BioNMR
NMR aggregator & online community since 2003
BioNMR    
Learn or help to learn NMR - get free NMR books!
 

Go Back   BioNMR > NMR community > News from NMR blogs
Advanced Search



Jobs Groups Conferences Literature Pulse sequences Software forums Programs Sample preps Web resources BioNMR issues


Webservers
NMR processing:
MDD
NMR assignment:
Backbone:
Autoassign
MARS
UNIO Match
PINE
Side-chains:
UNIO ATNOS-Ascan
NOEs:
UNIO ATNOS-Candid
UNIO Candid
ASDP
Structure from NMR restraints:
Ab initio:
GeNMR
Cyana
XPLOR-NIH
ASDP
UNIO ATNOS-Candid
UNIO Candid
Fragment-based:
BMRB CS-Rosetta
Rosetta-NMR (Robetta)
Template-based:
GeNMR
I-TASSER
Refinement:
Amber
Structure from chemical shifts:
Fragment-based:
WeNMR CS-Rosetta
BMRB CS-Rosetta
Homology-based:
CS23D
Simshift
Torsion angles from chemical shifts:
Preditor
TALOS
Promega- Proline
Secondary structure from chemical shifts:
CSI (via RCI server)
TALOS
MICS caps, β-turns
d2D
PECAN
Flexibility from chemical shifts:
RCI
Interactions from chemical shifts:
HADDOCK
Chemical shifts re-referencing:
Shiftcor
UNIO Shiftinspector
LACS
CheckShift
RefDB
NMR model quality:
NOEs, other restraints:
PROSESS
PSVS
RPF scores
iCing
Chemical shifts:
PROSESS
CheShift2
Vasco
iCing
RDCs:
DC
Anisofit
Pseudocontact shifts:
Anisofit
Protein geomtery:
Resolution-by-Proxy
PROSESS
What-If
iCing
PSVS
MolProbity
SAVES2 or SAVES4
Vadar
Prosa
ProQ
MetaMQAPII
PSQS
Eval123D
STAN
Ramachandran Plot
Rampage
ERRAT
Verify_3D
Harmony
Quality Control Check
NMR spectrum prediction:
FANDAS
MestReS
V-NMR
Flexibility from structure:
Backbone S2
Methyl S2
B-factor
Molecular dynamics:
Gromacs
Amber
Antechamber
Chemical shifts prediction:
From structure:
Shiftx2
Sparta+
Camshift
CH3shift- Methyl
ArShift- Aromatic
ShiftS
Proshift
PPM
CheShift-2- Cα
From sequence:
Shifty
Camcoil
Poulsen_rc_CS
Disordered proteins:
MAXOCC
Format conversion & validation:
CCPN
From NMR-STAR 3.1
Validate NMR-STAR 3.1
NMR sample preparation:
Protein disorder:
DisMeta
Protein solubility:
camLILA
ccSOL
Camfold
camGroEL
Zyggregator
Isotope labeling:
UPLABEL
Solid-state NMR:
sedNMR


Reply
Thread Tools Search this Thread Rate Thread Display Modes
  #1  
Unread 08-22-2010, 01:58 AM
nmrlearner's Avatar
Senior Member
 
Join Date: Jan 2005
Posts: 21,395
Points: 193,617, Level: 100
Points: 193,617, Level: 100 Points: 193,617, Level: 100 Points: 193,617, Level: 100
Level up: 0%, 0 Points needed
Level up: 0% Level up: 0% Level up: 0%
Activity: 50.7%
Activity: 50.7% Activity: 50.7% Activity: 50.7%
Last Achievements
Award-Showcase
NMR Credits: 0
NMR Points: 193,617
Downloads: 0
Uploads: 0
Default How native-like is a cold-denatured structure?

How native-like is a cold-denatured structure?

