Proteins in frozen solutions

The recoil-free fraction /a of transition metal complexes or proteins in frozen solution can be as small as 0.1-0.3, when measured just below the melting point, but the /-factor increases strongly when the temperature is lowered to fiquid nitrogen temperatures (77 K), and at fiquid helium temperatures (4.2 K) it may reach values of 0.7-0.9 [35]. This makes a substantial difference to the acquisition time of the spectra because of the square dependency on the signal (3.1). 

Early experimental studies of cryogenic radiolytic reduction of heme proteins in frozen solutions demonstrated that in most cases reduced proteins cannot undergo... 

G. B. Strambini and E. Gabellieri, Proteins in frozen solutions evidence of ice-induced partial unfolding, Biophys. J. 70 911-910 (1996). 

G. B. Strambini and E. GabeUieri. (1996) Proteins in Frozen Solutions Evidence of Ice-Induced Partial Unfolding, Biophysical Journal, 70(2), 971-976. 

Figure 15.2 Typical HX-MS experimental workflow for (a) protein adsorbed on solid surfaces, (b) protein In frozen solution, and (c) protein in lyophilized solid powders. The experimental conditions are shown in the figure...

In frozen solution EPR spectra of copper proteins, ligand and copper hf interactions are only partially resolved, or not at all. It is therefore striking that only few ENDOR studies on copper proteins have been published so far17,199-201). Nevertheless, these few ENDOR results already demonstrate the power of the double resonance technique to probe the coordination environment of the copper-containing sites in these types of proteins. 

Essentially nothing is known about tyrosine phosphorescence at ambient temperatures. In frozen solution, tyrosine residues have a phosphorescence decay of seconds. We would expect, however, a decay of milliseconds or shorter at ambient temperature. Observation of tyrosine phosphorescence from proteins in liquid solution will undoubtedly require efficient removal of oxygen. Nevertheless, it could be fruitful to explore ambient temperature measurements, since the phosphorescence decay could extend the range of observation of excited-state dynamics into the microsecond, or even millisecond, time range. 

Fourier Deconvolution. A synthetic 30-residue polypeptide was synthesized as an electron-transfer protein.42 A spin label was attached at position 21 and the distance between the two labels in a dimer was determined by Fourier deconvolution of the CW line-shape in frozen solution. Based on the known geometry of the label, it was calculated that the interspin distance of 22.5 A corresponded to 13.5 + 0.9 A between the Ca carbons on the two polypeptide chains. 

To model the moiety of glycoproteins which coimects the protein and carbohydrate parts of the molecule, amino acid - carbohydrate adducts (e.g. V-D-gluconylamino acids, thiazolidine-4-carboxylic acid derivatives of carbohydrates, " D-fructose amino acid derivates, etc.) have been synthetized. The protonation and metal ion (Cu2+, Ni2+, Co ", Zn2+ and Et2Sn2+) coordination equilibria of these model compounds have been studied by means of potentiometric equilibrium measurements. CD, EPR, NMR, EXAFS and MSssbauer investigations (the latter in frozen solutions) have been used to determine the structure and symmetry of the coordination sphere of the complexes. >6... 

Upon lyophilization the conformation of a protein can change too. From Mossbauer investigations of Hb (human) (41, 48) and Mb (horse heart) (48) in frozen solution and in dehydrated from it is known that the high-spin ferrous state in Hb is converted to an equal mixture of ferrous high-spin and low-spin iron in anhydrous Hb, whereas Mb and anhydrous Mb both contain ferrous high-spin iron only (Fig. 5). 

ESR evidence for this electron adduct of the carbonyl group in frozen solutions of acetylamino acids and di- and tripeptides is extensive (50). A typical spectrum is shown in Figure 6 for the anion radical of / -alanyl-glycine at — 153°C. Depending on the peptide and pH, deamination can occur by a process involving either inter- or intramolecular electron transfer. Dissociation at other C—N bonds is also possible, leading to an amide and a fatty acid radical (20,63) this process will be referred to as deamidation or secondary deamination. As will be mentioned below, this reaction and the chemistry that follows are important for peptides and proteins. 

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