Folding/Unfolding Kinetics of Apomyoglobin

Baryshnikova, E N; Melnik, Bogdan S.; Semisotnov, Gennady V.; Bychkova, Valentina E.

doi:10.1007/s11008-005-0109-6

Cited by 2 publications

(2 citation statements)

References 29 publications

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“…Based on the plasmid containing a wild-type (WT) apomyoglobin gene, plasmids possessing mutant apomyoglobin gene variants with selected substitutions were derived. Recombinant proteins were isolated and purified according to the methods described in our previous studies (Baryshnikova et al, 2005b;Baryshnikova et al, 2005a;Baryshnikova et al, 2005c;Baryshnikova et al, 2007).…”

Section: Apomyoglobinmentioning

confidence: 99%

Some useful ideas for multistate protein design: Effect of amino acid substitutions on the multistate proteins stability and the rate of protein structure formation

Majorina

Melnik

Glukhov

et al. 2022

Front. Mol. Biosci.

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The design of new protein variants is usually confined to slightly “fixing” an already existing protein, adapting it to certain conditions or to a new substrate. This is relatively easy to do if the fragment of the protein to be affected, such as the active site of the protein, is known. But what if you need to “fix” the stability of a protein or the rate of its native or intermediate state formation? Having studied a large number of protein mutant forms, we have established the effect of various amino acid substitutions on the energy landscape of the protein. As a result, we have revealed a number of patterns to help researchers identify amino acid residues that determine the folding rate and the stability of globular proteins states and design a mutant form of a protein with desired properties.

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Section: Apomyoglobinmentioning

confidence: 99%

Some useful ideas for multistate protein design: Effect of amino acid substitutions on the multistate proteins stability and the rate of protein structure formation

Majorina

Melnik

Glukhov

et al. 2022

Front. Mol. Biosci.

Self Cite

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“…Furthermore, the observation of two state unfolding of I to U is not unprecedented, since Baryshnikova et al found that unfolding of apoMb starting in the native state N proceeds through a single intermediate state I (N « I « U) under strongly denaturing conditions. 46 Thus it is possible to effectively bypass the L and E intermediate states under conditions that strongly destabilize the folded structure.…”

Section: Apomb Unfolding Kineticsmentioning

confidence: 99%

A simple three-dimensional-focusing, continuous-flow mixer for the study of fast protein dynamics

Burke

Parul²,

Reddish

et al. 2013

Lab Chip

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We present a simple, yet flexible microfluidic mixer with a demonstrated mixing time as short as 80 µs that is widely accessible because it is made of commercially available parts. To simplify the study of fast protein dynamics, we have developed an inexpensive continuous-flow microfluidic mixer, requiring no specialized equipment or techniques. The mixer uses three-dimensional, hydrodynamic focusing of a protein sample stream by a surrounding sheath solution to achieve rapid diffusional mixing between the sample and sheath. Mixing initiates the reaction of interest. Reactions can be spatially observed by fluorescence or absorbance spectroscopy. We characterized the pixel-to-time calibration and diffusional mixing experimentally. We achieved a mixing time as short as 80 µs. We studied the kinetics of horse apomyoglobin (apoMb) unfolding from the intermediate (I) state to its completely unfolded (U) state, induced by a pH jump from the initial pH of 4.5 in the sample stream to a final pH of 2.0 in the sheath solution. The reaction time was probed using the fluorescence of 1-anilinonapthalene-8-sulfonate (1,8-ANS) bound to the folded protein. We observed unfolding of apoMb within 760 µs, without populating additional intermediate states under these conditions. We also studied the reaction kinetics of the conversion of pyruvate to lactate catalyzed by lactate dehydrogenase using the intrinsic tryptophan emission of the enzyme. We observe sub-millisecond kinetics that we attribute to Michaelis complex formation and loop domain closure. These results demonstrate the utility of the three-dimensional focusing mixer for biophysical studies of protein dynamics.

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Folding/Unfolding Kinetics of Apomyoglobin

Cited by 2 publications

References 29 publications

Some useful ideas for multistate protein design: Effect of amino acid substitutions on the multistate proteins stability and the rate of protein structure formation

Some useful ideas for multistate protein design: Effect of amino acid substitutions on the multistate proteins stability and the rate of protein structure formation

A simple three-dimensional-focusing, continuous-flow mixer for the study of fast protein dynamics

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