Mapping fast protein folding with multiple-site fluorescent probes

Maxim B. Prigozhin, Shu Han Chao, Shahar Sukenik, Taras V. Pogorelov, Martin Gruebele

Research output: Contribution to journalArticlepeer-review

Abstract

Fast protein folding involves complex dynamics in many degrees of freedom, yet microsecond folding experiments provide only low-resolution structural information. We enhance the structural resolution of the five-helix bundle protein λ6-85 by engineering into it three fluorescent tryptophan-tyrosine contact probes. The probes report on distances between three different helix pairs: 1-2, 1-3, and 3-2. Temperature jump relaxation experiments on these three mutants reveal two different kinetic timescales: a slower timescale for 1-3 and a faster one for the two contacts involving helix 2. We hypothesize that these differences arise from a single folding mechanism that forms contacts on different timescales, and not from changes of mechanism due to adding the probes. To test this hypothesis, we analyzed the corresponding three distances in one published single-trajectory all-atom molecular-dynamics simulation of a similar mutant. Autocorrelation analysis of the trajectory reveals the same "slow" and "fast" distance change as does experiment, but on a faster timescale; smoothing the trajectory in time shows that this ordering is robust and persists into the microsecond folding timescale. Structural investigation of the all-atom computational data suggests that helix 2 misfolds to produce a short-lived off-pathway trap, in agreement with the experimental finding that the 1-2 and 3-2 distances involving helix 2 contacts form a kinetic grouping distinct from 1 to 3. Our work demonstrates that comparison between experiment and simulation can be extended to several order parameters, providing a stronger mechanistic test.

Original languageEnglish (US)
Pages (from-to)7966-7971
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume112
Issue number26
DOIs
StatePublished - Jun 30 2015

Keywords

  • Fluorescence
  • Helix bundle
  • Molecular dynamics
  • Protein folding
  • Thermal denaturation

ASJC Scopus subject areas

  • General

Fingerprint

Dive into the research topics of 'Mapping fast protein folding with multiple-site fluorescent probes'. Together they form a unique fingerprint.

Cite this