Lecture 4 - Macromolecule Characterization (pt. 2) Flashcards by Madeline Squires

SDS PAGE Gels:

protein mixtures can be separated by ___ ___ ____ (____) based on ___-___-___ ___ (__)

polyacrylamide gel electrophoresis (PAGE) based on mass-to-charge ratio (m/z)

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SDS PAGE Gels:

proteins care coated with ___ ____, so that ___ is proportional to ____

this ____ them and gives them a uniform ___ ___

because of the uniform __ ___, proteins migrate in the gel solely based on ____, not ___

SDS detergent, so that m/z is proportional to mass

denatures them and gives them a uniform negative charge

negative charge, proteins migrate in the gel solely based on size, not charge

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SDS PAGE Gels:

proteins migrate from the ___ (top) to the ____ (bottom) electrode

the gel acts as a molecular sieve, with smaller proteins moving ___ and traveling ___

negative (top) to the positive (bottom) electrode

faster and traveling farther

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SDS PAGE Gels:

gels can be used to check the ___ of ____

comparison to a set of ___ can be used to estimate ___ ___

purity of proteins

standards can be used to estimate molecular weight

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protein isolation by size or affinity:

proteins can be separated by ____ based on ___-____ or using ___ ____

chromatography based on size-exclusion or using affinity tags

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protein isolation by size or affinity:

size-exclusion chromatography (SEC):

gel beads in the column contain ____

large molecules cannot enter the ____, so they flow ____

small molecules enter the ___, so they take ___ to travel through the column

pores

pores, so they flow faster

pores, so they take longer to travel through the column

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protein isolation by size or affinity:

____ _-____ (___) is often used as an ___ ___ with a ___ that can be ___ by ____

glutathione S-transferase (GST) is often used as an affinity tag with a linker than can be cleaved by proteases

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protein isolation by size or affinity:

affinity chromatography using GST:

GST is used as an affinity tag fused to a ___ ____

the GST tag binds to ____, which is ____ in the column

after washing away ___ proteins, the ___-____ protein is eluted

a ___ can be used to remove the GST tag, leaving only the ___ ___ ____

target protein

glutathione, which is immobilized in the column

unbound proteins, the GST-tagged protein is eluted

protease can be used to remove the GST tag, leaving only the purified target protein

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proteases:

proteases are used to generally ___ ___ for ____ (___) or to specifically ___ off ___ ____ like ___ (___)

fragment proteins for sequencing (trypsin) or to specifically cleave of linked tags like GST (thrombin)

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proteases:

proteases are enzymes that cleave ___ ___ in proteins

they can be used to:
___ ____ for ____ (e.g. ___ digestion in ___ ___)

remove ___ ___ (e.g. ___ removes ___ ___)

peptide bonds in proteins

fragment proteins for sequencing (e.g. trypsin digestion in mass spectrometry)

affinity tags (e.g. thrombin removes GST tags)

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proteases:

trypsin cleavage:

cuts after ____ (___) or ____ (___) residues

does NOT cut if the next amino acid is ___ (___)

uses ___ to ___ the peptide bond

lysine (lys) or arginine (arg) residues

proline (pro)

water to hydrolyze the peptide bond

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2D gel separation:

gel columns with ___ ____ can separate proteins by ____ (___ of ___ ___ ___) when subjected to an ___ ____

polypeptide stops migrating on the IEF axis (____ axis) when ___ = ____

the gel column can be used to load an ____ ____ for ___ ___ (____ axis)

pH gradients can separate proteins by pI (pH of zero net charge) when subjected to an electric field

(horizontal axis) when pH = pI

SDS PAGE for 2D separation (vertical axis)

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identifying protein sequences by MS/MS:

the protein mixture is first separated using a ____ ____ (____)

proteins migrate until they reach their ___ ___ (___), where their net charge is ____

pH gradient (IEF)

isoelectric point (pI), where their net charge is zero

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identifying protein sequences by MS/MS:

the proteins from the IEF gel are subjected to ____ ____ to further separate them by ___ ___

this results in ___ ___ ____, where proteins appear as distinct spots

SDS PAGE to further separate them by molecular weight

2D gel electrophoresis where proteins appear as distinct spots

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identifying protein sequences by MS/MS:

individual protein sports can be ___ from the ___ and ___

____ bonds are ___ using a ___ ___ to break them apart

excised from the gel and purified

disulfide bonds are reduced using a reducing agent to break them apart

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identifying protein sequences by MS/MS:

the purified proteins are ___ with ____, an enzyme that cleaves at ___ (__) and ___ (__) residues, generating smaller peptide fragments

digested with trypsin, an enzyme that cleaves at lysine (K) and arginine (R) residues, generating smaller peptide fragments