A protein has several different levels of structure. The primary structure is the arrangements of atoms and bonds, and it is formed in the ribosome by the assembly of amino acids as directed by an RNA template. The secondary structure is the local topology, the helices and strands, and this forms mostly because of the release of energy through the formation of hydrogen bonds. The tertiary structure is the actual fold of the protein, the way helices, strands, and loops are arranged in space. The fold forms primarily because of the favorable entropy of burying the protein's hydrophobic groups where water cannot access them, analogous to the formation of an oil droplet in water. This suggests that, in addition to the well-known phenomenon of proteins denaturing, or losing their higher-order structure, under conditions of high heat, proteins might also denature when they get too cold.

As you might remember from your chemistry classes, the change in free energy due to a reaction under conditions of constant pressure is given by:
?G = ?H - T ?S
Where ?H is the change in enthalpy (i.e. the heat released or absorbed by a reaction), ?S is the change in entropy, and T is the temperature of the system in Kelvin. Here, the change we are talking about is the transition from the folded state to some unfolded state. Simplistically, since the entropic contribution is scaled by the temperature, one can imagine that for a reaction with favorable entropy and unfavorable enthalpy, lowering the temperature could cause the reaction to reverse. Protein folding is only marginally favorable at biological temperatures, so one could easily imagine that lowering the temperature enough could cause a protein to prefer the unfolded state.

Of course, this is an oversimplification: the entropy and enthalpy of a particular protein state do not remain constant over all temperatures. Rather, they vary in a way determined by the heat capacity (Cp), such that ?G as a function of temperature is (1):
?G(T) = ?H(Tr) + ?Cp(T-Tr) - T [?S(Tr) + ?Cp ln(T/Tr)]
Where Tr is some reference state at which the thermodynamic parameters have been determined, and ?Cp is defined with respect to the native (folded) state. Because the various states of the protein have different Cp (unfolded chains typically have higher Cp), at certain temperatures above and below the biological optimum we can expect proteins to lose their higher levels of structure. Even this is still an oversimplification, of course, because it does not directly account for changes in water structure and cosolute properties with temperature. These features may cause ?Cp itself to vary with temperature rather than remain constant.

Unfortunately, for most proteins the temperature that favors unfolding lies below the freezing point of water, which makes this phenomenon difficult to study unless you do something unusual to your system. In 2004, Babu et al. (1) reported results from experiments that used reverse micelles to study the denaturation of ubiquitin at temperatures below freezing. By encapsulating a protein-water droplet in inverted micelles dissolved in pentane, it was possible to reduce the temperature to 243 K without causing freezing. These micelles also had the convenient property of tumbling quickly in the pentane, which allowed for reasonable NMR spectra even at these low temperatures. The appearance of the spectra they obtained indicated that the protein underwent a slow unfolding process with many different unfolded states, and also that the protein did not unfold in a cooperative fashion. Rather, it appeared that one contiguous region of the protein unfolded while the rest remained folded (the main helix was particularly stable).

This wasn't expected, because ubiquitin apparently unfolds in a completely two-state manner when overheated. This being the case, what's expected is for the protein to either be all folded or all unfolded, not some mixture of the two. However, cold does not affect all intramolecular contacts the same way. Lowering the temperature is expected to make hydrophobic interactions less favorable while not significantly affecting polar interactions like hydrogen bonds. This being the case, one might expect an ?-helix to persist through a cold-denaturation transition, as happens in this case.

Something similar is observed in an upcoming paper in JACS from the Raleigh and Eliezer Labs (2), which approaches cold denaturation using a mutant form of the C-terminal domain of ribosomal protein L9. An isoleucine to alanine mutation at residue 98 of this domain doesn't appear to significantly alter the structure, but it causes the protein to denature somewhere in the high teens. At 12 C the unfolded state is about 80% of the visible population, and this is where Shan et al. performed their NMR experiments. They assigned the unfolded state using standard techniques and then decided to see what they could learn from the chemical shifts.

