargeted mutagenesis permits the identification of protein-coding genes from the change of the phenotype with subsequent determination of the DNA sequence of the phenotype of changing mutation .

Size and mass

A determination of the molecular weight of a protein can e.g. B. by gel permeation chromatography , by SDS-PAGE , by isopycnic centrifugation , by field-flow fractionation or by mass spectrometry.

shape

The protein folding is z. B. determined by an X-ray structure analysis (XRD), by electron microscope or by NMR . Changes in protein folding can be followed by FTIR spectroscopy or by measuring circular dichroism .

The protein folding can be partially limited in the course of a protection assay by limited proteolysis or thermal denaturation with protein dyes. The surface of a protein can be determined by a deuterium exchange of hydrogen atoms close to the surface.

function

Proteins can bind other molecules with the corresponding affinity , and enzymes also have catalytic activity.

interaction

In addition, protein-protein interactions and interactions with other molecules such. B. by affinity chromatography , co-immunoprecipitation , SPINE and other pulldown assays , molecular display , yeast two-hybrid system , chemical cross-linking and SDS-PAGE or mass spectrometry, native gel electrophoresis , Far Western blot , ligand binding tests (radioligand, reporter enzymes , fluorescent ligands, electric discharge), label transfer , proximity ligation assay , affinity electrophoresis , alanine scanning, microscale thermophoresis , isothermal titration calorimetry , surface plasmon resonance , bio-layer interferometry , fluorescence correlation spectroscopy , Förster resonance energy transfer , bioluminescence resonance energy transfer , bimolecular fluorescence complementation , protein fragment -Complementation , thermal shift assay , density gradient centrifugation or gel permeation chromatography . In addition, structure elucidation can be carried out by X-ray structure analysis or by NMR spectroscopy .

A gene knockout can also lead to cellular and phenotypic effects that provide an indication of the function of a protein. By restoring a function by gene knockin or by enhancing the function by overexpression , the effect of the opposite gene expression can be demonstrated. By comparing the amino acid sequence with databases such as BLAST and Pfam , sequence-related proteins with known functions can provide an indication of the possible functions of the protein to be examined.

Methods for determining protein-DNA interactions ( DNA sequence , DNA-binding proteins ) are e.g. B. the Electrophoretic Mobility Shift Assay , the ChIP , the ChIP-on-Chip , the ChIP-Seq , the DNase Footprinting Assay , the Yeast-One-Hybrid-System (Y1H), the Bacterial-One-Hybrid-System (B1H ) and the DamID .

Protein-RNA interactions can e.g. B. be detected by RNA-Seq , RIP-Chip , ICLIP , CLIP-Seq or by PAR-CLIP .

Methods for studying protein-lipid interactions are e.g. B. ESR spectroscopy , dual polarization interferometry, fluorescence microscopy with fluorescence- labeled lipids or proteins or with lipid dyes such as laurdan or filipin .

catalysis

If the protein function is unknown, enzymatic properties such as catalysis are investigated by determining the enzyme kinetics. Inhibitors of various proteins that may be involved in the function are often used here. An alanine scan can be used to determine the active site.

amount

The amount (quantification) of a protein is determined e.g. B. by photometry , ELISA , SDS-PAGE and Western Blot, 2D-PAGE or mass spectrometry ( iTRAQ , SILAC , ICAT , the isobar labeling , tandem mass tags ).

By photometry, the protein concentration can e.g. B. by Bradford test , by Lowry test , by biuret reaction , by BCA test or, in the case of higher protein concentrations, also by the absorption of the peptide bond , each method having its own interfering substances which, if present, exclude the use of this method.

Various dyes preferentially bind proteins, e.g. B. Coomassie brilliant blue , Fast Green FCF , amido or Ponceau S . As a fluorescent dye , for. B. Nile Red , Brilliant Sulfaflavin , 8-Anilinonaphthalene-1-sulfonic acid (8-ANS), Scopoletin , Iridium complexes, SYPRO Orange , SYPRO Red , SYPRO Ruby , SYPRO Tangerine, Flamingo , Krypton , Coomassie Fluor Orange, Lucy 506, Lucy 565, Lucy 569 or Epicocconon ( Lightning Fast , Deep Purple Stain ).

Due to the peptide bond, proteins absorb ultraviolet light at a wavelength of around 205 nm (190 nm to 230 nm), and phenylalanine , tyrosine and tryptophan also absorb UV light at wavelengths from 280 nm to 288 nm. This absorption can be used for photometric quantification and for Determination of the purification factors can be used.

modification

In contrast to carbohydrates and nucleic acids, some structural motifs only occur in proteins due to the various amino acids they contain, e.g. B. sulfhydryl-containing cysteines or phenol residues in tyrosines . These can be selectively marked with the appropriate reagents. Some protease inhibitors lead to a selective modification of proteins. By cross-linking mostly two reactive groups are connected in a protein with one another. Amino groups in proteins such as lysines or unmodified N -termini can be selectively mixed with N-hydroxysuccinimide esters, sulfosuccinimide esters [e.g. B. bis (sulfosuccinimidyl) suberate], imido esters (e.g. dimethyl adipimidate), isothiocyanates (e.g. PITC , FITC ), 1-fluoro-2,4-dinitrobenzene (DNFB), dansyl chloride or aldehydes (e.g. Glutaraldehyde ). Cysteines can be combined with maleimides (e.g. N- ethylmaleimide ), disulfides and sulfides (e.g. 2-mercaptoethanol , dithiothreitol , dithioerythritol , tris (2-carboxyethyl) phosphine , Ellman's reagent ) or iodoacetamides (e.g. IAEDANS ) react. Tyrosines can be labeled with iodine by oxidation . Fixatives mostly lead to cross-linking of different amino acids.