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identifying protein sequences by MS/MS:

electrospray ionization: peptides are ___ into the ___ phase

MS-1: measures the ___-___-___ ___ (___) of ___ ___ ___

collision cell: peptides are ___ into smaller pieces by ___ with ___ ___

MS-2: measures the ___ ___ ___, breaking them into ___ and ___ ____

b-fragments retain the __-___ end of the peptide

y-fragments retain the _-___ end of the peptide

ionized into the gas phase

mass-to-charge ratio (m/z) of intact peptide ions

fragmented into smaller pieces by collisions with gas molecules

fragmented peptide ions, breaking them into b and y fragments

N-terminal end of the peptide

C-terminal end of the peptide

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identifying protein sequences by MS/MS:

the detected fragment ions generate a ___ ___, showing peaks corresponding to different ___ ____

computational tools reconstruct the ___ ___ ___ by analyzing the ____ data

mass spectrum, showing peaks corresponding to different peptide fragments

original protein sequence by analyzing the fragment data

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identifying protein sequences by MS/MS:

computational analysis reconstructs ___ ___ from ___/___ fragments

B fragments are associated with the __ ___ of the cleaved peptide, and will show up at ____ m/z ratios

protein sequence from MS/MS fragments

N terminus of the cleaved peptide, and will show up at lower m/z ratios

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solid phase peptide synthesis:

the first AA is attached to a ___ ___ via its ___-____

this resin acts as a ____, keeping the growing peptide ___ during synthesis

solid resin via its c-terminus

support, keeping the growing peptide anchored during synthesis

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solid phase peptide synthesis:

the _-___ ___ (___) of the anchored AA is protected by an ____ group

a base like ___ is used to remove the ___ ___, exposing the __-___ ___, allowing for the next AA to attach

N-terminal amine (NH2) of the anchored AA is protected by an Fmoc group

piperidine is used to remove the Fmoc group, exposing the n-terminal amine, allowing for the next AA to attach

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solid phase peptide synthesis:

the next AA (aan) is ____ using ____ to make it reactive for __ ___ formation

activated using DCC to make it reactive for peptide bond formation

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solid phase peptide synthesis:

the activated AA forms a ___ ___ with the exposed ___-___ ____ on the ___-bound peptide

a new ___-protected aa is now attached

peptide bond with the exposed N-terminal amine on the resin-bound peptide

Fmoc-protected aa is now attached

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solid phase peptide synthesis:

steps 2-4 are repeated to build the ____, adding ___ AA at a time

repeated, adding one AA at a time

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solid phase peptide synthesis: 6. once the full peptide is synthesized, the final ____ group is ____ the ___ treatment is used to: - remove the ___ from the ____ - remove any ___ ___ on the AA __ ___ the free peptide is now ____

Fmoc group is removed peptide from the resin protecting groups on the AA side chains released

solid phase peptide synthesis: some AA side chains (like ___, ___, and ___) must be ___ during synthesis a __-___ protecting group is used, which is later removed with ____

serine, threonine, tyrosine t-Butyl protecting group is used, which is later removed with TFA

solid phase peptide synthesis: peptides (<= ___ AA) can be synthesized by iterative ____ on ___ ____

(<= 50 AA) can be synthesized by iterative couplings on solid phase

computational modeling: molecular mechanics (MM): molecular mechanics (MM) is used to model ___ ___ by considering different ___ ___

protein folding by considering different energy contributions

computational modeling: molecular mechanics (MM): proteins fold from an ____ (____) state into their ___ ___ through several intermediate conformations

unfolded (denatured) state into their native structure through several intermediate conformations

computational modeling: molecular mechanics (MM): unfolded (denatured) state: the protein is fully ____ and lacks defined ___ or ____ structure

extended and lacks defined secondary or tertiary structure

computational modeling: molecular mechanics (MM): molten globule states: these are ___ folded states where ___ __ (such as ___-___) start forming, but the full ____ structure is not yet established

partially folded states where secondary structures (such as alpha-helices) start forming, but the full 3d structure is not yet established

computational modeling: molecular mechanics (MM): folding intermediates: the protein continues folding into more ____ structures

stable structures

computational modeling: molecular mechanics (MM): native structure: the ___ ___ form of the protein

