Exam 2 Flashcards
Amino Acid residues that are O-linked glycosylation sites
Ser, Thr, Hydroxy-Lys, Hydroxy-Pro
N-linked Glycosylation sites
Asn- x-Ser, Asn-x-Cys, Asn-x-Thr
NO PRO
what is glycosylation
common post-translational modification
functions of glycosylation
- affects proteins structure and stability because it changes the primary structure by adding covalently adding organic materials
- increases protein lifetime
- change shape and charge (role in molecular recognition)
effects on absorbance
solvents, ions, h-bonding
Difference effects on absorbance
something that affects the electronic structure and changes the energy for excitation
red max wavelength shift
bathochromic
shifts to the right of an absorbance vs wavelength graph
lower energy -> gap deltaE smaller
Blue max wavelength shift
hypsochromic
shifts to the left of an absorbance vs wavelength graph
higher energy
x-ray
-size of unit cell
-strength or intensities of diffraction spots show electron density
-spacing of molecules in structure
-crystal structures and structure of molecules in crystal lattice
Mass Spectrometry
-identify protein structure (primary structure)
-identify post translations modifications
MALDI
observe singly charged ions (polymers and proteins)
Tandem Mass Spectrometry
-identify protein sequence
-peptide fragmentation
-peptide identification
-(B-mercatoethanol reduces proteins trypsin cleaves peptides)
omics
global profiling to identify concentration of biomolecules in a given population of cells
hydrodynamic methods
-estimate particle mass, size, shape, density, and oligomeric state
-study transport and diffusion
sedimentation
-determine molecular weight, density, approximate shape of macromolecule
-isolate certain species from a mixture
Dynamic Light Scattering
-info on particle size and distribution of macromolecules in solution
-(big particles -> slower, big disturbance, Small Particles -> faster, small disturbance)
-info on shape and how macromolecule moves through solution
electrophoresis and chromatography (general)
-separation of biomacromolecules by size, shape, charge, Polarity, or affinity for a given binding partner
Electrophoresis
-describes how charges molecules and complexes migrate in an electric field (Separate based on size, charges, and shape)
-Assess purity
Agarose gel electrophoresis
-Separates DNA fragments by mass
-Charge is more negative on larger DNA pieces and mass is larger
DNA gel
-smaller particles -> fast
-larger particles -> slow
staining with ethidium bromide
visualize DNA band
DNA/RNA Autoradiography
-DNA blots = find presences of certain sequences
-northern blot = RNA detected by 32P
-southern blot = DNA detected by 32P
-western blot = proteins detected by antibodies
SDS PAGE gels
-separates protein mixtures based on m/z
-SDS detergent used to make m/z proportional to mass
-check purity of proteins
-estimate molecular weight by comparing to standards
Chromatography
-separate proteins by size (smaller proteins are slower to exit, larger proteins are faster to exit)
-separate protein by affinity (separated by how they interact with bead)
-adjust pH of solvent to get protein off
GST
affinity tag with linker that can be cleaved by protease
2D gel
-gel columns with pH gradients that can separate proteins by pI in an electric field
-pH=pI -> protein is neutral
proteases
-used to generally fragment proteins for sequencing (trypsin)
-cleave off linkage tags like GST (Thrombin)
centrifuge findings
- particles with larger masses move faster
- particles with smaller volumes (more dense) move faster
- the denser the solution the slower the particle will move
- the greater the frictional coefficient, the slower the particle
Mass analyzers
quadrupole, magnetic sector, time of flight
How to find the number of charges in ESI-MS
The amount of m/z between peaks
separated by 1 - single
separated by 1/2 - double
separated by 1/3- triple
what happens to m/z when you increase charge in ESI-MS
when charges increase, m/z decreases
Electrospray ionization (ESI)
removal of solvent =less sample damage
exact mass
(mono isotopic mass)sum of most abundant masses
1D NMR
peaks assigned to amino acid types
2D NMR
peak heights indicate strength of nuclear overhauser interactions (NOE)
NOE
Nuclear Overhauser
used to identify atoms in space
Multi-dimensional NMR
used to determine the structure of proteins in solution
x-ray crystallography
used to obtain near atomic resolution structures of proteins
molecular mechanics
uses energy terms describing both covalent and non covalent interactions
CD Spectroscopy
-(Circular Dichroism) measure the molar absorbance difference of left and right circularly polarized light
-detects the asymmetric interaction of chiral molecules with circularly polarized light
GFP Chromophore
spontaneously forms from the sequences S-Y-G in the presence of oxygen
time dependent FRET
used to observe structural changes on the us-ms timescale on which receptors move
quantum yield (Q)
of protons emitted / # of protons absorbed
Kasha’s rule
molecules rapidly relax to the ground vibrational state of the lowest lying excited singlet (internal conversion)
what side chains hav pi to pi* transitions
H R E Q N D
uv/vis spectroscopy
observes electronic transitions between energy levels