As I've mentioned before, the chemical shift of a nucleus depends on the probability distribution of the surrounding electrons, and therefore is sensitive to the strength, composition, and angles of the atom's chemical bonds. Because the dihedral angles of the protein backbone are a good proxy for the secondary structure, one can use the chemical shifts of particular atoms to determine whether a given residue is in a helix or strand. When they performed this analysis, Shan et al. noticed two major differences between the native and cold-denatured states of the protein. The first was that the helix and strand propensities of the denatured protein were much lower than the folded form, as expected. In addition, however, they noticed that one loop of the protein had gained ?-helical character. That is, it seemed like an ?-helix had actually gotten longer as a result of the unfolding.

This doesn't mean that denaturing the protein added secondary structure. The low values in the output from the algorithm Shan et al. used suggest that the secondary structure in this denatured state forms only transiently. However, the chemical shifts suggest, and other structural data appear to confirm, that this region of the protein has an increased propensity to inhabit a helical structure as a consequence of the unfolding.

These results emphasize the fact that the "unfolded state" isn't as simple as it's often described. Residual structure persists in unfolded states of many proteins, and unfolded ensembles of one protein generated through different means (heat, cold, pH, cosolutes) may not resemble each other. Unlike unfolding at high temperature, cold denaturation of ubiquitin appears to be non-cooperative. In both ubiquitin and L9, it appears that helices are robust to the unfolding process, persisting and even propagating as the protein denatures. While some of these features may be held in common between different kinds of denatured states, others may be unique to particular denaturation conditions. The lingering question is which of these unfolded ensembles best resembles the denatured state that exists under biological conditions, giving rise to misfolded states and their associated diseases.

(1) Babu, C., Hilser, V., & Wand, A. (2004). Direct access to the cooperative substructure of proteins and the protein ensemble via cold denaturation Nature Structural & Molecular Biology, 11 (4), 352-357 DOI: 10.1038/nsmb739

(2) Shan, B., McClendon, S., Rospigliosi, C., Eliezer, D., & Raleigh, D. (2010). The Cold Denatured State of the C-terminal Domain of Protein L9 Is Compact and Contains Both Native and Non-native Structure Journal of the American Chemical Society DOI: 10.1021/ja908104s



