The cellular interior, where most biological processes occur, is unlike the dilute solutions where most proteins are studied. The large volume excluded by high macromolecule concentrations in cells, from 20-40% [1
], is predicted to change many protein properties compared to dilute solution. We used a synthetic microgel composed of poly(N
-acrylic acid) [p
-AAc (Figure )], as a crowding agent to study the backbone dynamics and the stability of the globular test protein, chymotrypsin inhibitor 2 (CI2).
Structure and Size of p-NIPAm-co-AAc: A) The monomeric repeat of NIPAm. B) Overall shape and size of p-NIPAm-co-AAc microgels.
-AAc is of interest in pharmaceutical applications because it forms environmentally sensitive microgels [2
]. Each microgel particle (Figure ) is a lightly cross-linked single polymer molecule of molecular weight 109
Da with an average of 70 monomer units between each cross link. The polymer absorbs a large amount of water resulting in spherical particles of 300 nm radii that exclude large amounts of solution volume. Their porosity arises from the balance between the external (solution) osmotic pressure and the internal osmotic pressure. This internal pressure is the result of the solvated cations that neutralize the deprotonated polymer side chains. We chose this crowding agent because its status as a drug delivery molecule makes it pharmaceutically relevant, and its ability to take up water provides a model for volume exclusion by a molecule much larger than our test protein.
CI2 is a small globular protein (7.4 kDa, PDB ID: 2CI2
) that exhibits two-state folding [3
]. NMR relaxation experiments [4
] allowed us to assess backbone rotational dynamics for CI2 in the presence and absence of p
-AAc. Amide proton exchange experiments [5
] allowed us to assess the stability of CI2 in dilute and crowded conditions.
Globular proteins are often treated like hard spheres, but they have measurable amounts of internal motion. Analysis of relaxation parameters from NMR experiments - longitudinal and transverse relaxation times, T1
, and the 15
H nuclear Overhauser effect (NOE) of backbone 15
N atoms - offers a residue-level window into this ps-ns backbone motion. The analysis involves a model-free method established by Lipari and Szabo [7
]. Analysis is performed by fitting the spectral density function I
) as calculated from measured T1
, and NOE values [8
], to the equation [7
The overall correlation time τ
is linked to the correlation time for isotropic tumbling, τm
, and internal motion timescale, τe
, by the equation
with the internal motions faster than the overall isotropic tumbling. The order parameter, S2
, can have values between 0 and 1, and is related to the degree of internal mobility for a particular 1
N vector. An S2
value of 0 corresponds to complete freedom of motion. In this instance, relaxation is related solely to internal motion. An S2
value of 1 corresponds to complete restriction of the vector with respect to overall molecule motion, and relaxation is related solely to isotropic tumbling of the protein. These parameters can be linked to models for motion, in our case, the "wobble-in-a-cone" model [7
]. Variations of Lipari-Szabo analysis exist for cases involving ms timescale conformational exchange, but no CI2 residue (except Thr40) has significant contributions from slow exchange [9
]. It is also possible to study the equilibrium thermodynamic stability of globular proteins by using NMR.
Amide proton exchange experiments can be used with NMR to assess protein stability. The technique relies on the exchange of amide protons for deuterons in a D2
O solution. We have recently reviewed the requirements for its application in crowded solution by using NMR [10
Exchange occurs via
with opening rate kop
, closing rate kcl
, and rate of exchange from the open state kint
. If the protein is stable (kcl
) and exchange from the open state is rate limiting, the stability of an amide proton against exchange (
) can be determined with the equation,
is the gas constant and T
is the absolute temperature. The value of kobs
, the overall rate of exchange for any particular backbone amide proton, is assessed by acquiring 1
N heteronuclear single quantum correlation (HSQC) spectra as a function of time after initiating exchange. As with dynamics,
can be quantified on a per-residue basis. The largest
values match the global protein stability values determined by other methods (e.g.
, calorimetry, circular dichroism spectropolarimetry) [11