Laura,

Short of x-ray diffraction the gold-standard in protein structural analysis (although it also has some limitations and is time consuming), there are several spectroscopic techniques that will only provide partial information.  There is also neutron diffraction, usually providing lower resolution structures.

For the last couple decades there has been increasing success using two-dimensional NMR (COSY and NOESY) to provide x-ray structural-like information in the solution-state.  It is typically limited to small proteins and will provide primary, secondary and tertiary structural information.

Circular dichroism (CD) spectroscopy (between 175 nm to 240 nm) will yield secondary structural information.  The chromophore being the peptide bond, the recorded spectrum will display specific signatures that can be related to certain peptide backbone arrangements.

Fluorescence spectroscopy can be used to determine if tryptophan or tyrosine residues are buried or exposed.  Time resolved fluorescence can be used to measure molecular distances within proteins using FRET.  

Room-temperature phosphorescence can provide information on protein rigidity.

Other spectroscopic techniques will provide other small pieces of the structural puzzle.

I hope this is some help.

Cheers,

John

Dear Laura,

you can do circular dichroism experiments (CD) to analyze the protein secondary/tertiary structure changes after temperature and pH treatments of your proteins.

We recently published a paper describing the analysis of protein folding by CD after heat treatments (Tuppo et al.Structure, stability, and IgE binding of the peach allergen Peamaclein (Pru p 7), Biopolymers: Peptide Science, 2014). Few years ago we described the analysis of protein secondary structure changes as a function of pH and apolar solvents by CD (Bernardi et al. Physico-chemical features of the environment affect the protein conformation and the immunoglobulin E reactivity of kiwellin (Act d 5), Clin Exp Allergy 40 (2010) 1819-1826).

Laura,

I have provided you with links to two papers.  One where I have used CD to measure the kinetics of the thermal aggregation of a membrane protein (aquaporin 0),  and the other where I measured the equilibrium denaturation of a protein (azurin) in the presence of guanidinium HCl.  In both of these studies I was following changes in secondary structure.

With kind regards,

John

Article The kinetics of thermal stress induced aggregation of Aquaporin 0

Article A pH Dependence Study on the Unfolding and Refolding of Apoa...

Two ways for rapidly determining some information on the tertiary structure of a protein short of actually determining the structure:

1D NMR: The dispersion of the peaks in the aliphatic region of a 1D NMR spectrum is related to how well-packed the core of the protein is ( http://bitesizebio.com/8252/what-can-nmr-do-for-you-part-one ) . If the protein is partially unfolded but has secondary structure (the molten globule state http://en.wikipedia.org/wiki/Molten_globule ), the peaks are broad but in a narrow region of the spectrum. On the other hand, a well folded protein with a well-packed core will have many sharp peaks  covering a larger range of chemical shifts. See Figure 7F and I in https://www.researchgate.net/publication/258031760_An_Evolution-Based_Approach_to_De_Novo_Protein_Design_and_Case_Study_on_Mycobacterium_tuberculosis

for example of molten globules and Fig 7 G, H, and J for examples of well folded proteins. This procedure can be made into a quantitative test for stable tertiary structure http://www.ncbi.nlm.nih.gov/pubmed/15808220 . There is no need for labeling or assignment of peaks and each sample can be done in a few minutes.

SAXS Small Angle Xray Scattering can also provide information on the tertiary structure of proteins and can be run in a 96 well plate format. I haven't personally used it yet so I leave it to others to describe their experiences.

Article An Evolution-Based Approach to De Novo Protein Design and Ca...

Many thanks for all your responses, I am really surprised. Yours responses help me a lot, now I have t o study the best for my research.

Thanks