cleavage

By cyanogen bromide , a protein can chemically to contained methionine residues are cleaved from the methionine arises the homoserine - lactone . With some isothiocyanates such as PITC, the N-terminal amino acid can be split off, which can also be used repeatedly in the course of Edman degradation. Selective cleavages can be effected enzymatically by endopeptidases , which require longer recognition sequences compared to non-enzymatic cleavages.

Expression pattern

The different protein expression in a proteome can e.g. B. by gel electrophoresis ( 2D-PAGE ), protein arrays , Difference Gel Electrophoresis , MeCAT or ICAT .

Sequence analysis

The amino acid sequence of a protein can be used to identify various properties of a protein. Certain secondary structures can be identified by a Ramachandran plot or a Janin plot . The localization in a cellular compartment can be determined from the signal sequences and the transmembrane domains . The hydropathic index describes the tendency of certain sequences to be embedded in a biomembrane . The instability index provides an indication of the biological half-life of a protein. The sequence can be used to determine possible locations for post-translational modifications .

literature

Individual evidence

  1. ↑ YH Cai, H. Huang: Advances in the study of protein-DNA interaction. In: Amino acids. Volume 43, Number 3, September 2012, pp. 1141-1146, ISSN  1438-2199 . doi : 10.1007 / s00726-012-1377-9 . PMID 22842750 .
  2. ↑ T. Sahr, C. Buchrieser: Co-immunoprecipitation: protein-RNA and protein-DNA interaction. In: Methods in molecular biology (Clifton, NJ). Volume 954, 2013, pp. 583-593, ISSN  1940-6029 . doi : 10.1007 / 978-1-62703-161-5_36 . PMID 23150422 .
  3. ↑ B. Deplancke, A. Mukhopadhyay, W. Ao, AM Elewa, CA Grove, NJ Martinez, R. Sequerra, L. Doucette strain, JS Reece-Hoyes, IA Hope, HA Tissenbaum, SE Mango, AJ Walhout: A gene-centered C. elegans protein-DNA interaction network. In: Cell. Volume 125, Number 6, June 2006, pp. 1193-1205, ISSN  0092-8674 . doi : 10.1016 / j.cell.2006.04.038 . PMID 16777607 .
  4. ↑ RC YashRoy: Protein heat denaturation and study of membrane lipid-protein interactions by spin label ESR. In: Journal of biochemical and biophysical methods. Volume 22, Number 1, January 1991, pp. 55-59, ISSN  0165-022X . PMID 1848569 .
  5. ↑ A. Mashaghi, M. Swann, J. Popplewell, M. Textor, E. Reimhult: Optical anisotropy of supported lipid structures probed by waveguide spectroscopy and its application to study of supported lipid bilayer formation kinetics. In: Analytical chemistry. Volume 80, Number 10, May 2008, pp. 3666-3676, ISSN  1520-6882 . doi : 10.1021 / ac800027s . PMID 18422336 .
  6. ^ Y. Chen, J. Yang, Z. Wang, X. Wu, F. Wang: Scopoletine as fluorescence probe for determination of protein. In: Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy. Volume 66, Number 3, March 2007, pp. 686-690, ISSN  1386-1425 . doi : 10.1016 / j.saa.2006.04.012 . PMID 16859971 .
  7. ↑ Junli Jia, Hao Fei, Ming Zhou: Luminescent iridium (III) complexes as novel protein staining agents. In: Electrophoresis (2012), Volume 33, Issue 9-10, pp. 1397-1401, doi : 10.1002 / elps.201100693 .
  8. ↑ C. Li, M. Yu, Y. Sun, Y. Wu, C. Huang, F. Li: A nonemissive iridium (III) complex that specifically lights-up the nuclei of living cells. In: Journal of the American Chemical Society. Volume 133, Number 29, July 2011, pp. 11231-11239, ISSN  1520-5126 . doi : 10.1021 / ja202344c . PMID 21682270 .
  9. ↑ Aleksandr Petrovich Demchenko: Advanced Fluorescence Reporters in Chemistry and Biology III: Applications in Sensing and Imaging Volume 3 of Advanced Fluorescence Reporters in Chemistry and Biology . Springer 2011. ISBN 9783642180354 . P. 179ff.
  10. ↑ T. Rabilloud, JM Strub, S. Luche, A. van Dorsselaer, J. Lunardi: A comparison between Sypro Ruby and ruthenium II tris (bathophenanthroline disulfonate) as fluorescent stains for protein detection in gels. In: Proteomics. Volume 1, Number 5, May 2001, pp. 699-704, ISSN  1615-9853 . doi : 10.1002 / 1615-9861 (200104) 1: 5 <699 :: AID-PROT699> 3.0.CO; 2-C . PMID 11678039 .