final functional form of the protein

computational modeling: molecular mechanics (MM): energy landscape diagram: energy wells: as the protein folds, it goes through ___ ___ states and ___ ___ intermediates, where it is ___ before reaching its final form

molten globule states and discrete folding intermediates, where it is stabilized before reaching its final form

computational modeling: molecular mechanics (MM): energy landscape diagram: the ___ ____ is at the bottom of the energy well, representing the most ___ and ___ energy conformation

native structure is at the bottom of the energy well, representing the most stable and lowest energy conformation

computational modeling: molecular mechanics (MM): molecular mechanics methods use ___ ___ describing both ____ (e.g. ___-___) and __-___ (e.g. ____) interactions

energy terms describing both covalent (e.g. bond-stretching) and non-covalent (e.g. electrostatic) interactions

x-ray crystallography: x-ray crystallography can be used to obtain near-___ resolution structures of ____, even ___-____ assemblies with ___ ____ bound

near-atomic resolution structures of proteins multi-subunit assemblies with small molecules bound

x-ray crystallography: a. x-ray ___ pattern obtained from ____ (a) the process begins with the formation of a ___ ___ this ___ is exposed to ___ ____, producing a ___ ___, where spots represent ___ ___ information from the atoms in the protein

diffraction pattern obtained from crystals protein crystal crystal is exposed to x-ray radiation, producing a diffraction pattern, where spots represent electron density information from the atoms in the protein

x-ray crystallography: b. calculation of ___ ___ (__) map and initial threading of ___ ___ (b) the diffraction pattern is used to calculate an ___ ___ __, a 3D representation of where ____ are likely to be in the structure a ___ __ is initially threaded into the ___ ___ ___ to start building the ___ ___

electron density (ED) map and initial threading of peptide backbone electron density map, a 3D representation of where electrons are likely to be in the structure peptide backbone is initially threaded into the electron density map to start building the protein model

x-ray crystallography: c. refinement of ___ __ to optimize fit to ___ and minimize ___ ____ of structure (c) the ____ ____ is refined to better fit the ___ ___ ___ computational methods optimize the fit by minimizing ___ ___ (___) ___ to improve accuracy

backbone fit to optimize fit to ED and minimize MM energy of structure backbone structure is refined to better fit the electron density data molecular mechanics (MM) energy to improve accuracy

x-ray crystallography: d. ______ fit to ___ to obtain full ___ ___ (d) ____ of AA are added to the model, completing the full ___ ___ structure of the protein the final ___ structure represents the protein with ___ ___ bound

sidechains fit to ED to obtain full protein structure sidechains of AA are added to the model, completing the full 3D structure of the protein ribbon structure represents the protein with small molecules bound

x-ray crystallography: ___ ___ info is essential to solving crystal structures the ___ alone is often not sufficiently high resolution to identify/distinguish ____ reliably knowing the expected __ __ ___ helps researchers correctly fit ___ ___ into the density

primary sequence info is essential to solving crystal structures ED alone is often not sufficiently high resolution to identify sidechains reliably amino acid sequence helps researchers correctly fit sidechains into density

two-dimensional NMR: multi-demenstional NMR can be used to determine the ____ of ___ in ___ typically used for ___ ___ (<= ___ AA)

structures of proteins in solution small proteins (<= 100 AA)

two-dimensional NMR: a. 1D spectrum - peaks assigned to ___ ___ 2D spectru - peak ___ indicate strength of ___ ___ ___ (___) interactions (a)

AA types heights indicate strength of nuclear Overhauser effect (NOE) interactions

two-dimensional NMR: b. NOEs used to identify ___ ___ in ____ use NOEs as ___ (b) NOE interactions provide ___ ___ between hydrogen atoms, helping to infer ___ ___ NOE-derived distances guide ___ ___ of atoms, especially for defining ___ ___ (e.g. ___ ___ and __ __)

atoms near in space constraints distance constraints between hydrogen atoms, helping to infer protein folding spatial arrangements of atoms, especially for defining secondary structures (e.g. alpha-helices and beta sheets)

two-dimensional NMR: c. ____ ____ used to identify __ ___ structure(s) within NOE constraints

molecular mechanisms used to identify minimum energy structure(s) within NOE constraints

cryo-EM: cryo-electron microscopy can be used to solve ___ ___ structures of ___, ____ molecules without the need to form ____

high resolution structures of large, complex molecules without the need to form crystals

cryo-EM: 1. ___ samples are imaged using an ___ ___, capturing multiple ____ of the molecule rapid ___ preserves ___ ___ structures in near-natural state

frozen samples are imaged using an electron beam, capturing multiple orientations of the molecule freezing preserves native protein structures in near-natural state

cryo-EM: 2. computation reconstruction refines 2D images into a ___ ___, using ___ and ___ techniques

