Protein unfolding is a key step in several cellular processes, including protein translocation across some membranes and protein degradation by ATP-dependent proteases. ClpAP protease and the proteasome can actively unfold proteins in a process that hydrolyzes ATP. Here we show that these proteases seem to catalyze unfolding by processively unraveling their substrates from the attachment point of the degradation signal. As a consequence, the ability of a protein to be degraded depends on its structure as well as its stability. In multidomain proteins, independently stable domains are unfolded sequentially. We show that these results can explain the limited degradation by the proteasome that occurs in the processing of the precursor of the transcription factor NF-kappaB.

Utilizing surface plasmon resonance (SPR), we have developed novel methodology for the detection of conformational change(s) in immobilized proteins. A genetically altered E. coli dihydrofolate reductase (DHFR-ASC) was attached to a carboxymethyldextran matrix layer covering the sensor surface of an SPR biosensor through a disulfide linkage at the engineered protein's C-terminus. The DHFR-ASC-immobilized surface exhibited a larger response to acid treatment than reference surfaces lacking immobilized proteins. The SPR signal of the tethered protein and the molar ellipticity of DHFR-ASC in solution responded similarly to pH changes, consistent with the interpretation that changes in the SPR signal reflect conformational changes occurring during acid denaturation. A pH shift observed between the SPR signal and ellipticity changes may reflect a difference between surface and bulk pH. The tethered protein sensor surface was stable to repeated acid treatment using solutions in the pH range of 0.12-7.80 and yielded reproducible measurements. This is the first demonstration of detection of conformational changes in an immobilized protein using an SPR biosensor. This technique has potential for developing novel sensors and/or switching devices in response to protein conformational changes.

The importance of chain connectivity in determining protein function and stability can be examined by breaking the peptide backbone using a technique such as circular permutation. Cleavage at certain positions results in a complete loss of the ability of the protein to fold. When such cleavage sites occur sequentially in the primary structure, we call the region a 'folding element', a new concept that could assist in our understanding of the protein folding problem. The folding elements of dihydrofolate reductase have been assigned by conducting a systematic circular permutation analysis in which the peptide backbone was sequentially broken between every pair of residues in the protein. The positions of folding elements do not appear to correspond to secondary structure motifs, substrate or coenzyme binding sites, or accessible surface area. However, almost all of the amino acid residues known to be involved in early folding events are located within the folding elements.

No abstract

In vitro molecular evolution is regarded as a hill-climbing on a fitness landscape in sequence space, where the 'fitness' is a quantitative measure of a certain physicochemical property of a biopolymer. We analyzed a 'cross-section' of the enzymatic activity landscape of dihydrofolate reductase (DHFR) by using a method of analysis of a fitness landscape. We limited the sequence space of interest to the five-dimensional sequence space, where the coordinate corresponds to the 1st, 16th, 20th, 42nd and 92nd site in the DHFR sequence. Thirty six mutants mapped into the limited sequence space were taken in the analysis. As a result, the cross-section is of the rough Mt Fuji type based on the mutational additivity. The ratio of the mean slope to the roughness is 2.8 and the Z-score of the original ratio against a distribution of random references is 7.0, which indicates a large statistical significance. The existence of such a cross-section was discussed in terms of the occurrence probability of sets of five sites distantly separated from each other on the DHFR 3D structure. Our results support the effectiveness of the evolution strategy which exploits the accumulation of advantageous single point mutations in such a cross-section.