Get complete info from mwclarkson blog
Reply With Quote


Did you find this post helpful? Yes | No

Reply
Similar Threads
Thread Thread Starter Forum Replies Last Post
[NMR paper] Solution NMR structure of the cold-shock protein from the hyperthermophilic bacterium
Solution NMR structure of the cold-shock protein from the hyperthermophilic bacterium Thermotoga maritima. Related Articles Solution NMR structure of the cold-shock protein from the hyperthermophilic bacterium Thermotoga maritima. Eur J Biochem. 2001 May;268(9):2527-39 Authors: Kremer W, Schuler B, Harrieder S, Geyer M, Gronwald W, Welker C, Jaenicke R, Kalbitzer HR Cold-shock proteins (Csps) are a subgroup of the cold-induced proteins preferentially expressed in bacteria and other organisms on reduction of the growth temperature below the...
nmrlearner Journal club 0 11-19-2010 08:32 PM
[NMR paper] Microscopic stability of cold shock protein A examined by NMR native state hydrogen e
Microscopic stability of cold shock protein A examined by NMR native state hydrogen exchange as a function of urea and trimethylamine N-oxide. Related Articles Microscopic stability of cold shock protein A examined by NMR native state hydrogen exchange as a function of urea and trimethylamine N-oxide. Protein Sci. 2000 Feb;9(2):290-301 Authors: Jaravine VA, Rathgeb-Szabo K, Alexandrescu AT Native state hydrogen exchange of cold shock protein A (CspA) has been characterized as a function of the denaturant urea and of the stabilizing agent...
nmrlearner Journal club 0 11-18-2010 09:15 PM
[NMR paper] Hydrodynamic radii of native and denatured proteins measured by pulse field gradient
Hydrodynamic radii of native and denatured proteins measured by pulse field gradient NMR techniques. Related Articles Hydrodynamic radii of native and denatured proteins measured by pulse field gradient NMR techniques. Biochemistry. 1999 Dec 14;38(50):16424-31 Authors: Wilkins DK, Grimshaw SB, Receveur V, Dobson CM, Jones JA, Smith LJ Pulse field gradient NMR methods have been used to determine the effective hydrodynamic radii of a range of native and nonnative protein conformations. From these experimental data, empirical relationships...
nmrlearner Journal club 0 11-18-2010 08:31 PM
[NMR paper] NMR assignments for acid-denatured cold shock protein A.
NMR assignments for acid-denatured cold shock protein A. Related Articles NMR assignments for acid-denatured cold shock protein A. J Biomol NMR. 1998 May;11(4):461-2 Authors: Alexandrescu AT, Rathgeb-Szabo K
nmrlearner Journal club 0 11-17-2010 11:06 PM
[NMR paper] Hydrogen exchange properties of proteins in native and denatured states monitored by
Hydrogen exchange properties of proteins in native and denatured states monitored by mass spectrometry and NMR. http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--www3.interscience.wiley.com-aboutus-images-wiley_interscience_pubmed_logo_FREE_120x27.gif http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--www.pubmedcentral.nih.gov-corehtml-pmc-pmcgifs-pubmed-pmc.gif Related Articles Hydrogen exchange properties of proteins in native and denatured states monitored by mass spectrometry and NMR. Protein Sci. 1997 Jun;6(6):1316-24 Authors: Chung EW,...
nmrlearner Journal club 0 08-22-2010 03:31 PM
[NMR paper] Hydrogen exchange properties of proteins in native and denatured states monitored by
Hydrogen exchange properties of proteins in native and denatured states monitored by mass spectrometry and NMR. http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--www3.interscience.wiley.com-aboutus-images-wiley_interscience_pubmed_logo_FREE_120x27.gif http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--www.pubmedcentral.nih.gov-corehtml-pmc-pmcgifs-pubmed-pmc.gif Related Articles Hydrogen exchange properties of proteins in native and denatured states monitored by mass spectrometry and NMR. Protein Sci. 1997 Jun;6(6):1316-24 Authors: Chung EW,...
nmrlearner Journal club 0 08-22-2010 03:03 PM
[NMR paper] Solution NMR structure of the major cold shock protein (CspA) from Escherichia coli:
Solution NMR structure of the major cold shock protein (CspA) from Escherichia coli: identification of a binding epitope for DNA. http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--www.pubmedcentral.nih.gov-corehtml-pmc-pmcgifs-pubmed-pmc.gif Related Articles Solution NMR structure of the major cold shock protein (CspA) from Escherichia coli: identification of a binding epitope for DNA. Proc Natl Acad Sci U S A. 1994 May 24;91(11):5114-8 Authors: Newkirk K, Feng W, Jiang W, Tejero R, Emerson SD, Inouye M, Montelione GT Sequence-specific...
nmrlearner Journal club 0 08-22-2010 03:33 AM
[NMR paper] Solution NMR structure of the major cold shock protein (CspA) from Escherichia coli:
Solution NMR structure of the major cold shock protein (CspA) from Escherichia coli: identification of a binding epitope for DNA. http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--www.pubmedcentral.nih.gov-corehtml-pmc-pmcgifs-pubmed-pmc.gif Related Articles Solution NMR structure of the major cold shock protein (CspA) from Escherichia coli: identification of a binding epitope for DNA. Proc Natl Acad Sci U S A. 1994 May 24;91(11):5114-8 Authors: Newkirk K, Feng W, Jiang W, Tejero R, Emerson SD, Inouye M, Montelione GT Sequence-specific...
nmrlearner Journal club 0 08-22-2010 03:33 AM


Thread Tools Search this Thread
Search this Thread:

Advanced Search
Display Modes Rate This Thread
Rate This Thread:

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are Off
Pingbacks are Off
Refbacks are Off



BioNMR advertisements to pay for website hosting and domain registration. Nobody does it for us.



Powered by vBulletin® Version 3.7.3
Copyright ©2000 - 2021, Jelsoft Enterprises Ltd.
Copyright, BioNMR.com, 2003-2013
Search Engine Friendly URLs by vBSEO 3.6.0

All times are GMT. The time now is 03:32 PM.


Map