3D structure, using classification and averaging techniques

reversible protein denaturation: for a reversibly unfolding protein, ____ = ____ (___) unfolding can be accomplished ___ or ____ conditions: folding is effectively a ___-___ process (no ___) fraction unfolded can be monitored by changes in ____ ____, ____ ____, or ____

native = denatured (equilibrium) thermally or chemically 2-state process (no intermediates UV/Vis spectroscopy, circular dichroism, or fluorescence

UV/Vis spectroscopy: ___ ___ ___ (___, ____, ___)and all ____ ___ absorb light in the ___ range

aromatic amino acids (trp, tyr, phe) and all nucleic acids absorb light in the UV range

UV/Vis spectroscopy: molar extinction coefficients (E) can be used to determine the ___ of ___ or ____

concentration of protein or DNA

UV/Vis spectroscopy: the extinction coefficient changes as the molecule/protein ____ as the protein unfolds, its aromatic ring AA are placed in a ___ ___ / become ____ to the ___, altering ____ absorption this allows monitoring of ___ ___ (e.g. ___-induced ____)

unfolds different environment / become exposed to the solvent, altering UV absorption protein denaturation (e.g. heat-induced unfolding)

circular dichroism: circular dichroism (CD) measures the ___ ___ ___ (delta E) of ___- and ___- ____ ____ light delta E = El - Er measures the ___ structure of proteins

molar absorption difference (delta E) of left- and right-circularly polarized light secondary structure of proteins

circular dichroism: proteins contain ____ chromophores (___ ___ in peptide backbone) that absorb polarized light differently, depending on their ___ structure --> produce ____ signals

chiral chromophores (amide bonds in peptide backbone) that absorb polarized light differently, depending on their secondary structure --> produce characteristic signals

circular dichroism: CD signals from peptide bonds depend on the ____ ____ () CD signal is influenced by ___ ___ ___, which varies based on whether the protein is in an ___-___, ___-____, or ___ ___ conformation

chain conformation amide bond alignment, which varies based on whether the protein is in an alpha-helix, beta-sheet, or random coil conformation

fluorescence: the amino acids ___ and ____ fluoresce in the ____nm range when irradiated with ___nm light

Trp and Tyr fluoresce in the 350nm range when irradiated with 280nm light

fluorescence: fluorescence emission intensity increases when the ____ is placed in a ____ medium (___ ___)

fluorophore is placed in a hydrophobic medium (protein interior)

fluorescence: measuring fluorescence at various ____ ____ or ____ can be used to monitor ____

urea concentrations or temperatures can be used to monitor folding

fluorescence: folded state: Trp is buried inside the ___ ___ (___ fluorescence) unfolded state: Trp is exposed to ___, ___ fluorescence or shifting emission to ___ wavelengths

protein core (high fluorescence) water, reducing fluorescence or shifting emission to longer wavelengths

fluorescence: urea disrupts ___ ___ and weakens ____ ___, causing the protein to unfold at low urea concentrations: protein is ____, and fluorescence intensity is ____ at high urea concentrations: protein is ___, and fluorescence intensity ___ or ____

hydrogen bonds and weakens hydrophobic interactions, causing the protein to unfold folded, and and fluorescence intensity is high unfolded, and fluorescence intensity drops or shifts

folding analysis by mutation: potentially important ___ interactions are identified in ___ ____ AA involved in the interaction are ___ to other ___; the free energy of ___ is measured (e.g. by ___ ___) observed change in free energy of folding (___ ___ ___) is compared to expectations of hypotheses to confirm ___ of ____

non-covalent interactions are identified in protein structures mutated to other AA; the free energy of folding is measured (e.g. by urea denaturation) (delt delta G) is compared to expectations of hypotheses to confirm relevance of interaction

folding analysis by mutation: mutational analysis helps identify residues critical for ___ ____ higher delta delta G value means ___ ____ (____ energy needed to fold, ___ stable protein) confirming that ___ contributes significantly to ___

protein folding greater destabilization (more energy needed to fold, less stable protein) Asp157 contributes significantly to stability

monitoring single substrate binding to an enzyme: Ys is the fraction of ___ ___ __ ___ aka the proportion of the total enzyme molecules that have a ____ ____ to them at a given ___ concentration

enzyme bound to substrate substrate bound to them at a given substrate concentration

monitoring single substrate binding to an enzyme: after separation of unbound substrate, the amount of E + E.S can be determined and used to determine ___ by plotting as a function of ___

Kd by plotting as a function of [S]

monitoring single substrate binding to an enzyme: Kd is the ___ ____ or the substrate concentration at which ____ of the ___ ____ molecules are bound (Ys = ___)

dissociation constant or the substrate concentration at which half of the available enzyme molecules are bound (Ys = 0.5)

monitoring single substrate binding to an enzyme: E = ___ ___ E0 = ___ of ___ ___

measured absorbance absorbance of enzyme alone

monitoring single substrate binding to an enzyme: the binding curve plots ___ (___ ___) vs. __ (___ ____)

Ys (fraction bound) vs. [S] (substrate concentration)

Fully Cooperative Multiple Substrate Binding: cooperative substrate binding in enzymes is when the ___ of ___ ___ affects the ___ of ____ ___ the ___ ___ models this behavior, and the ___ ___ helps determine the ___ of ____

binding of one substrate affects the binding of additional substrates hill equation models this behavior, and the hill plot helps determine the degree of cooperativity

Fully Cooperative Multiple Substrate Binding: fully cooperative (___ or ___) binding is when the enzyme binds ___ ___ ____ or not at all this binding can be fit by the ___ ___

multiple substrates simultaneously or not at all hill equation

Fully Cooperative Multiple Substrate Binding: for multiple substrates, the Kd is not clear from a simple plot of ___ vs. ____ a hill plot is used to find ___ and ___

Ys vs. [S] n and Kd

Fully Cooperative Multiple Substrate Binding: n=1, binding is ___ (___-____) n>1, ___ cooperativity (binding ___ affinity for more substrate) n<1, ___ cooperativity (binding ___ affinity for additional substrate higher n leads to a ___ binding curve n is the ___ of the binding curve

independent (non-cooperative) positive cooperativity (binding increases affinity for more substrate) negative cooperativity (binding decreases affinity for additional substrate steeper binding curve slope

substrate binding specificity: protease: trypsin protease is specific for cleaving after ___ or ___ (at ___)

Lys or Arg (at Rn-1)

substrate binding specificity: protease: specificity information obtained by carrying out a series of ___ ___ to determine ___ binding experiments determine ___, which quantifies how strongly the enzyme ___ to its ___ higher Kd = ____ binding (substrate ___ ___) lower Kd = ___ binding (substrate __ ___ ___)

binding experiments to determine Kd Kd, which quantifies how strongly the enzyme binds to its substrate weaker binding (substrate dissociates easily) stronger binding (substrate stays bound longer)

substrate binding specificity: protease: ____ of certain AA can be used to ____ interactions observed in crystal structures

mutations of certain AA can be used to confirm interactions observed in crystal structures

monitoring an enzymatic reaction: chromatography: reactants and products can be separated by ____ and quantified using a ____ detector (a chromatogram)

chromatography and quantified using a UV/Vis detector (a chromatogram)

monitoring an enzymatic reaction: chromatography: chromatograms that are run at varying ___ can be used to determine the ___ ___ (k)

times can be used to determine the reaction rate (k)

Enzymatic Substrate Specificity: Protease: trypsin protease is specific for ____ or ____ at ___, but not when ____ is at ____

Lys or Arg at Rn-1, but not when Pro is at Rn

Enzymatic Substrate Specificity: Protease: specificity info is obtained by testing ___ ___ with various ___ ____ under identical conditions, WRAA is ___, WRPA is ___ ___

cleavage rates with various synthetic peptides cleaved, WRPA is not cleaved

Testing Protease Mechanism: ____ ____ may suggest certain AA carry out a reaction ____ of those ___ can be used to test that hypothesis by comparing ___ ___ of ___ ___ and ___ enzymes e.g. if ser 195 acts as a nucleophile, Ala mutant should be ___

crystal structures may suggest certain AA carry out a reaction mutations of those AA can be used to test that hypothesis by comparing reaction rates of wild type and mutant enzymes inactive

Testing Protease Mechanism: crystal structures suggest ___ ___, but ____ confirm their function ___ ___ is critical for protease activity, as mutating it eliminates ___ ___

catalytic residues, but mutations confirm their function Ser 195 is critical for protease activity, as mutating it eliminates substrate cleavage